ir.amu.ac.inir.amu.ac.in/11781/1/T10303.pdf · 2018. 6. 12. · i Acknowledgements All praise is almighty Allah who bestowed me with ample and perseverance that paved the path to

BIOPHYSICAL STUDIES ON AGGREGATION AND INHIBITION OF SOME PROTEINS

BY VARIOUS MOLECULES

THESIS

SUBMITTED FOR THE AWARD OF DEGREE OF

Doctor of Philosophy

IN

BIOTECHNOLOGY

By

MASIHUZZAMAN

Under the supervision of

PROF. RIZWAN HASAN KHAN

INTERDISCIPLINARY BIOTECHNOLOGY UNIT, ALIGARH MUSLIM UNIVERSITY,

ALIGARH-202002 (INDIA) 2017

i

Acknowledgements

All praise is almighty Allah who bestowed me with ample and perseverance that paved the path to

keep me going through thick and thin for the accomplishment of this endeavor….

Though only my name appears on the cover of this thesis, many great people have contributed to its

production. I owe my gratitude to all those people who have made this thesis possible and because

of whom my research experience is the one that I will cherish forever.

My deepest gratitude is to my supervisor Prof. Rizwan Hasan Khan. I have been amazingly

fortunate to have an advisor who gave me the freedom to explore on my own and at the same time

the guidance to recover when my steps faltered. His scientific acumen, strong determination,

excellent knowledge and immense valuable ideas may my work easier. He taught me the patience,

sustained efforts and always advised to have faith in Allah. I have been lucky to work under his

unique guidance that develops a sense of sincerity, tackle the harsh situation and self confidence. I

sincerely thank him for giving me immense freedom to work during the course of this doctoral

programme and training in manuscript writing. This thesis would not have been in its present form

without his guidance and utmost careful review.

I am indebted to the faculty members Prof. Asad Ullah Khan, Prof. Saleemuddin, Prof. Mohd

Owais, Dr. Hina Younus and Dr. Shahper Nazeer khan of Interdisciplinary Biotechnology Unit

for their valuable support and encouragement.

I also like to thanks to non-teaching staff Mr. Syed Faisal Maqbool, Mr. Amir, Mr. Lal Mohd.

Khan, Mr. Aqtedar Husain, Dr. Parveen Salahuddin, Mr. Ramesh Chandra, Mr. Chandra Pal,

Mr. Mohd. Nasir, Mr. Isham Khan, Mr. Ashraf, Mr. Sanjay, Mr. Karamvir, Mr. Naresh for their

help and cooperation. Without their help I could not procure the required chemicals and complete

my experimental work.

It would be not fair if I will not owe thanks to the Saba mam who has been extremely generous

and helpful for me.

I am deeply grateful to my seniors Dr. Sumit bhai and Dr. Nida aapa for the long discussions that

helped me to sort out the technical details of my work. I am also sincerely thankful to my senior

Dr. Atiya that helped me to learn the basics of biophysical techniques during my initial days.

Special thanks also go to Dr. Ejaz, Dr. Rabbani bhai, Dr. Javed bhai, Dr. Mairaj bhai, Dr. Faraz

bhai , Dr. Ishtikhar Bhai and Dr. Rehan for guiding me at various steps of my work.

I am genuinely indebted to my senior Mr. Mohsin Vahid Khan that helped me to learn the basics

of life beside research. No words are enough to thank my dearest Syed Mohammad Zakariya who

always lends a hand for experimental work and day to day problem. I wish both of you ‘best of

luck’ for future endeavors. I cannot forget to mention Kazmi bhai, Dr. Mehboob bhai, Dr. Fazle

bhai and Shadab bhai for consistently help, support, care and valuable advices.

I cannot put in words the contribution of my junior Ms. Saima Nusrat who has been truly helpful,

inspiring and supportive throughout of my research. Special thanks should also be given to Ms.

Tajalli Ilm Chandel who always altruistically helps me in every situation from experimental hurdle

to common issues.

ii

My special thanks also go to Mr. Parvez Alam for timely help and great sense of humor that kept

me enthusiastic and cheerful all the time. Special thanks should also be given to Jyoti, Syed faisal

khursheed and Aaquib who always selflessly help me. I am also thankful to Asra, Ayesha, Esha,

Sadiya, Aiman and Haris for their humble support and cooperation. I truly acknowledge the

sincere help and efforts of my juniors Aamj, Sayeed, Ibrar, Azhar, Sajid Nadeem, Ravi, Haris,

Abdullah, Arsalaan and Faijan during the most busy and difficult moments. Thanks are also

extended to Dr. Farzana aapa, Dr. Neelofer, Sidra and Naureen for their help and cooperation.

It would be not fair, if I will not owe thanks to the most gorgeous people I come across in my life

and P.hD tenure Dr. Sayeed, Dr. Tarique Dr. Kasif Jamal, Dr. Imran Asghar, Akil Akhtar, Shahid

Habib, Dr. Abad Khan, Dr. Mohammad Aadil, Dr. Ahmad Yazdaan, Mohammad Shoeb and Dr.

Ahmad Abdur Rehman, and who always supporting me and giving the valuable suggestion, help,

encouragement and guidance in spite of their busy working schedules. I also thankful to old seniors

of IBU like Dr. Akram, Raghib Ashraf, Dr. Javed Iqbal, Dr. Shazi, Dr. Arun, Dr. Anis, Dr.

Azmat and Dr. Asif.

The place Aligarh itself has taught me several excellent lessons here, I have met friends for life. I

express heartiest thanks to my friends Dr. Faiz Haque, Dr. Sharique Ansari, Dr Beenish, Dr.

Danish, Syed Shams Tabrez, Hasaan, Mohammad Saquib, Nizam, Bharat, Saad, Ahmad ul bari,

Dr. Ashad, Salman Shahid, Dr. Abishekh, Dr. Shadab, Dr. Waseem, Dr. Obaid, Dr. Sarfaraz, Dr.

Aamir Hussain, Dr, Maroof Alam, Dr Sameer, Aadil afroz, Furqaan, Dr. Firoz ansari, Mobaid,

Dr. Rizwanul Haque, and Dr. Sajid Khan, for their love, care and moral support. The

acknowledgement is incomplete to mention the name of Amim Ahmad, Yasir Iqbal Khan and

Ahmad Zeeshan Javaid who made family like environment for me to survive the student life from

senior secondary school to Ph.D. with cosy and comfort.

I wish my warm thanks to two special people in my life for their love, care, encouragement,

blessings and understanding that kept me moving on, for me they are gateway to heaven. I am

feeling incredibly emotional in acknowledging them (my parents) who laid seeds of enthusiasm in

me for knowledge and give meaning of life. The unfathomable blessings of my abbu and ammi are

the spiritual strength with which I have persuaded this thesis. I have short to words to thanks my

brothers Takiuzzaman, Wahiduzzaman and khaliquzzaman, who always be my best guide in

every situation. They are with me in bearing the brunt of the frustrations and sharing in the joy of

the successes. I have short of words to thank my elder sister, brother in law (Sohail Akhtar) and

bhabhi for being my strength and moral teacher. They always nourished me by their du’as and

advices which cannot be justified merely by mentioning here. The special mention goes to my sweet

nephews Anas, Aamir, Basit and Maaz.

I attribute my sincere thanks to the Interdisciplinary Biotechnology Unit, Aligarh Muslim

University, Aligarh. I acknowledge the financial assistance provided by University Grant

Commission, Department of science and technology and Department of Biotechnology, New Delhi

during the course of this study. I am deeply thankful to the Aligarh Muslim University to learn

several excellence lessons in my life.

Masihuzzaman

Abstract

1

ABSTRACT

To understand the mechanism of protein misfolding and aggregation and then

development of therapeutics to overcome the pathological condition related to these

issues is topic of utmost investigation; however there is much more to investigate. One of

the major challenges in development of therapeutics against the aggregation associated

pathological disorder is to study the process/mechanism in details. Thus in this thesis, we

give detailed study of the protein aggregation process/mechanism under various

conditions and then checked the inhibitory potential of some molecules on the protein

aggregation. The mechanism by which these molecules inhibit the formation of amyloid

fibrils is also discussed in details. This work enhanced understanding of protein

misfolding, give ways to prevent misfolding and suggestion of molecules that generate

new ideas for biotechnology and pharmaceutical industries and medical sciences. Chapter 1

Protein and peptides are converted from their soluble forms into highly ordered

fibrillar aggregates under various conditions inside the cell. Such transitions confer

diverse pathological conditions such as prion diseases, Huntington’s, Parkinson’s

disease, Alzheimer’s disease, and type II diabetes. Presently, major advances are

witnessed to understand the structural basis of detailed insight into the mechanism of

amyloid formation, their cytotoxicity along with various therapeutic approaches

towards it. This chapter classifies and summarizes the detailed overview of protein

misfolding and aggregation at their molecular level including the factors that

promotes protein aggregation under in vivo and in vitro conditions. In addition, we

describe the recent technologies that aid characterization of amyloid aggregates along

with several models that might be responsible for amyloid induced cytotoxicity to

cells. Finally, we have discussed some of the important approaches that are being

employed to combat and reverse the amyloidogenesis.

Chapter 2

Stem bromelain, a cysteine proteases from Ananas comosus is a widely accepted

therapeutic drug with broad medicinal application. It exists as intermediate states at

pH 2.0 and 10.0, where it encountered in gastrointestinal tract during adsorption

Abstract

2

(acidic pH) and in gut epithelium (alkaline pH), respectively. In this study, we

monitored the thermal aggregation/amyloid formation of SB at different pH

intermediate states. Thermal treatment of stem bromelain at pH 10.0 favors the

fibrillation in which the extent of aggregation increases with increase in protein

concentration. However, no fibril formation in stem bromelain at pH 2.0 was found at

all the concentration used at pH 10.0. The fibril formation was confirmed by various

techniques such as turbidity measurements, Rayleigh light scattering, dye binding

assays and far UV circular dichroism. The Dynamic light scattering confirmed the

formation of aggregates by measuring the hydrodynamic radii pattern. Moreover,

microscopic techniques were performed to analyze the morphology of fibrils. The

aggregation behavior may be due to variation in number of charged amino acid

residues. The less negative charge developed at pH 10.0 may be responsible for

aggregation. This work helps in to overcome the aggregation related problems of stem

bromelain during formulations in pharmaceutical industry.

Chapter 3

Negatively charged species such as nucleic acids have commonly been found associated

with the proteinaceous deposits in the tissue of patients with amyloid diseases.

Numerous studies have demonstrated that various environmental and intracellular

factors affect the fibrillation property of proteins, by accelerating the process of

assembly. Thus in present study, the effect of calf thymus DNA (CT-DNA) on stem

bromelain, a proteolytic phytoprotein, is investigated at pH 2.0, using multiple

approaches that includes turbidity measurements, Rayleigh light scattering, dye binding

assay (ThT and ANS), far-UV circular dichroism, dynamic light scattering fluorescence

microscopy and transmission electron microscopy. Large sized β-sheet aggregates of

SB are found in presence of CT-DNA at pH 2.0. The propensity of aggregation

concomitantly increases with increasing concentration of CT-DNA (0-100 μM) and

level off at higher concentration of CT-DNA (beyond 100 µM). Isothermal titration

calorimetric results confirmed that electrostatic interaction between positively charged

SB at pH 2.0 and negatively charged phosphate group of CT-DNA is the probable

mechanism behind aggregate formation. However, the hydrophobic interaction between

CT-DNA and SB cannot be neglected. Survival of aggregates even after treatment with

DNase indicates that aggregates are DNase resistant.

Abstract

3

Chapter 4

Neurodegenerative disorders are mainly associated with amyloid fibril formation of

different proteins. Stem bromelain, a cysteine protease, is known to exist as a molten

globule state at pH 10.0. It passes through the identical surrounding (pH 10.0) in the

gut epithelium of intestine upon oral administration. Protein-surfactant complexes are

widely employed as drug carriers, so the nature of surfactant towards protein is of

great interest. The present work describes the effect of cationic surfactants (CTAB &

DTAB) and their hydrophobic behavior towards amyloidogenesis behavior of stem

bromelain at pH 10.0. Multiple approaches including light scattering, far UV-CD,

turbidity measurements and dye binding assay (ThT, Congo red and ANS) were

performed to measure the aggregation propensity of SB. Further, we monitored the

hydrodynamic radii of aggregates formed using dynamic light scattering techniques.

Structure of fibrils was also visualized through fluorescence microscopy as well as

TEM. At pH 10.0, low concentration of CTAB (0-200 µM) induced amyloid

formation in SB as evident from a prominent increase in turbidity and light scattering,

gain in β-sheet content and enhanced ThT fluorescence intensity. However, further

increase in CTAB concentration suppressed the fibrillation phenomenon. In contrast,

DTAB did not induce fibril formation at any concentration used (0-500 µM) due to

lower hydrophobicity. Net negative charge developed on protein at high pH (10.0)

might have facilitated amyloid formation at low concentration of cationic surfactant

(CTAB) due to electrostatic and hydrophobic interactions.

Chapter 5

Protein aggregation and misfolding have been allied with numerous human disorders

and thus inhibition of such occurrence has been center for intense research efforts

against these diseases. Here, we investigated anti-fibrillation activity of cysteine and

its effect on amyloid fibril formation kinetics of stem bromelain. We established the

anti-fibrillation and anti-aggregation activities of cysteine by using multiple

approaches like turbidity measurements, dye binding assays (ThT and ANS) and

structural changes were monitored by circular dichroism (CD) followed by electron

microscopy. Our experimental study inferred that cysteine inhibits temperature

induced fibrillation of protein in a concentration dependent way. In addition, MDA-

MB-231 cell viability of pre-formed amyloid was increased in presence of cysteine as

Abstract

4

compared to the fibrils alone . Furthermore, dynamic light scattering studies of native,

aggregated as well as incubated (amyloids in presence of cysteine) samples indicates

that cysteine restores native like structures of stem bromelain. Isothermal titration

calorimetric results revealed that hydrogen bonding between cysteine and stem

bromelain plays a significant role during inhibition of stem bromelain aggregation.

However, thiophilic interaction between thiol group of cysteine and aromatic amino

acid residue of stem bromelain may also have noteworthy role in inhibition of

amyloid formation.

Chapter 6

Protein misfolding and aggregation lead to amyloid generation that in turn may induce

cell membrane disruption and leads to cell apoptosis. In an effort to prevent or treat

amyloidogenesis, large number of studies has been paying attention on breakthrough

of amyloid inhibitors. In the present work, we aim to access the effect of two drugs

i.e, acetylsalicylic acid and 5-amino salicylic acid on insulin amyloids by using

various biophysical, imaging, cell viability assay and computational approaches. We

established that both drugs reduce the turbidity, light scattering and fluorescence

intensity of amyloid indicator dye thioflavin T and maximum reduction was observed

in presence of 5-aminosalicylic acid. Premixing of drugs with insulin inhibited the

nucleation phase and inhibitory potential was boosted by increasing the concentration

of the drug. Cicular dichroism results indicated that both drugs stabilized insulin and

help it to attain a native or native like conformation. Moreover, addition of drugs at

the studied concentrations attenuated the insulin fibril induced cytotoxicity in breast

cancer cell line MDA-MB-231. Our results highlight the amino group of salicylic acid

exhibited enhanced inhibitory effects on insulin fibrillation in comparison to acetyl

group. It may be due to presence of amino group that helps it to prolong the

nucleation phase with strong binding as well as disruption of aromatic and

hydrophobic stacking that plays a key role in amyloid progression. The inhibitory

potential of these drugs beside insulin fibrillation have considerable biological

implications, because insulin fibrillation is recognized as a causative agent to injection

amyloidogenesis.

Contents

LIST OF CONTENTS Page No.

Acknowledgement

Abstract

i-ii

iii-vi

Abbreviations and Symbols vii-ix

List of figures x-xvi

List of tables xvii-xviii

Chapter 1

Review of literature

Protein folding, misfolding, aggregation and mechanism of amyloid

cytotoxicity: an overview and therapeutic strategies to combat

aggregation

1.1. Protein folding 1

1.2. Protein misfolding and aggregation 2

1.3. Possible mechanism of amyloid induced cytotoxicity 19

1.4. Therapeutic strategies for aggregation/amyloid inhibition 22

1.5. Proteins used in this study 28 1.6. Conclusion 33

Chapter 2

Amyloidogenic behavior of different intermediate state of stem

bromelain: a biophysical insight

2.1. Introduction 35

2.2. Materials and methods 37

2.3. Results and discussion 41

2.4. Conclusion 51

Contents

Chapter 3

DNA induced aggregation of stem bromelain: a mechanistic insight




3.4. Conclusion 67

Chapter 4

Surfactant-mediated amyloidogenesis behavior of stem bromelain: a

biophysical insight




4.4. Conclusion 85

Chapter 5

Cysteine act as a potential anti-amyloidogenic agent with protective

ability against amyloid induced cytotoxicity for stem bromelain




5.4. Conclusion 101

Chapter 6

Amino group of salicylic acid exhibits enhanced inhibitory potential

against insulin amyloid fibrillation with protective aptitude towards

amyloid induced cytotoxicity



6.3. Results and Discussion 109

6.4. Conclusion 121

Contents

BIBLIOGRAPHY 122

LIST OF PUBLICATION 153

AUTOBIOGRAPHY

List of Abbreviations and Symbols

vii

ABBREVATIONS AND SYMBOLS

% percentage

= equal to

µ micro

µl microlitter

µM micromolar

mM milimolar

Å angstrom

ANS 1-anilino-8-naphthlene sulfonic acid

BI bovine insulin

CD circular dichroism

cm centimeter

CR congo red

DLS dynamic light scattering

EM electron microscopy

ε specific extinction coefficient

Fd fraction denaturation

FI fluorescence intensity

FTIR fourier transform infrared

h hour

ITC isothermal titration calorimetry

J Joule

kcal kilocalorie

kDa kiloDalton

Ka association constant

Kb binding constant

kq quenching constant/biomolecular rate constant

l path length of the cell

M molar

mdeg millidegree

mg milligram


viii

min minute

ml milliliter

mM millimolar

mm millimeter

MRE mean residual ellipticity

N native state

n number of amino acid

NaCl sodium chloride

NaOH sodium hydroxide

Gly glycine

nm nanometer

NA nucleic acid

ºC degree Celsius

R universal gas constant

Rh hydrodynamic radii

RLS Rayleigh light scattering

s second

SB stem bromelain

FM fluorescence microscopy

TEM transmission electron microscopy

ThT thioflavin-T

Trp tryptophan

Tyr tyrosine

U unfolded state

υ0 initial velocity

Vmax maximum velocity

v/v volume by volume

UV ultra violet

w/v weight by volume

α alpha

β beta

ΔG standard free energy change


ix

ΔH standard enthalpy change

ΔS standard entropy change

η viscosity of water

θ theta

θobs observed ellipticity

λ lambda

λmax wavelength maximum

List of Figures

x

LIST OF FIGURES

Figure No. Title Page No.

Figure 1.1 Schematic representation showing protein aggregation

mediated by partially folded intermediate. 4

Figure 1.2

(A) Schematic illustration of protein aggregation through

reversible association of monomers.

(B) Schematic illustration of aggregation of

conformationally-altered protein.

(C) Schematic illustration of aggregation of chemically-

modified proteins.

(D) Schematic illustration of protein aggregation through

nucleation-dependent pathway.

(E) Schematic illustration of aggregation of protein on

container surfaces and air-liquid interface.

11

12

13

14

15

Figure 1.3 Factors affecting the aggregation of proteins under

various conditions. 16

Figure 1.4 Schematic illustrations of pathway that inhibit amyloid

fibril formation (A)-(E) represent the molecules used for

either inhibition or stabilization of protein. (A) Native

state stabilization (B) Refolding of polypeptide (C)

Diversion from oligomerization pathway (D) Inhibition

of fibril elongation by β-sheet breakers (E)

Disaggregation of amyloid aggregate.

23

Figure 1.5 Three dimensional structure of stem bromelain (modeled

using Phyre software) showing presence of two domains.

The figure is generated in Chimera software.

28

Figure 1.6 Crystal structure of bovine insulin as obtained from PDB

having PDB id 2zp6. 32

Figure 2.1 Turbidity measurements of SB samples at pH 2.0 & pH

10.0 at two different concentrations of SB with respect to

time at 65°C.

42

List of Figures

xi

Figure 2.2 Rayleigh light scattering intensity at 350 nm of thermally

induced SB samples at pH 2.0 & pH 10.0 at two different

concentrations of SB with respect to time.

43

Figure 2.3 Time-dependent changes in ThT fluorescence spectra

and fluorescence intensity at 485 nm of SB at pH 2.0 &

pH 10.0. 5 μM (A & B) and 10 μM (C & D).

44

Figure 2.4 ANS fluorescence spectra of (A) 5 μM and (B) 10 μM

thermally induced stem bromelain at pH 2.0 and pH 10.0

at various time intervals. (C) ANS fluorescence intensity

at 480 nm of SB (5 μM & 10 μM) at pH 10.0. (D) Shift

in λmax of SB at pH 10.0 (5 μM & 10 μM).

46

Figure 2.5 Far-UV CD spectra of SB at pH 7.4 (native SB), pH 2.0

(PFI state) & pH 10.0 (MG state) at two different

concentration exposed at different temperature.

48

Figure 2.6 DLS measurements to determine the hydrodynamic radii

of stem bromelain at 65ºC. The hydrodynamic radii of

stem bromelain (A) 5 μM (pH 7.4), (B) 5 μM (pH 2.0),

(C) 10 μM (pH 2.0), (D) 5 μM (pH 10.0), (E) 10 μM (pH

10.0).

49

Figure 2.7 Fluorescence microscopy of thermally induced

aggregation of SB (A) 5 μM (pH 2.0), (B) 10 μM (pH

2.0), (C) 5 μM (pH 10.0), (D) 5 μM (pH 10.0) and

transmission electron microscopic images of heat

induced SB (E) 5 μM (pH 2.0), (F) 10 μM (pH 2.0), (G)

5 μM (pH 10.0), (H)10 μM (pH 10.0).

50

Figure 2.8 Schematic representation of thermal induced fibrillation

of stem bromelain at different intermediate states. 50

Figure 3.1 Turbidity at 350 nm of SB in absence and presence of

varying concentration of CT-DNA (0-120 µM) at pH 2.0. 58

Figure 3.2 Rayleigh light scattering at 350 nm of SB in the absence

and presence of varying concentration of CT-DNA (0-

120 µM) at pH 2.0.

59

Figure 3.3 (A) ThT fluorescence intensity of SB at 485 nm at

varying concentration of CT-DNA (0-100 µM) at pH 2.0.

(B) ThT fluorescence spectra of SB (pH 2.0) in the

absence and presence of 50 µM and 100 µM CT-DNA.

60

List of Figures

xii

Figure 3.4 (A) ANS fluorescence spectra of SB in the absence and

presence of varying concentrations of CT-DNA at pH

2.0. (B) Changes in emission maxima (λmax) and

fluorescence intensity of SB as a function of varying CT-

DNA concentration.

61

Figure 3.5 Far-UV CD spectra of SB at pH 7.4, and in the absence

and presence of CT-DNA at pH 2.0. 62

Figure 3.6 Dynamic light scattering measurement of SB in the

absence and presence of various concentration of CT-

DNA (A) SB at (pH 7.4), (B) SB at pH 2.0, (C) SB (pH

2.0) + 50 µM CT-DNA, (D) SB (pH 2.0) + 100 µM CT-

DNA.

63

Figure 3.7 Fluorescence microscopic images for (A) Native SB (pH

7.4) (B) SB (pH 2.0) (C) SB (pH 2.0) + 100 µM CT-

DNA, and transmission electron microscopic images for

(D) Native SB (pH 7.4) (E) SB (pH 2.0) (F) SB (pH 2.0)

+ 100 µM CT-DNA.

64

Figure 3.8 Aggregated assembly of SB treated with DNase (A)

Rayleigh light scattering (B) ThT fluorescence intensity

at 485 nm (C) Far-UV CD spectra and (D) Transmission

electron microscopic image.

66

Figure 3.9 Schematic representation of DNA mediated fibrillation

of stem bromelain. 67

Figure 4.1 Chemical structures of CTAB and DTAB. 69

Figure 4.2 Turbidity measurements of SB samples at 350 nm in the

presence of 0-500 µM CTAB and DTAB at pH 10.0.

Prior to measurements all samples were incubated

overnight at 25°C.

74

Figure 4.3 Rayleigh light scattering of SB at 350 nm in the absence

and presence of CTAB and DTAB (0-500 µM) at pH

10.0. Before measurements all samples are incubated

overnight at 25°C.

76

Figure 4.4 (A) ANS fluorescence spectra of SB in the absence and

presence of 0-500 µM CTAB and DTAB at pH 10.0. (B)

ANS fluorescence intensity of SB at 480 nm in the

presence of different concentration of CTAB.

77

List of Figures

xiii

Figure 4.5 (A) ThT fluorescence spectra of SB in the absence and

presence of 0-500 µM CTAB and DTAB at pH 10.0. (B)

ThT fluorescence intensity at 485 nm of SB under

various conditions.

78

Figure 4.6 Intrinsic fluorescence measurements (A) Changes in Trp

emission maxima of SB in native state (pH 7.4) and MG

state (pH 10.0) as a function of CTAB concentration. (B)

Changes in fluorescence intensity of SB at pH 7.4 and

pH 10.0 as a function of CTAB concentration.

80

Figure 4.7 (A) Far UV-CD spectra of SB at pH 7.4, pH 10.0 and in

the presence of different concentration of CTAB and

DTAB. (B) Near UV-CD spectra of SB under different

conditions.

81

Figure 4.8 Dynamic Light Scattering of SB under different

conditions. (A) Native state (pH 7.4), (B) SB (pH 10.0),

(C) SB (pH 10.0) + 200 µM CTAB, (D) SB (pH 10.0) +

500 µM DTAB.

83

Figure 4.9 Fluorescence microscopic images of SB (A) SB at pH

7.4, (B) SB pH 10.0, (C) SB + 200 µM CTAB, (D) SB +

500 µM DTAB. Transmission electron microscopic

images of (E) SB at pH 7.4, (F) SB + 200 µM CTAB,

(G) SB + 500 µM DTAB.

84

Figure 4.10 Schematic representation of effect of CTAB and DTAB

on MG state of SB at pH 10.0. 85

Figure 5.1 (A) Turbidity measurements of SB at 350 nm with

increasing time (0-12 h) and (B) in the presence of

various concentrations of cysteine (0-2 mM). (C)

Rayleigh light scattering measurement of SB in absence

and presence of various concentration of cysteine (0-2

mM).

93

Figure 5.2

Figure 5.3

(A) ThT fluorescence spectra of SB incubated at 65°C in

the absence and presence of cysteine (0-2 mM). (B) ThT

fluorescence intensity of SB at 485 nm incubated in the

absence and presence of cysteine (0-2 mM) at 65°C. (C)

Effect of cysteine on SB ThT fluorescence kinetics in the

absence and presence of different concentration of

cysteine.

(A) ANS fluorescence spectra of SB incubated at 65ºC in

the absence and presence of various concentration of

cysteine. (B) ANS fluorescence intensity of SB at 480

nm incubated at 65ºC in the absence and presence of

different concentration of cysteine.

95

96

List of Figures

xiv

Figure 5.4 Far-UV CD spectra of SB in native state (pH 7.4), MG

state (pH 10.0) and aggregated state in the absence and

presence of cysteine (2 mM).

97

Figure 5.5 Dynamic light scattering measurement of SB in absence

and presence of cysteine (A) SB in native state (pH 7.4,

25°C), (B) SB in MG state (pH 10.0, 25°C), (C) SB at

pH 10.0, 65°C, (D) SB at pH 10.0, 65°C + 2 mM

cysteine.

98

Figure 5.6 Fluorescence microscopic images of (A) SB at 25°C, (B)

SB incubated at 65°C in absence of cysteine, (C) SB

incubated at 65°C in the presence of 2 mM cysteine.

Transmission Electron Microscopic images of (D) SB at

25°C, (E) SB incubated at 65°C in absence of cysteine

(F) SB incubated at 65°C in the presence of 2 mM

cysteine.

99

Figure 5.7 MDA-MB-231 cell viability after being exposed to SB

fibrils formed in the absence and presence of cysteine

(1mM and 2mM). *Statistically significant from the

control group, p ≤ 0.01 and # statistically significant

from the SB, p ≤ 0.01.

101

Figure 6.1 (A) Turbidity measurements of bovine insulin samples

incubated at 65°C over a period of 24h in the absence

and presence of ASA and 5-ASA (0-1000 µM). (B)

Rayleigh scattering measurements of bovine insulin

samples incubated at 65°C over a period of 24h in the

absence and presence of ASA and 5-ASA (0-500 µM).

109

Figure 6.2 Effects of ASA and 5-ASA on amyloid formation of

bovine insulin. (A) ThT fluorescence spectra of bovine

insulin in the absence and presence of ASA at various

concentrations. (B) ThT fluorescence spectra of bovine

insulin in the absence and presence of 5-ASA at various

concentrations. (C) ThT fluorescence intensity at 485 nm

of bovine insulin in the absence and presence of ASA

and 5-ASA at various concentrations. (D) Kinetics of

bovine insulin fibrillogenesis in the presence of various

concentrations of (ASA & 5-ASA). Results represent

means ± s.d (n=3).

110

Figure 6.3 (A) ANS fluorescence spectra of bovine insulin

incubated at 65ºC in the absence and presence of ASA.

(B) ANS fluorescence spectra of bovine insulin

incubated at 65ºC in absence and presence of 5-ASA. (C)

(A) ANS fluorescence intensity at 480 nm of bovine

insulin incubated at 65ºC in the absence and presence of

112

List of Figures

xv

ASA and 5-ASA. Experimental data represent the

average ± s.d (n= 3).

Figure 6.4 (A) Far UV-CD spectra of bovine insulin in the absence

and presence of 500 µM ASA at different conditions. (B)

Far UV-CD spectra of bovine insulin in the absence and

presence of 500 µM 5-ASA at different conditions.

113

Figure 6.5 DLS pattern of bovine insulin in the absence and

presence of ASA and 5-ASA. (A) Bovine insulin alone

(pH 7.4, 25°C) (B) Bovine insulin in presence of 500 µM

ASA (pH 7.4, 25°C), (C) Bovine insulin in the presence

of 500 µM 5-ASA (pH 7.4, 25°C), (D) Bovine insulin

alone (pH 2.0, 65°C), (E) Bovine insulin in the presence

of 500 µM ASA (pH 2.0, 65°C) (F) Bovine insulin in the

presence of 500 µM 5-ASA (pH 2.0, 65°C).

114

Figure 6.6 TEM and FM images of bovine insulin amyloid

formation in the absence and presence of salicylic acid

derivatives. (A) Native bovine insulin (B) Bovine insulin

incubated at 65°C. (C) Bovine insulin co-incubated with

500 µM ASA at 65°C, (D) Bovine insulin co incubated

with 500 µM 5-ASA at 65°C, (E) FM image of native

bovine insulin, (F) FM image of bovine insulin incubated

at 65°C, (G) FM image of bovine insulin co-incubated

with 500 µM ASA at 65°C, (H) FM image of bovine

insulin co incubated with 500 µM 5-ASA at 65°C.

116

Figure 6.7

MTT reduction assay for cell cytotoxicity of 24h aged

bovine insulin amyloid fibrils in MDA-MB-231 breast

cancer cell lines in absence and presence of different

concentration of ASA and 5-ASA. (A) Incubation time

24h (B) incubation time 48h. Control represents cell lines

without prior exposure to bovine insulin fibrils.

*Statistically significant from the control group, p ≤

0.01and # statistically significant from the bovine insulin,

p ≤ 0.05 for ASA and 5-ASA.

118

Figure 6.8 Molecular docking results of drugs (ASA & 5-ASA) +

bovine insulin complex. (A) acetylsalicylic acid is shown

in a stick representation, and bovine insulin represented

with ribbon model. (B) Detailed view of the docking

poses of acetylsalicylic acid + bovine insulin complex.

(C) 5-amino salicylic acid is shown in a stick

representation, and bovine insulin represented with

ribbon model. (D) Detailed view of the docking poses of

5-amino salicylic acid + bovine insulin complex.

119

List of Figures

xvi

Figure 6.9 Schematic representation of bovine insulin aggregation

and its inhibition by acetylsalicylic acid (ASA) and 5-

aminosalicylic acid (5-ASA).

120

List of Tables

xvii

LIST OF TABLES

Table No. Title Page No.

Table 1.1 Proteins involved in human diseases caused by amyloid

formation.

3

Table 1.2 Analytical methods generally used for studying protein

structure, folding and self assembly.

10

Table 1.3 Anti amyloidogenic agents and related proteins of study. 26

Table 1.4 Amino acid composition of stem bromelain. 29

Table 1.5 Physiochemical properties of proteins used in this study. 33

Table 2.1 Spectroscopic Properties of SB at different conditions

following heat treatment.

45

Table 2.2 ANS fluorescence intensity and shift in λmax. 47

Table 2.3 Hydrodynamic radii (Rh) and polydispersity (Pd) of SB

at different pH.

49

Table 3.1 Spectroscopic properties of SB at different conditions. 61

Table 3.2 Secondary structure properties of SB at different

concentration of CT-DNA.

63

Table 3.3 Hydrodynamic radii and polydispersity of SB with

varying concentration of CT-DNA.

64

Table 3.4 Thermodynamic parameters obtained by Isothermal

Titration Calorimetric measurements of SB with CT-

DNA.

65

Table 4.1 Spectroscopic properties of SB at different conditions. 78

Table 4.2 Shift in λmax of SB at pH 10.0 upon binding with CTAB

and DTAB.

79

Table 4.3 Hydrodynamic radii (Rh) and polydispersity (Pd) of SB

at different conditions.

83

List of Tables

xviii

Table 5.1

Table 5.2

Table 6.1

Table 6.2

Table 6.3

Hydrodynamic radii (Rh) and polydispersity (Pd) index

of Stem bromelain (SB) in absence and presence of

cysteine.

Thermodynamic parameters obtained by isothermal

titration calorimetric measurements of SB with cysteine.

Spectroscopic properties of Bovine insulin (BI) in

absence and presence of ASA and 5-ASA at various

conditions.

Hydrodynamic radii (Rh) and polydispersity (Pd) index

of Bovine insulin (BI) in absence and presence of

varying concentration of ASA and 5-ASA.

Molecular docking parameters for drugs (ASA and 5-

ASA) - bovine insulin interaction.

98

100

111

115

120

CHAPTER 1

Review of literature Chapter 1

1

Protein folding, misfolding, aggregation and mechanism of

amyloid cytotoxicity: an overview and therapeutic strategies

to combat aggregation

Proteins are significant biomolecules, consisting of one or more long chain of amino

acids that are fundamental to the proper functioning of cells. It may be folding;

unfolding, misfolding and aggregation resulted into either functional or non-

functional states. A major cause for various neurodegenerative diseases is conversion

of peptides and proteins into characteristic amyloid fibrils. In such diseases, structural

transitions occur from soluble functional states (protein or peptides) to highly

organized fibrillar aggregates. All together, the gathering of aggregates (abnormal

protein and peptide) exerts toxicity by overwhelming protein degradation pathways,

disturbing intracellular transport, and/or disturbing vital cell functions. Protein

aggregation or amyloid formation is governed by various in vivo and in vitro factors.

Here in, we have discussed details of aggregation process, neurological disorders,

recent advances, mechanism of aggregation and cytotoxicity, their characterization,

emerging technologies towards exploration, detection at molecular level and various

strategies to combat the aggregation phenomenon under both in vivo and in vitro

conditions.

1.1. Protein folding

Proteins are most functional players of living cells and are crucial component of

biological machinery that runs living organisms. These biomolecules have diverse

structure and functions, are synthesized in cellular machinery known as ribosomes.

The linear covalent structure of polypeptide produced by the ribosomes is ultimately

headed towards specific native functional conformation (Finkelstein et al., 2017).

This process which is guided by the amino acid sequence of polypeptide is known as

„protein folding‟. All biological processes governed by proteins that are accompanied

by conformational changes in proteins under various conditions. Understanding how

this process occurs is one of the greatest challenges in protein science. It may be

folding, unfolding and misfolding that results into either a functional or non-

functional state of a protein. Prokaryotic and eukaryotic systems possess several

Chapter 1 Review of literature

2

thousand different kinds of proteins. If all of them try to exist in primary structure, it

will be difficult to accommodate all these within a limited area of a cell. Therefore,

protein folding provides geometry of minimum volume for proteins to exist in a cell.

1.2. Protein misfolding and aggregation

Protein is one of most abundant molecules in biological world and their correct

folding into native functional conformation is a challenging task inside the crowded

milieu of the living cell. The failure to do so may result in pathological conditions

collectively known as “protein misfolding diseases”. These are found as extracellular

plaques (amyloids) or intracellular inclusions and are characterized by presence of

organized fibrillar structures whose main constituent is a specific protein or peptide

which vary for different types of diseases (Westermark et al., 2005). Each disease is

associated with unique protein or peptide and a distinctive set of tissues is affected.

Loss of cellular protein quality-control, ineffective execution of molecular chaperone

machinery, incapability of ubiquitine-proteosome complex to demean and abolish

misfolded aggregation prone molecules, hampering of normal cellular transport of

protein, inappropriate protease activity producing amyloidogenic fragments of

protein, destabilizing mutations, etc. are some factors that are responsible for such an

unwanted aggregation in proteins (Falk and Skinner, 1999). The diagnostic aspect

familiar with protein aggregation disease is the deposition of insoluble aggregates

called amyloid fibrils and hence the generic term amyloidogenesis. Some of

neurodegenerative diseases are Parkinson‟s, cystic fibrosis, Huntington‟s, Marfan

syndrome, Fabry disease, Alzheimer‟s, type II diabetes, some cancers, Creutzfeldt-

Jakob disease (table 1.1) that arises due to different pathological mechanism of

dramatic social impact (Stefani, 2004; Ono et al., 2014; Huang et al., 2015; Sami et

al., 2017). Earlier well-ordered fibrillar structure was accredited to only pathological

conditions, but some functional amyloids presents in fungi, insects and bacteria seems

to play significant role in maintaining the homeostatis of the organism (Romero and

Kolter, 2014). In addition, maximum amyloids are toxic in nature to cells and

propensity of amyloid formation vary from sequence to sequence suggesting that

some residue favors more aggregation (Eisenberg and Jucker, 2012; Lee et al., 2017).


3

Table 1.1 Proteins involved in human diseases caused by amyloid formation.

Proteins involved Disease conditions

Proteins involved Disease conditions

Alpha-synuclein Parkinson‟s disease

Lactoferrin Corneal amyloidosis

Amyloid beta

Peptide

Inclusion body

mysotis Lysozyme Hereditary non-

neuropathic systemic

amyloidosis

Amylin (IAPP) Diabetes mellitus

type 2 Medin Aortic medial

Amyloidosis

β2 microglobulin Dialysis related

amyloidosis

Prion protein Spongiform

encephalopathies

Beta amyloid Alzheimer‟s

disease

Prolactin Prolactinomas

Calcitonin Medullary

carcinoma of

thyroid

PrPsc

Fatal familial

insomnia

Gelsolin Finnish

amyloidosis

Superoxide

dismutase 1

Amyotrophic lateral

sclerosis

Huntingtin Huntington‟s

disease

Serum amyloid A Rheumatoid arthritis

Insulin Injection localized

amyloidosis

Transthyretin Familial amyloid

polyneuropathy

Immunoglobulin

light chain AL

Systemic

amyloidosis Tau Frontotemporal

dementia

1.2.1. Classification

Due to lack of precise description, various authors classified protein aggregates into

different categories. It is appropriate to categorize protein aggregates into following

categories that include amorphous and amyloid, in vitro and in vivo. Amyloid fibrils

are examples of ordered aggregates that occurs in both (in vitro and in vivo)

conditions and are fibrillar in nature, whereas unordered and amorphous aggregates

formed under in vivo conditions are termed as inclusion bodies (Chiti et al., 1999).


4

Likewise during refolding, proteins (high concentration) forms disordered aggregates

under in vitro conditions. In addition, non-covalent (physical) versus covalent

(chemical) aggregates, irreversible versus reversible aggregates, soluble versus

insoluble oligomers are some other examples of protein aggregates (De Young et al.,

1993; Fink, 1998). Protein aggregation has been usually implicit to engage native or

unfolded state, where as extracellular aggregates come up from native-like

conformations. In addition, aggregation is probably arise from some specific partially

folded intermediates (figure 1.1) and favored by several factors and environment that

enhances such type of populations.

Figure 1.1 Schematic representation showing protein aggregation mediated by partially

folded intermediate.

This is due to presence of bulky patches of adjacent surface hydrophobicity that

makes them more prone to aggregate under various conditions.

1.2.2. Protein aggregation in vitro

The misfolding and aggregation of proteins does not only have pathological

consequences but is also a major and serious problem encountered in the field of

biotechnology and pharmaceutical industries during the in vitro study. Protein

aggregates under various conditions and higher concentration of proteins acts as a

barrier in the refolding process (Benjwal et al., 2006). Thermodynamic parameters

which provide the stability of proteins are hindered by enhanced temperature that

causes denaturation of proteins followed by aggregation. Many proteins also


5

aggregates, when they are put under relatively weak native conditions (Oberg et al.,

1994). The purification of recombinant proteins is hampered by their aggregation in

the form of inclusion bodies (Stempfer and Rudolph, 1996). Moreover, protein

folding is also a complication during industrial manufacturing of therapeutic proteins.

During purification and processing, proteins encounters several stress such as high

temperature, high protein concentration, surface adsorption, shear strain,

fermentation, freeze-thawing, and drying etc which is expected to change their

structure and making them more prone to aggregation (Mahler et al., 2009). Further,

in vitro studies have provided several clues with which we can explore the mechanism

underlying the amyloid fibril formation.

1.2.3. Structure and morphology

The morphology of proteins is mainly determined by the conditions of solutions

irrespective of protein sequence. This is due to formation of amorphous as well as

fibrillar structure by same proteins under different conditions. pH of the solution plays

a decisive role during aggregates morphology determination, as if affects the extent of

structural perturbation as well as distribution of charges in proteins. Various studies

revealed that high net charge to protein favors the fibrillar structure, whereas low net

charge on proteins likely to formed aggregates with amorphous morphology and

sometimes not favor the aggregation process (Krebs et al., 2007; Krebs et al., 2009;

Zaman et al., 2016b). Due to poor solubility, larger size and non-crystalline nature, it

is hard to recognize the molecular level description of aggregated structure,

particularly of amyloid fibrils. However, recent advancement in analytical techniques

like solid-state NMR, X-ray diffraction, X-ray crystallography and electron

microscopic techniques enhances the knowledge of fibrillar structure at molecular

level. Despite of having dissimilarities in structural properties of proteins those results

in amyloidogenesis; amyloid fibrils share some basic similar features. Amyloid fibrils

formed by homotypic polymerization of monomers of size ranging from 3-30 kDa.

Normally, amyloid fibrils are unbranched, 5-15 nm wide (bigger in few cases),

straight, produced by variable number of protofilaments known as elementary

filaments (1.5-2 nm in diameter), as studied by various techniques. However, the main

structural hallmark of amyloid fibrils are the cross beta structures that are responsible

for tinctorial properties of these assemblies. The distance among each beta strand is 4


6

A° and separated by a distance of ~ 10 A°. The beta sheets are found to be parallel or

anti-parallel orientation, but in most cases it possesses parallel orientation (Kumar et

al., 2016). In addition, it is well-known fact that fibrils generated under in vitro

surroundings differs in their morphology depending upon the conditions of solution

(Chaturvedi et al., 2016b). For example of insulin have different fates, as it acquires

soluble hexamers or insoluble fibrils as well as soluble dimmers and insoluble

disulfide-bonded aggregates, depending on the physical and chemical aggregation

conditions respectively (Sluzky et al., 1991; Sluzky et al., 1992; Darrington and

Anderson, 1995).

1.2.4. Why do protein aggregates

In the field of protein chemistry, it is an important question why do protein aggregates

rather than to achieve their minimum energy level landscape to be in functional state.

A complete retort to this problem play a significant role to understand the mechanism

underlying that a variety of human diseases and it also sheds light on the basic

properties associated with proteins. Undoubtedly, a few proteins turn into aggregated

form through „off-pathway‟ misfolding from their well-folded soluble forms that can

be considerably concealed by cellular chaperone systems. Various biophysical studies

have been exclusively conducted to understand the factors mediating the aggregation

process (Soto, 2003; Song, 2013). Partially folded or misfolded states of proteins,

generated by loss of quality control system in protein as well as by various external

factors (described earlier) possess exposed hydrophobic patches and unstructured

regions (buried inside the native state of protein) are often tend to aggregate formation

(Knowles et al., 2014). In addition, formation of stable aggregate structure were also

formed by hydrophobic collapse due to presence of external moieties in the solutions

that alters the conditions (Rosa et al., 2014). On the other hand, electrostatic

interaction along with hydrophobic forces plays a decisive role in the formation of

complex interplay which results into amyloid fibrils (Khan et al., 2014a).

1.2.5. Characterization of amyloid fibrils

The characterization of protein aggregates by using a number of biophysical and

microscopic techniques is not only important to understand the mechanism of

aggregation, but it also provides to designing tool for potential therapeutic strategy

towards amyloidogenesis related diseases. The characterization of amyloid fibrils


7

under in vitro conditions may be mostly categorized into three kinds of studies:

identification, structural characterization and mechanism of fibril development. The

first step towards characterization is identification of aggregates.

1.2.5.1. Turbidity and Rayleigh light scattering measurements

Turbidity measurements are useful to detect amyloid aggregates but its sensitivity is

too low as it is a good tool for lager aggregates (Kumar et al., 2016). Turbidity refers

to haziness or cloudiness of fluids that are caused due to different size of individual

particles. The optical property of a solution is function of number of particles present

to scatter or absorb light. Increased turbidity in solutions clearly demonstrates the

aggregation of proteins. However, Rayleigh theory of light scattering states that

particles scatter light when their diameter size is lesser than the incident light. It is an

essential tool to investigate the protein aggregation in solutions and monitored

through spectrophotometer.

1.2.5.2. Dye binding assays

1.2.5.2.1. Congo red binding assay

The most accepted method for identification of amyloid fibrils with their

characteristic features are binding with some specific dyes. Congo red (CR), ANS and

Thioflavin T (ThT) have specific binding with amyloid beta pleated sheet; however

the exact mechanism between interaction of amyloid fibrils and CR is yet to be

established. Congo red binds specifically against beta pleated sheet conformation and

does not bind with shorter beta pleated structures (Klunk et al., 1989). The change in

absorbance wavelength of CR in the visible region is also used to characterize fibrils

as different spectrum of CR were observed in the absence and presence of amyloid

fibrils at around 540 nm (Nilsson, 2004).

1.2.5.2.2. ThT binding assay

Thioflavin T (ThT) binds to amyloid under both conditions i.e, in vitro and in vivo

and a detailed molecular mechanism of ThT binding to amyloid fibrils are reported by

various groups (Khurana et al., 2005; Biancalana and Koide, 2010). Thioflavin T

(ThT) has a hydrophobic end with dimethylamino group linked to a phenyl group that

is associated to an additional polar benzothiazole group containing the polar N and S.


8

This phenomenon (grouping of hydrophobic and polar regions) creates possibility for

ThT molecules to form micelles in aqueous solution that have a hydrophobic interiors

with positively charged N headed for solvent. Form this, it has been suggested that

hydrogen bonds are formed by hydroxyl group of tissue and thiazole nitrogen of dye

offer specific binding of Tht to amyloid and other tissue structures (Kelieny, 1967).

1.2.5.2.3. ANS binding assay

ANS (1-anilino-8-naphthalene sulfonate) is another influential tool to check the

progression of fibril formation when studying the diseases mechanism under in vitro

conditions (Khurana et al., 2001; Lee, 2010). The preferential binding of ANS to

hydrophobic patches gives rise to an enhanced fluorescence emission accompanied by

a blue-shift of spectral maximum and therefore the surface hydrophobicity of protein

samples (which enhances during aggregation) can be monitored by ANS fluorescence

emission (Qadeer et al., 2014).

1.2.5.3. Cicular dichroic measurements

The next step towards characterization is to monitor secondary structural changes in

the protein by using various approaches. Fourier transform infrared spectroscopy

(FTIR) and circular dichroism (CD) are generally used to observe the secondary

structural changes in proteins. Circular dichroism has characteristic features for α-

helix and β-sheet due to difference in electronic environment of the amide groups that

are present in these structures (Radko et al., 2015). In addition, CD analysis is also

used to study the kinetics of fibrillogenesis under in vitro conditions (Tomski and

Murphy, 1992).

1.2.5.4. Fourier transform infrared spectroscopy method

To characterize the secondary structural changes of proteins, fourier transform

infrared spectroscopy (FTIR) is another technique that allow one to obtain vibrational

spectra of molecules which contains large number of absorption bands of different

intensity for random coils, α-helix and β-sheets (Sarver and Krueger, 1991). This

technique is based on the fact that transition of secondary structural conformation is

accompanied by spectral shift of the amide absorption band from 1650–1658 cm–1

to

1640–1648 cm–1

and to 1620–1640 cm–1

for α-helical conformation to random coil, or


9

β-sheet respectively (Fraser et al., 1991). Although, FTIR is widely used for

secondary structural analysis of proteins but it is rarely used to study the kinetics of

Aβ aggregation in the aqueous saline conditions (Radko et al., 2015).

1.2.5.5. Dynamic light scattering method

Dynamic light scattering (DLS) is a useful technique to characterize the aggregation

process in solutions (Khodabandehloo et al., 2017). DLS is possible to determine the

diffusion coefficient for dispersed particles in liquids by analyzing the correlation

function of the intensity fluctuations of scattered light (Lomakin et al., 2005). The

hydrodynamic radii of samples are further measured by using the observed diffusion

coefficient of dispersed particles. DLS can measure the size of different populations

of oligomers formed under same conditions that were composed of dimmers,

trimmers and tetramers. However, the major disadvantage of DLS is its low resolution

and difficulty in detection of monomers and oligomers in the presence of large

aggregates, because contribution of large aggregates to the light scattering dominates

(Bruggink et al., 2012).

1.2.5.6. Electron microscopic techniques

The structural analysis, morphology and shape of fibrils can be monitored by using

different microscopic techniques that include transmission electron microscopy

(TEM), fluorescence microscopy (FM), atomic force microscopy (AFM) and

scanning electron microscopy (Arimon et al., 2005; Lara et al., 2011; Adamcik and

Mezzenga, 2012; Das et al., 2017). AFM is used to detect the structural changes in

aggregates such as protofibrils, fibril elongation, globular aggregates and fibril

maturation as well as fibril formation on different surfaces

1.2.5.7. Other techniques

In addition, structural characterization of amyloids and their intermediates are

characterized by mass spectroscopy and nuclear magnetic resonance (NMR) and

atomic details of fibrils are characterized by X-ray diffraction method (Luhrs et al.,

2005; Kheterpal and Wetzel, 2006; Nelson and Eisenberg, 2006; Toyama and

Weissman, 2011). However, some more techniques are used for structural

characterization of amyloid fibrils and they are SPR (surface palsmone resonance),

EPR (electron paramagnetic resonance), gel electrophoresis, FCS (fluorescence


10

correlation spectroscopy) and gel filtration etc (Pedersen and Heegaard, 2013). All

techniques are summarized in table 1.2.

Table 1.2 Analytical methods generally used for studying protein structure, folding

and self assembly.

Monomer Oligomers Protofibrils/

Fibrillar aggregates

Amorphous

aggregates

Assembly size/

Size

distribution

Turbidity measurements/Rayleigh light scattering measurements

Size exclusion chromatography

Gel electrophoresis

Analytical centrifugation

Dynamic light scattering

Secondary

structure

Circular dichroism spectroscopy

Fourier transform infra red spectroscopy

Thioflavin T / Thioflavin S fluorescence/ Congo

red birefringence/ Congo red binding

ANS/bis-ANS/ Nile red fluorescence

Morphology

Transmission electron microscopy (TEM)

Atomic force microscopy (AFM)

Fluorescence microscopy (FM)

Scanning electron microscopy (SEM)

Atomic

structure

X-ray crystallographic diffraction (XRD)

Nuclear magnetic resonance spectroscopy (NMR)

Surface plasmon resonance (SPR)

1.2.6. Mechanism of protein aggregation

Protein aggregation occurs through not single pathways but distinct mechanism or

pathways are responsible for this phenomenon. These mechanisms are not

independent to each other but more than one can occur for the same product. Some

mechanistic understanding can help to point the way to prevent the formation of

amyloids, a method to suppress and remove the aggregates, or provide various

realistic approaches to develop some new formulations and drugs. Some mechanisms

are discussed below that are common against fibril formation.

1.2.6.1. Reversible association of the native monomer:

Native protein has an intrinsic tendency to self associate in a reversible manner to

form small oligomers. Different types of interfaces (produced by multiple sticky or


11

complementary patches presented by monomer surface) potentially produce numerous

conformations of oligomers of the similar stoichiometry with diverse patterns of

oligomer growth (figure 1.2A). Formation of large oligomers takes place with

increase in protein concentration that is determined by law of mass action. Further

these aggregates became irreversible with time by formation of covalent bonds like

disulfide linkages. In addition, this mechanism also illustrates that side by side or end

by end combination of protein molecules are responsible for aggregation and protein

folding is not a required condition for aggregation process (Barbu and Joly, 1953).

The best example of reversible association is association of a therapeutic protein

insulin which readily and normally associates with this mechanism (Pekar and Frank,

1972). Such involvement may have significant consequences for bioactivity like

producing innovate products by manipulation of this association via mutation as

illustrated by insulin association (Brems et al., 1992).

Figure 1.2 (A) Schematic illustration of protein aggregation through reversible association of

monomers.

The best example of product is formation of Interleukin-1 receptor antagonist (rhIL-

1RA), which forms irreversible dimers and trimers by undergoing reversible

dimerization at high concentrations (Alford et al., 2008).

1.2.6.2. Self assembly of conformationally altered monomer

In contrast to previous one, self assembly of conformationally altered monomer has a

very low propensity towards reversibly association. However, conformationally

altered or partially unfold monomer links robustly in a manner similar to the previous

one. To achieve native state, proteins (smaller and larger) crosses high kinetic

barriers. Thus, when left to fold on their own, these proteins (partially folded

intermediates) become “kinetically trapped” in local energetic minima and associate


12

to form aggregates. A few intermediate states may be obligatory ones, and when

significantly populated, they tend to form aggregates by specific interactions (figure

1.2 B). In addition, for some familial forms of disease in which the proteins involved

in aggregation normally adopt folded conformations. Further, this mechanism is

promoted by stresses that include high temperature, low pH, pressure, moderate

organic solvents and shear that might generate the early conformational change that

have an increased propensity towards aggregation (Chiti and Dobson, 2006). It is

apparent that deterioration of the native conformation increases the population of non

native states, is the key mechanism during which natural mutations arbitrate their

pathogenicity (Raffen et al., 1999; Canet et al., 2002; Hammarstrom et al., 2002).

Figure 1.2 (B) Schematic illustration of aggregation of conformationally-altered protein.

An important consequence is that conditions or excipients that alleviate the native

state of protein may be helpful in proposing inhibitory agent against aggregation. This

mechanism is reported and discussed for various proteins in many reviews and G-CSF

along with interferon-γ is two best examples of therapeutic proteins which favors this

mechanism (Kendrick et al., 1998; Krishnan et al., 2002; Raso et al., 2005).

1.2.6.3. Aggregation of chemically modified monomer

In this mechanism, aggregation proceeds through conformational change in proteins

(highly appropriate towards aggregation) that arises due to difference in covalent

structure of protein. This mechanism is thought to be preceding step of earlier

mechanism and deamidation, amino acid modification, fragmentation, oxidation of

methionine, proteolysis susceptibility are some chemical degradation factors that

makes differences in proteins. These chemical modifications either change the electric

charge on proteins or create new sticky patches on the surface of proteins in such a


13

way that reduces electrostatic repulsion between monomers and favors the

aggregation process (figure 1.2 C). A notable and diagnostic characteristic of this

mechanism is their enrichment with modified forms of monomers. The best example

for this is glycoproteins as there might be un-glysosylated or under-glycosylated

fraction that is prone to aggregation and chemically altered aggregates can be

particularly immunogenic (Hermeling et al., 2006).

Figure 1.2 (C) Schematic illustration of aggregation of chemically-modified proteins.

1.2.6.4. Aggregation via nucleation dependent

It is extensively recognized that aggregation (fibril formation) has many

characteristics of nucleation controlled mechanism and it is the most common

mechanism in the formation of visible aggregates or precipitates. This nucleated

method has been well studied (exponentially and theoretically) in numerous additional

contexts, usually for the process of crystallization of both large and small molecules

(Chayen, 2005). In this mechanism, preliminary step is formation of nucleation

(typically rich in β-sheet structure and presumably oligomeric) that is sparingly

occupied with high energy species that has a different structure of the soluble protein.

Native monomer has low propensity towards oligomers (low or moderately sized)

formation as well as thermodynamically un-favored for smaller aggregates. However,

sufficient size of aggregate leads to formation of critical nucleus by addition of

monomers. One a nucleus is formed, aggregation proceed rapidly (thermodynamically

strongly favored) by further association of either oligomers or monomers to the

growing non-covalent polymer to generate larger aggregates that can also act as seeds

(figure 1.2 D). A distinctive aspect of a nucleation-controlled procedure is that the

rate of formation of the bulky precipitates or particles generally includes a lag phase

that is followed by a rapid exponential growth phase. Certain type of mutations,


14

change in environmental conditions may reduce or eliminate the lag phase assumed to

result from a situation wherein nucleation is no longer rate limiting.

Figure 1.2 (D) Schematic illustration of protein aggregation through nucleation-dependent

pathway.

This may simply states that fibril growth is sufficiently slow to the nucleation process

in absence of lag phase, but still nucleated growth mechanism is operating. It is

increasingly clear that lag phase, in which fibrils do not appear to a significant extent,

is an important stage of fibril formation. This may be due to formation of different

oligomers during this phase that includes β-sheet rich species which present nuclei for

formation of mature fibrils. Two types of nucleation have been reported depending

upon the formation of critical nucleus. When critical nucleus is itself a product of

protein aggregate it is termed as homogeneous nucleation, but when the critical

nucleus is a product of particle contaminant or impurity instead of protein itself, it is

termed as heterogeneous nucleation. Silica and steel particles shed by product vials

and piston pump used for filling vials respectively are two best examples of

contaminants that have been reported so far (Philo and Arakawa, 2009).

1.2.6.5. Surface induced polymerization

These mechanisms are driven by hydrophobic as well as electrostatic interactions

depending upon the surface that are being used for aggregation, such as air-liquid

interface is driven by hydrophobic interaction. Interaction of monomer with surface

brings about conformational change that increases the propensity of aggregation

(figure 1.2 E). Surprisingly, this mechanism is alternative to the nucleated dependent

mechanism in which surface of critical nucleus induces aggregation. Aggregation at

the surfaces of ice crystals or crystal of excipients results into freeze/thaw damage

that occurred through this mechanism (Philo and Arakawa, 2009). Apart from these


15

coarse grain model, two step model of nucleation and secondary nucleation are some

more recent mechanisms that are proposed by different groups for aggregation of

proteins (Chaturvedi et al., 2016b).

Figure 1.2 (E) Schematic illustration of aggregation of protein on container surfaces and air-

liquid interface.

Understanding aggregation mechanisms possibly assist into development and/or

formulation attempt, help in mounting an optimal chromatography step to eradicate

aggregation as well as to trim down aggregation by addition of additives.

1.2.7. Factors affecting for aggregation

Protein aggregation is supposed to be inherent assets of polypeptides; however the

susceptibility of aggregation varies on the environmental conditions both in vitro and

in vivo (figure 1.3). These aggregates may exhibit less desirable characteristics like

reduced or no biological activity with other side effects. Some of the factors that

cause protein aggregation under in vitro conditions are described below.

1.2.7.1. Effect of temperature

Proteins are stable at native conditions due to equilibrium between large stabilizing

and destabilizing forces. High temperature has direct impact on the conformation of

proteins (secondary, tertiary and quaternary level), and it causes denaturation of

proteins that is irreversible in nature (reversible in some cases), as a result protein gets

aggregated (Wang et al., 2006; Chaudhuri et al., 2014). Further, oxidation and

deamination reaction becomes rapid at high temperature that could also lead to

aggregation in proteins. In addition, high frequency of molecular collision, enhanced

hydrophobic interactions, lowering of activation energy and increased diffusion of

molecules at elevated temperature are also responsible for protein aggregation.


16

Figure 1.3 Factors affecting the aggregation of proteins under various conditions.

1.2.7.2. Effect of pH

It is well documented that proteins acquire net positive charge below its pI and net

negative charge above its pI. Net charge in proteins as determined by pH will lead to

partial unfolding of proteins and affects electrostatic interactions that are present in

the proteins. Most of the proteins are stable in a very slight range of pH and any

alteration in these pH leads to aggregation of proteins (Chen et al., 2014).

Aggregation occurs due to neutralization of charged molecules along with increased

hydrophobic interactions. Repulsive interaction between protein molecules (when

proteins are highly charged) stabilizes it and making aggregation process energetically

non-favorable. However, anisotropic charge distribution give rise to dipoles in

proteins where proteins possess both negatively and positively charged proteins. In

such type of cases aggregation process were favored by protein-protein interaction.

1.2.7.3. Effect of surfactants

Surfactants are amphiphilic molecules that tend to orient in such a way that the

exposure of hydrophobic portions to the aqueous solution is minimized. These are the

most common molecules, used for inducing amyloid in various proteins. They have

strong impact on protein conformation by either destabilizing or stabilizing and

inducing aggregation in proteins (Khan et al., 2012). There are various types of

surfactant molecules i.e, cationic (CTAB, CPC, DTAB), anionic (SDS, SLES, AOT)

and nonionic (Brij). Protein aggregation takes place by interaction of surfactants with

opposite charge center of protein molecules along with repulsion of water molecules

by their hydrophilic tails. Aggregation induction by CTAB and DTAB requires

development of net negative charge as protein incubated below its isoelectric point


17

(where they acquire net positive charge) usually don‟t aggregate in presence of

cationic (CTAB, DTAB) surfactants and vice versa for anionic detergents

1.2.7.4. Effect of pressure

It is well understood that proteins are marginal stable in solutions under high pressure

and hydrophobic interactions are perturbed that are necessary for the stability of

proteins in native state. This results in the formation of partially folded intermediates

that are more prone for aggregation (Silva et al., 2014). Various groups reported the

effect of pressure to understand the role of hydration and hydrophobic cavities

towards aggregation of proteins (Ferrao-Gonzales et al., 2000).

1.2.7.5. Effect of ionic strength

Ionic strength is another important parameter that significantly impacts the

aggregation behavior of proteins. The nature of protein plays a key role during

ionic strength aggregation process. If protein folding is facilitated by salts (charge

neutralization), then aggregation is inhibited by low ionic strength as it provides

stability of proteins. In contrast, high salt concentration unfolds and destabilizes

the proteins, as a result promotes aggregation. Moreover, morphology of amyloids

is also altered by ionic strength. When the solution is at low ionic strength,

monomers associated only at growing ends of protofibrils and prevents the

interaction between axial sides (electrostatic repulsion), thus resulting in long and

thin fibres. However, at higher ionic strength, monomers interact with axial sides

too due to predominance of hydrophobic interactions resulting into short and thick

fibres.

1.2.7.6. Effect of salts

Proteins stability and solubility are highly influenced by salts which determine the

rate of formation of non-native aggregation. Electrostatic interactions between

charged groups of proteins are strengthening by salts. High salt concentration results

in preferential binding of ions to proteins, as a result decrease in thermodynamic

instability of proteins favors the aggregation process. However, no such type of

phenomenon is observed in presence of low salt concentration as it favors charge

shielding.


18

1.2.7.7. Effect of nucleic acids

Nucleic acids (NAs) are biopolymers, or large biomolecules, essential for all known

forms of life which include RNA and DNA that are made from monomers known

as nucleotides (Zaman et al., 2016a). The interaction of prion protein (PrP) with nucleic

acids has been shown to occur both ex vivo and in vitro. The reaction product, the

PrP−NA complex, is sometimes proteinase K-resistant and undergoes amyloid

oligomerization (Macedo et al., 2012b). One of the more intriguing characteristics of

the interaction between PrP and NAs is the finding that some NA sequences can act as

catalysts for the formation of a scrapie-like PrP conformation (Silva et al., 2008).

Depending on the binding conditions, this interaction can also convert PrP into an

aggregated β-sheet rich structure (Macedo et al., 2012b). Host nucleic acids catalyze the

conversion between PrPC and PrPSc by reducing the protein‟s mobility and by favoring

protein−protein interactions (Macedo et al., 2012b). In addition, transient binding to

short (11 bp) specific plasmid-derived ds-DNA sequences in vitro modulated the

assembly of the protein into amyloid fibres (Giraldo, 2007). NAs may exert their effects

by reducing the protein‟s mobility and by making protein – protein interactions more

likely has been reinforced recently by immunization experiments using dimeric PrP

(tandem PrP) and CpG–oligonucleotide (Kaiser-Schulz et al., 2007).

1.2.7.8. Chemical modification

Chemical modification is another method that leads to aggregation in proteins.

Chemical reactions such as hydrolysis, oxidation, isomerization, deamidation might

be responsible for destabilization to protein structure and thus promoting aggregation.

In addition, protein aggregation induction is also favored by glycation (Wei et al.,

2009). Insulin in its reduced and glycated form has been found to be more prone to

aggregation than its non-reduced form (Alavi et al., 2013). Moreover, aggregation

induction will be also induced by photolytic degradation of proteins which involve

oxidation of aromatic residues, His, Cys and Met (Mahler et al., 2009).

1.2.7.9. Effect of protein concentration

Protein concentration is critical factor in amyloid formation since formation of

nucleus may not occur below a critical concentration of protein. The increased protein

concentration may exert contrasting effects on aggregation/amyloid formation due to

the existence of different aggregation pathways (by self-association of native

https://en.wikipedia.org/wiki/Biomolecule

https://en.wikipedia.org/wiki/Life

https://en.wikipedia.org/wiki/RNA

https://en.wikipedia.org/wiki/Monomers

https://en.wikipedia.org/wiki/Nucleotide


19

monomers or initiation by partial-unfolding). High protein concentration may increase

or decreases aggregation due to increased chances of self-association and crowding

effect respectively. Furthermore, aggregation occurring in high concentration

formulations is also a matter of concern for storage of protein.

1.2.7.10. Miscellaneous factors

Apart from all these here we put some more factors that influence protein aggregation

which includes agitation, freezing, contact materials such as rubbers, glass, steel,

plastic silicones, UV illumination, organic solvents, metal ions, freezing, thawing,

drying, spraying etc (Mahler et al., 2009).

1.3. Possible mechanism of amyloid induced cytotoxicity

The reasons why amyloid fibrils are toxic to cells are front line of research by various

researchers by using an extensive array of biochemical, cytological and physiological

perturbations. Several possible mechanisms have been suggested for the observed

cytotoxicity of amyloidogenic assemblies on living systems. The present models are

divided into two major categories that include direct and indirect effect of amyloid

fibrils on cellular mechanisms which ultimately lead to cellular death. The suggested

direct effects are formation of pores in cellular membranes and permeation effect of

amyloidogenic bundles that leads to instability of membranes. However,

abnormalities in redox systems, loss of protein function due to aggregation, increased

formation of reactive nitrogen and oxygen species as well as hyper-phophorylation of

proteins are some indirect models that have been suggested for amyloid induced

cytotoxicity to cells.

1.3.1. Amyloid induced cytotoxicity via membrane perturbation

Till date, we are still lacking the mechanism by which amyloid peptides induces

cytotoxicity in cells. One of the key premises is that amyloids cause membrane

perturbation through amyloid peptide channel formation and changes in membrane

fluidity. Plasma membrane as well as mitochondrial membrane (internal membrane)

was affected. However, channel activity of several amyloid forming peptides have a

great concern that is modulated by lipid composition, electrostatic interaction along

with several amyloid inhibitors. Various studies revealed that these peptides


20

(amyloid) causes membrane leakage via formation of 8-12 nm channels with lipid

membranes (Green et al., 2004). It was suggested that peptides physically disrupted

membrane after achieving threshold deposition. Further, micelles were formed

(reorientation of hydrophobic patches) which leave the bilayer, as a result membrane

disruption occurs. Most likely these defects are due to loss of lipid molecules by the

membrane. However, the other possibility is insertion of peptides (directly) between

lipid molecules might increase the membrane packing density.

1.3.2. Amyloid toxicity via a common oxidative mechanism

Beta amyloid protein has been implicated in neurodegenerative diseases and the

molecular details of mechanism by which it causes cell death is still lacking. It may

increase H2O2 accumulation in cells that results into free radical induced lipid

peroxidation following cell death (Schubert et al., 1995). By using various cell line

systems it has been shown that a number of antioxidants protects cell from Aβ

cytotoxicity, suggesting that free radical damage is one of the possible pathway for

Aβ cytotoxicity. Catalase (H2O2- degrading enzyme) and antioxidants such as

vitamins protect cells from Aβ toxicity. In addition to Aβ, amyloid peptides of

calcitonin, ANP and human amylin causes cell death by similar mechanism. LDH-

release and MTT assay suggests that ANP (which is deposited in the atria of aged

individuals) and calcitonin (which is found in amyloid deposits of medullary

carcinomas) are toxic to B12 cells (Schubert et al., 1995).

1.3.3. Toxicity due to calcium dysregulation and membrane disruption

Soluble Aβ oligomers are found in the cerebrospinal fluid and are supposed to be the

main cause for neurotoxicity. Increasing evidence imply that elevated intracellular

Ca2+

and disruption of intracellular Ca2+

homeostasis in various amyloidogenic

diseases are known to trigger apoptosis or/and excessive phosphorylation of key

proteins that eventually guide to cell death (Demuro et al., 2005). Pre-fibrillar

aggregates are known to elevate the cytosolic Ca2+

in neurons (Bucciantini et al.,

2004). Mobilizing action of Ca2+

on amyloids oligomeric states includes several

mechanisms such as direct interaction with membrane components, activation of cell

surface receptors coupled to Ca2+

influx and direct interaction with membrane

components to destabilize the membrane structure (Mattson, 2004). Among these

Demuro et al. suggested that increase in membrane permeability were induced


21

specially by spherical amyloid oligomers. The increase in cytosolic Ca2+

along with

addition of oligomers is rapid to prevent it being a secondary effect resulting from

metabolic impairment and reduced Ca2+

pumping. However, long term pathogenesis

of amyloid diseases might be possible due to perturbations of other ions and

molecules.

1.3.4. Toxicity due to β-sheet structure

In human amyloidogenesis, all the protein deposits possess β- sheet structure that may

be sufficient to causes cytotoxicity in cells. A series of repeating copolymers of lysine

and leucine with different α-helical, random coil and β-sheet structure contents were

tested towards B12 cells for their relative toxicity. Further, direct correlation was

measured between toxicity and β-sheet content of the peptide. The ability of

diphenylene iodonium (DPI) a NADPH oxidase inhibitor, towards β- sheet structures

(low or high) suggests the degree of peptide toxicity. The direct influence of β-sheet

peptide is not required to cause cell cytotoxicity, but, it might be a necessary

configuration to causes cell death in cells via DPI inhibitable mediated mechanism

(Schubert et al., 1995).

1.3.5. Toxicity due to conductivity of membranes

There are various reports regarding membrane perturbation cytotoxicity of amyloid

peptides, but their effects are not specific. Kayed et al. suggested that homogeneous

populations of spherical amyloids protofibrils and oligomers increases membrane

conductivity through non- channel formation mechanism (Kayed et al., 2004).

Increase in membrane conductivity may be the primary mechanism of oligomers

pathogenesis and conductivity is roughly proportional to the concentration of

oligomers. However, such type of increment is not observed for low molecular weight

monomers and oligomers. In addition, this increase occurs in absence of any

substantiation of discrete ion pore or channel formation. Bilayer conductivity will also

increase in a concentration dependent way for various spherical oligomers of different

proteins such as α-synuclein, poly (Q) and IAPP. Further, cellular toxicity due to

shared common structure of soluble oligomers suggests that it share a common

primary mechanism of pathogenesis.


22

1.3.6. Toxicity due to gain of function

In this mechanism, toxicity occurs due to altered protein conformation that is formed

under different conditions. Apolipoprotein (APOE) a lipid transport molecule is one

of the best examples that confer toxicity by this mechanism (Valastyan and Lindquist,

2014). The APOE4 polymorphism stabilizes the altered conformation of proteins and

is also associated with increased level of Aβ (a peptide associated with aggregation).

Changes in lipid affinity and disruption of mitochondrial function strongly implicates

APOE role in the pathogenesis of AD. The formation of an extra salt bridge (that

stabilizes the misfolded form) in APOE4 by different alleles might be also responsible

for cellular toxicity (Valastyan and Lindquist, 2014). However, the exact mechanism

for this structural transition is still not known.

1.4. Therapeutic strategies for aggregation/amyloid inhibition

The inhibition of protein self-assembly is studied in two perspectives; developing

strategies for combating protein aggregation in general (situation encountered during

manufacturing, protein refolding and expression, handing out of biopharmaceuticals),

and scheming or searching for inhibitors that exclusively target amyloid aggregates

(linked through numerous pathogenic conditions). Suppression of aggregation during

the formulation of protein-based therapeutics is attempted by various kinds of

additives and excipients through various mechanisms such as, stabilization of native

state, destabilization of incorrectly folded state, reduced solvent accessibility and

increased rate of protein folding etc (figure 1.4). Nature has adopted various strategies

to avoid unwanted protein aggregation and amyloid formation inside a living system

where local protein concentration is quite high. In addition, several therapeutic

approaches have been suggested to combat amyloidogenic diseases that include

increase in the clearance rate of misfolded or aggregated proteins, reduction in the

production of the amyloidogenic form of proteins in amyloidogenic proteins.

1.4.1. Aggregation inhibition by denaturants and chaperons

Denaturants such as urea and guanidine are the most conventionally used additives

that assist refolding of protein and suppress aggregation. Sodium dodecyl sulfate, an

anionic detergent is also found to inhibit aggregation at post micellar concentration in

some proteins which is well known amyloid inducer at submicellar concentration

(Giehm et al., 2010). Moreover, non-detergent surfactants such as sulfobetaines


23

(NDSBs) and ionic liquids such as ethyl ammonium nitrate (EAN) have confirmed

effectual inhibition of thermal and chemical induced aggregation. During inhibition or

suppression of protein aggregation, an important technique employed is formulation

of artificial chaperone that increases the refolding effectiveness of numerous proteins

including lysozyme and serum albumins (Potempa et al., 2010; Gull et al., 2011).

GroEL (bacterial chaperonin), devoid of their structural context, binds to proteins in

non-native states to both (α-helical proteins and β-sheet–rich ones) and successfully

competes for hydrophobic sites which provided surface for aggregation induction

(Horwich, 2002). Apart from this, inhibition of amyloid formation can also be

achieved by various means of ligand (small or large) that can bind and arrest

peptides/proteins at different steps of amyloid formation pathway viz oligomers,

amyloid seeds, fibrils, etc.

Figure 1.4 Schematic illustrations of pathway that inhibit amyloid fibril formation (A)-(E)

represent the molecules used to either inhibition or stabilization of protein. (A) Native state

stabilization (B) Refolding of polypeptide (C) Diversion from oligomerization pathway (D)

Inhibition of fibril elongation by β-sheet breakers (E) Disaggregation of amyloid aggregate.

1.4.2. Aggregation inhibition by nanoparticles

In order to design an efficient inhibitor as well as the nanomolecular species which

are suitable for this purpose, there is a need to focus on forces that are responsible for

fibrillation. Inhibition by nanoparticles could be done at nucleation phase (by

increasing the lag phase), polymerization phase (by decreasing the elongation phase)


24

or diversion of peptide from the polymerization pathway to reduce the end point at

equilibrium. Nanoparticles slow down the rate of the fibrillation by altering the

amount of free monomeric peptides that are present but at the same time fibril

formation still could not be prevented (Cabaleiro-Lago et al., 2010). Experimental

data show that nanoparticles added at beginning of the kinetic experiment arrest the

fibrillation process in lag phase. The effect of PAMAM G4 dendrimers on α-

synuclein were observed by and their structural changes were compared in the

presence of nanoparticles (dendrimers) with the help of CD. The higher percentage of

α-structure and presence of positive signal in the range of 195-206 nm after

incubation with nanoparticles indicated reduction in the β-sheet structure (Milowska

et al., 2011). Dendrimer nanoparticles can be used as thermodynamic and kinetic

inhibitor based on size and concentration (Klajnert et al., 2006). In case of kinetic

inhibitors the final amount of fibrils is unchanged with varying lag time, while

thermodynamic inhibitors do not affect amyloid formation rate but the final amount of

fibrils is reduced. Insulin fibril formation is a physical process in which nonnative

insulin aggregates and further deposition of insulin fibrils causes type II diabetes. A

significant inhibition of insulin fibrils were observed by Skaat et. al. in presence of γ -

Fe2O3/PHFBA nanoparticles (Skaat et al., 2009). These nanoparticles slow down the

transition of α-helix to β-sheets during insulin fibril formation.

1.4.3. Aggregation inhibition by amino acids

Amino acids are building block of proteins and are reported to exhibit potent

inhibitory action against various protein aggregation and amyloid formations induced

thermally. Among all amino acids arginine, cysteine and their derivatives shows most

effective against protein fibrillation. Arginine have been studied as a suppressor of

protein aggregation in a variety of protein systems (Varughese and Newman, 2012).

However, depending on the binding of guanidinium group the role of arginine differs.

For example arginine promotes aggregation when guanidinium group of arginine

binds to the acidic group of proteins and it inhibits aggregation of insulin when the

guanidinium group binds to aromatic groups of protein (Lyutova et al., 2007; Shah et

al., 2011). In addition, arginine containing small molecules exhibits remarkable

inhibitory potential against the formation of globulomer and the fibril of Aβ

aggregation process. Arginine rich short peptides (4-7 residues) bind soluble 42-

residue amyloid β-peptide (Aβ-42) and inhibit globular formation using phase display


25

(Kawasaki and Kamijo, 2012). Cysteine, an amino acid had influential role during Aβ

peptide fibrillation as it reduced the amount of fibrils as well as decelerated the fibril

formation. Interestingly, the thiophilic interaction of a sulphur-containing ligand is

much stronger with bicontinuous aromatic residues than a single aromatic residue and

efficiently inhibit the formation of amyloid fibrils of an β-amyloid peptide that has a

core sequence of 16

KLVFF20

(Takai et al., 2014b). Apart for this, D-amino acids that

can mimic naturally occurring peptides were also capable to bind with Aβ and prevent

amyloid formation (Kumar and Sim, 2014).

1.4.4. Aggregation inhibition by polyphenols

In past few years, various in vitro assays were performed to check the inhibitory

potential of polyphenols against amyloid fibril formation. Polyphenols are most

numerous and ubiquitous groups of natural and synthetic small molecules that are

composed of one or more aromatic phenolic rings. Natural polyphenols are a class of

phytochemicals found in high concentrations in berries, tea, wine, nuts, cocoa, and a

wide variety of other plants. Vitamins such as b-carotene and a-tocopherol, flavonoids

such as flavanone and isoflavone and phenolic acids like benzoic acid and

phenylacetic acid along with thymol, sesamol, ellagic acid, eugenol, are some

examples of natural phenols (table 1.3). However, synthetic polyphenols such as

butylated hydroxylanisole (BHA), phenolsulfonphthaleine and butylated

hydroxytoluene (BHT) are generally used as synthetic food additives as well as pH

indicators in cell culture media (Porat et al., 2006). However, antioxidants are a class

of compounds that can inhibit or delay the oxidation of materials that can be oxidized

by scavenging free radicals and diminishing oxidative stress (Porat et al., 2006).

Tannic acid is a water-soluble polymeric polyphenol (mixture of phenolcarboxylic

and gallic acids) act as a potential inhibitor of β-amyloid fibrillization and the

inhibition was found to be dose dependent (Ono et al., 2004a). The compact and

symmetric structure of curcumin and rosmarinic acid might be suitable for specific

binding of free β-amyloid and inhibits the formation of amyloid beta oligomers and

fibrils (Ono et al., 2004b; Yang et al., 2005). In addition, inhibition of islet amyloid

polypeptide, insulin and β-amyloid by phensulfonphthaleine as well as α-synuclein

inhibition by baicalein and squalamine also falls under polyphenolic inhibition

(Hoppener et al., 2000; Zhu et al., 2004; Perni et al., 2017).


26

Table 1.3 Anti-amyloidogenic agents and related proteins of study.

Category

Anti-amyloidogenic

agents

Proteins

Polyphenols

(-)-Epigallocatechingallate

Curcumin

NDGA

Rosamarinic acid

Myricetin

Kaempferol

Hntingtin (Ferreira et al., 2011)

Transthyretin (Hudson et al., 2009)

β-amyloid 1-42 (Ono et al., 2004b)

β-amyloid 1-42 (Ono et al., 2004b)

β-amyloid 1-40 (Ono et al., 2003)


Proteins Albumin

Catalase

β-amyloid (Luo et al., 2014)

β-amyloid (Luo et al., 2014)

Peptides K4 (KLVFF)

LVFFA

GN peptide (GNLLTLD)

Apo A-I mimetic peptide

β-amyloid (Chafekar et al., 2007)

β-amyloid 1-42 (Neddenriep et al., 2011)

β-amyloid (Paula-Lima et al., 2009)

β-amyloid (Handattu et al., 2009)

Amino acids L-arginine

Cysteine

Insulin (Varughese and Newman, 2012)

Lysozyme (Takai et al., 2014a)

Chaperons Alpha S

β Casein

Casein (Thorn et al., 2005)


Nanoparticles

Copymeric NiPAM:BAM

Gold nanoparticles

Fe3O4 nanoparticles

PAMAM G4 dendrimers

β-amyloid (Cabaleiro-Lago et al., 2008)

β-amyloid (Liao et al., 2012)

Lysozyme (Bellova et al., 2010)

α-synuclein (Milowska et al., 2011)

Surfactants SDS (commercial)

Gemini (synthetic)

Papain (Qadeer et al., 2014)

Serum albumin (Gull et al., 2011)

Sugars Trehalose

Glycopolymers

β-amyloid 1-42 (Liu et al., 2005)

β-amyloid (Miura et al., 2007)

Dyes ANS

Congo red


β-amyloid (Lorenzo and Yankner, 1994)

Flavonoids

Drugs

Quercitin

Baicalein

Apigenin

Naringenin

Geniestein

Ketoprofen

Naproxen


α-synuclein (Meng et al., 2009)

Transthyretin (Baures et al., 1998)

Acetylcholinasesterase (Heo et al., 2004)

Transthyretin (Green et al., 2005)

β-amyloid 1-42 (Hirohata et al., 2005)

β-amyloid 1-42 (Hirohata et al., 2005)

Certain polyphenols do not interfere with early nucleation events and inhibition was

observed on elongation phase of fibril assembly or the assembly of large oligomers


27

(Porat et al., 2006). Additionally, β-amyloid 1-40 and tau protein were also inhibited by

various polyphenols that includes exifon, myricetin, hypericin, gossypetin,

purpurogallin, pentahydroxybenzophenon and epicatechin gallate (Porat et al., 2006).

1.4.5. Aggregation inhibition by peptides

Short peptides sequences comprise of naturally occurring amino acids have been

shown to inhibit amyloid fibril formation are classified into two major groups.

Peptides which are similar in sequence to wild type proteins are termed as rationally

designed peptides and peptides which are identified from libraries that may or may

not show sequence similarity to wild type are termed as randomly generated peptides.

In past decades, biotechnology and molecular biology advancement make it possible

to construct and screen large peptide libraries. LVFFA in Aβ is considered to be

primary aggregation prone site and often termed as self recognition site (Neddenriep

et al., 2011). Peptides that hold the LVFFA sequence are established to be a sturdy

group of inhibitory agents for Aβ42, through precise binding with parallel sequence of

natural Aβ-42 (Neddenriep et al., 2011). Phase display and GFP-based screening are

selected methods that are being used to select an epitopes of interest for proper

binding during in vivo aggregation. In addition, numerous peptides have been

recognized (rich in arginines) that showed aptitude to hinder Aβ 42 aggregation under

in vitro conditions (Kawasaki et al., 2010).

1.4.6. Inhibition by anti-inflammatory drugs

The pathophysiology of Alzheimer's disease is generation of amyloid beta peptide

which in turn induces neurodegeneration. This process may involve inflammation and

suppression of this by anti-inflammatory drugs which can provide protection as well

as improve the neuropathy (Aisen, 2002). However, these types of drugs reduce the

risk of Alzheimer's disease as suggested by various epidemiological studies (Aisen,

2002). NSAIDs such as aspirin, diclofenac sodium salt, ketoprofen, flurbiprofen,

ibuprofen and naproxen has been found to preferentially reduce the secretion of the

highly amyloidogenic amyloid Aβ-42 by Rho associated kinase that regulate the

amount of produced Aβ-42 in vitro (Zhou et al., 2003; Hirohata et al., 2005).

Celestrol (a triterpene from plant derivative) possesses antioxidant and anti-

inflammatory activities. Its effects on cognitive functions suggests that this drug may

http://topics.sciencedirect.com/topics/page/Alzheimer's_disease

http://topics.sciencedirect.com/topics/page/Anti-inflammatory

http://topics.sciencedirect.com/topics/page/Alzheimer's_disease


28

be useful in the treatment of neurodegenerative diseases accompanied by

inflammation (Allison et al., 2001). Acetylsalicylic acid, a derivative of salicylic acid

is the most widely used drug for the treatment of various diseases and has a broad

spectrum of biological activities including, neuroprotective, anti-inflammatory,

antitumor and analgesic (Abdu-Allah et al., 2016).

1.5. Proteins used in this study

1.5.1. Stem bromelain (SB)

Bromelain a collective name for closely related proteolytic enzymes found in tissues

of plant family, Bromeliaceae, of which pineapple, Ananas comosus is the best

known. Two distinct types of pineapple bromelain are recognized stem bromelain (EC

3.4.22.32), the major proteolyticcysteinyl protease in pineapple stem and fruit

beomelain (EC3.4.22.33) formerly called bromelain which is a major proteolytic

component in pineapple fruit (figure 1.5). Stem bromelain has a single polypeptide

chain with 212 amino acid residues and contains a single covalently attached

oligosachhrides moiety per molecule liked with amino acid sequences- Asn-Asn117

-

Glu-Ser. The terminal sugar chain is attached to asparagine present at 117 sites

(Murachi, 1976; Ritonja et al., 1989).

Figure 1.5 Three dimensional structure of stem bromelain (modeled using Phyre software)

showing presence of two domains. The figure is generated in Chimera software.

Stem bromelain and other cysteine proteases of plant origin such as ficin and

chymopapain possesses single polypeptide chain and are relatively basic in nature

with a pI > 8 (Lei et al., 2007). Amino acids sequence similarities and


29

crystallographic studies suggest that these proteins are composed of two domains of

equal size (roughly) with active site located in between (Ritonja et al., 1989). SB

belongs to α + β class of proteins in which helical secondary structure was primarily

found in one domain and second domain comprises frequently β-sheet structure.

Table 1.4 Amino acid composition of stem bromelain.

Amino acid Residues per mole of bromelain

Alanine 25

Valine 14

Leucine 05

Isoleucine 18

Proline 11

Phenylalanine 06

Tryptophan 05

Methionine 03

Cysteine 07

Glycine 23

Threonine 09

Serine 17

Tyrosine 14

Aspartate 08

Asparagine 09

Glutamate 09

Glutamine 07

Lysine 15

Arginine 06

Histidine 01

Total 212

These proteins generally possess three internal disulfide bonds along with a free Cys

residue which is responsible for enzyme activity. The catalytic activity generally

requires the reactive thiol (-SH) group of these enzyme to be in reduced form.

Bromelain is adsorbed through the gastrointestinal tract and detected in blood after


30

oral administration. Its concentration was found maximally after one hour of

administration and it also adsorbed from the intestine as reported earlier.In addition,

bromelain can be absorbed in human intestines without degradation and without

losing its biological activity (Castell et al., 1997; Chobotova et al., 2010).

1.5.1.1. Application of stem bromelain

1.5.1.1.1. Beverage industry

Immobilized stem bromelain has been found to be a food safe and promising

biocatalyst for unstable real wine future applications (Ilaria et al., 2012). In addition,

for clear and bright appearance of beer they are treated with proteolytic enzymes such

as crude stem bromelain and papain.

1.5.1.1.2. Food processing

Stem bromelain and papain are specially used in preparation of protein hydrolysates

having excellent taste as well as absence of bitterness. Besides, these are also used for

hydrolyzing milk proteins as well as for meat tenderization (Khanna and Panda,

2007).

1.5.1.1.3. Baking industry

Due to rapid rate of reaction, optimum temperature, lack of side reactions and broad

pH, proteases like bromelain is widely used in baking industry. Treatment with

proteases helps in preventing shrinkage and facilitates faster bakery throughput.

1.5.1.1.4. Medicinal and pharmaceutical uses

A variety of medicinal benefits are claimed form bromelain which was introduced as

a therapeutic compound since 1957. The established activities of stem bromelain

includes anti-inflammatory action, anti-tumor activity, anti-tuberculosis activity,

enhanced absorption of drugs, reversible inhibition of platelet aggregation,

fibrinolytic activity, skin debridement of burns, the modulation of cytokines and

immunity, mucolytic properties, improvement in cardiovascular activity etc (Maurer,

2001). It has been proposed that these effects of SB may originate from its unusual


31

ability to traverse membranes (Seifert et al., 1979; Grabovac and Bernkop-Schnurch,

2006).

1.5.1.1.5. Animal feed

The variation of the nutritional value of forages is highly influenced by the climate,

forage species and cultivars, and the preservation method. Therefore, the estimation of

soluble nitrogen compounds in rumen is pivotal to assess the forage attributes to

maximize its utilization in ruminants diets (Abdelgadir et al., 1997). The

determination of protein degradation in the rumen by the actions of proteases can be

an alternative to replace the conventional method, which is highly expensive and

time-consuming. The addition of protease to animal feed can increase protein

inversion and availability, decrease the cost of animal feed, and increase the source of

protein.

1.5.1.1.6. Cosmetic industry

The cosmetic products that have papain and bromelain as active ingredients can

effectively alleviate skin problems such as wrinkles, acne, and dry skin. These

enzymes gently digest the protein of dead cells in the upper layer of the skin, resulting

in their replacement by younger skin cells from lower layers (Ozlen, 1995).

Bromelain can also reduce bruising and swelling of skin after cosmetic injection

treatments (Levy and Emer, 2012). Apart from it, cysteine proteases have a wide

range of application in textile industry, leather industry as well as in organic

chemistry.

1.5.2. Bovine insulin

Insulin is a peptide hormone produced by the β-cells of pancreatic islets of

Langerhans. Infact, it is a dipeptide hormone containing 51 amino acid residue and

comprises of two chains (A & B) linked together by a disulphide bridges (figure 1.6).

A chain possesses 21 amino acid residue and B chains is made up of 30 amino acids

residue. It has a molecular weight of ~ 58 kD with an isoelectric point near to pH 5.5

(Wilcox, 2005). N-terminal helix of A chain is linked to an anti parallel C terminal

helix and both are joined to B chain (central helical segment) by two disulphide bonds

(Dodson and Steiner, 1998). Bovine insulin is structurally homologous to human


32

insulin (differ by only three amino acid residue) which has been associated with the

clinical syndrome injection localized amyloidogenesis (Wang et al., 2010a). Insulin is

synthesized as pro-insulin in the endoplasmic reticulum and is processed to the

biologically active form inside the secretory granules. Pro-insulin is synthesized on

ribosomes of rough endoplasmic reticulum from mRNA as pre-proinsulin.

Figure 1.6 Crystal structure of bovine insulin as obtained from PDB having PDB id 2zp6.

Pro-insulin were transferred from RER to Golgi apparatus (rich in zinc and calcium

environment) with the help of secretory vesicles which favors the formation of soluble

zinc containing pro-insulin hexamers. Further, enzymes outside the Golgi complex

convert the pro-insulin to insulin and C-peptide (Wilcox, 2005). However, alterations

at the level of post translational modification, translation, and gene transcription

influenced the insulin secretion. In addition, secretion may also influenced by various

factors that include acetylcholine, fatty acids, amino acids, glucagon-like peptide-1

(GLP-1), glucose-dependent insulinotropic polypeptide (GIP), pituitary adenylate

cyclase-activating polypeptide (PACAP) and several other agonists (Bratanova-

Tochkova et al., 2002). Insulin regulates and maintains normal blood glucose levels

by facilitating cellular glucose uptake, regulating carbohydrate and lipid and amino

acid metabolism. It acts at multiple steps in carbohydrate metabolism that include

increase in glycogen synthesis and decrease in glycogen breakdown by

dephosphorylation of glycogen synthase and glycogen phosphorylase kinase

respectively (Wilcox, 2005). It also stimulates fatty acid synthesis in liver and adipose

tissue with formation and storage of triglycerides in liver and adipose tissue.

Additionally, translation and phospholipid metabolism are also influenced by insulin

(Hunter and Garvey, 1998; Liu and Barrett, 2002). Metabolic syndrome and type 2


33

diabetes is the most common clinical syndrome associated with insulin resistance.

Apart from this, certain form of cancers, hypertension, PCOS and non-alcoholic fatty

liver disease are also associated with insulin resistance (Reaven, 2004; Wilcox, 2005).

Table 1.5 Physiochemical properties of proteins used in this study.

Properties Stem bromelain Bovine insulin

Source Ananas comosus Bovine pancreas

Molecular weight (kDa) 23.8 5.8

Number of amino acids 212 51

Number of tryptophan residue 5 0

Number of S-S bonds 3 3

pH optima 6-8.5 6.5-8.5

Extinction coefficient at 280 nm 20.1 10.0

1.6. Conclusion and future prospective

Undoubtedly, a large number of human diseases are allied with protein misfolding

and their aggregation. Protein misfolding and aggregation into polymeric assemblies,

predominantly of amyloid form, can be considered a somewhat generic behavior of it.

This is based on the physicochemical properties of the common polypeptide backbone

and its intrinsic property to bring together into intermolecular β-sheets capable of

proteolytic resistant with high stability. Hydrophobic collapse aromatic interactions

are the main key forces that are involved in the maturation of amyloid fibrils. During

amyloid formation, monomers of protein enters in to the nucleation step (early stage)

and convert it in to oligomers which further elongate either by head to head or side by

side to form a mature fibril. The key reason behind toxicity of aggregates is mostly

involvement of oligomeric species that are formed during the early stage of

aggregation. Attempts towards mechanism of protein folding and aggregation as well

as their inhibition have been many, but still insufficient. The misfolding and

subsequent aggregation of proteins is an important area of intensive research as it is

directly related to the several pathological conditions. This improvement relates

principally to our perceptive of the nature and impact of protein aggregation/amyloid

formation plus establishes their relation among aberrant and normal behavior of living


34

organisms. Significant advances through combination of theoretical and experimental

studies endow with the principles, to establish the progression of protein aggregation

as well as the aggregates species structures. Now a day much sophisticated techniques

are being applied to elucidate this phenomenon in greater detail. However, much more

techniques be discovered that will shed light on the relationships between protein

misfolding and aggregation. Remarkable efforts have been committed to obtaining an

improved indulgent of fibril formation mechanisms and, in parallel, finding methods

for intervention. Even our present understanding regarding protein aggregation and

their mechanism of amyloid formation is leading to more reliable methods of early

diagnosis and more rational therapeutic strategies. Although, extensive improvement

has been made, our present understanding of protein aggregation is still incomplete.

Various strategies are presently being assayed to prevent the formation of amyloids

and toxic oligomers. These include, preventing the production of amyloids at first

place or removing the amyloid protein after it has been produced. Both of these

strategies hold an immense potential for slowing and/or preventing the onset of

amyloid-based diseases. However, whatever prevention or moderate inhibition has

been obtained is mostly experimental. Therefore in the current scenario, improving

our knowledge about the mechanism of protein folding and misfolding is crucially

required for rational development of therapeutics against devastating diseases as well

as in various biopharmaceutical processes. More concisely, an enhanced

understanding of protein misfolding, and ways to prevent misfolding will undoubtedly

provide high intellectual satisfaction and novel insight into the nature and evolution of

biological molecules, and also generate new ideas for biotechnology and

pharmaceutical industries, and for medical sciences.

CHAPTER 2

Published in Int J Biol Macromol. 2016 Oct;91:477-85.

Amyloidogenic behavior of SB Chapter 2

35

Amyloidogenic behavior of different intermediate state of

stem bromelain: a biophysical insight

2.1. Introduction

Protein aggregation is associated with various neurodegenerative disorders including

prion disease and several form of amyloidosis as Alzheimer's disease (Aguilar-Calvo

et al., 2014; Andreasen et al., 2015; Sohail et al., 2015). One of the major hurdle in

the field of biotechnology is protein aggregation, during the process of protein

purification, processing, adsorption and storage (Kudou et al., 2004). Additionally,

aggregation of proteins remains an important factor behind thermo-inactivation

(Kudou et al., 2003), which is undesirable especially for industrially important

enzymes. Aggregation occurs when unassembled or misfolded proteins improperly

expose hydrophobic surfaces that are normally buried in the protein interior‟s region

or takes place at the interface of subunits on exposure of extreme pH and temperature

(Majhi et al., 2006). Under in vitro conditions aggregation occurs by employing harsh

conditions such as acidic or alkaline pH, by the use of cosolvents, high temperature,

high pressure, metal ions, lipid assemblies and surfactants (Loveday et al., 2011;

Khan et al., 2014b; Chaturvedi et al., 2016a). Among these, temperature and pH are

most recurrent factor that affects the progression of aggregation process (Rondeau et

al., 2010; Tutar et al., 2010; Fallah-Bagheri et al., 2013; Mession et al., 2013; Belton

and Miller, 2103). This is because of unfolding and hydrophobic exposure at high

temperature (Yan et al., 2004; Chaturvedi et al., 2015b). Conformational changes also

occur in proteins due to change in pH along with heat treatment. They are commonly

recognized to play a pivotal role in the aggregation process, since they may promote

the onset of new intermolecular interactions. Besides these, any conformational or

structural alterations in the free –SH group also lead to aggregate formation, as they

are more exposed to new intermolecular interaction (Vetri et al., 2007b). At elevated

temperature, hydrophobic interactions as well as frequency of molecular collision

increase that lead to enhancement of protein aggregation process (He et al., 2015).

Large aggregates formed as a consequence of new intermolecular interactions, arises

due to increase in flexibility of nature and compact structure of proteins at high

temperature (Militello et al., 2003).

Chapter 2 Amyloidogenic behavior of SB

36

Stem bromelain [EC 3.4.22.32], a cysteine proteases obtained from pineapple Ananas

comosus. It is widely accepted as a potential phytotherapeutic drug due to its broad

medicinal application such as reversible inhibition of platelet aggregation, bronchitis,

sinusitis, angina pectoris, enhances adsorption of drugs specially antibiotics (Maurer,

2001; Yuan et al., 2006). In addition to antithrombotic and fibrinolytic effects it has

also anti-inflammatory and analgesic actions. Anti-inflammatory activity of SB is

mediated by various factors like increased serum fibrinolytic activity, reduced plasma

fibrinogen levels and reduced vascular permeability due to decreased bradykinin

levels (Livio et al., 1978; Pirotta and De Giuli-Morghen, 1978; Kumakura et al.,

1988). Like other cysteine proteases SB belongs to the α+β protein class with 23%

alpha helix, 5% parallel beta sheet, 18% anti-parallel β-sheet, 28% turns and rest other

secondary structures (Arroyo-Reyna et al., 1994; Arroyo-Reyna and Hernandez-

Arana, 1995). Stem bromelain, unlike most enzymes, has a very wide effective range

of activity at both acidic and alkaline conditions that allows it to remain active in a

variety of biological environments (Ahmad et al., 2006). During their adsorption,

when administered orally, SB encounters low pH in the stomach followed by an

alkaline pH in the intestine. Previous studies have demonstrated that SB adopts

partially folded intermediate in acidic (Haq et al., 2002), as well as specific molten

globule state at basic pH (Dave et al., 2010b). At acidic pH the occurrence of PFI

state of SB was inferred by various approaches like conformational changes in the

vicinity of surface of the exposed hydrophobic tryptophan that results in

internalization in the hydrophobic environment (Haq et al., 2002). Further, retention

of some secondary structural features as well as presence of large number of solvent

accessible non-polar clusters in the protein molecules indicates occurrence of PFI as

reported in earlier reports (Haq et al., 2002). At basic pH (10.0) stem bromelain

undergoes conformational rearrangement in which the hydrophobic core becomes

more compact and accessible (Dave et al., 2010b). Further, retention of most of the

secondary structural features with disruption of tertiary contacts also indicates that SB

acquires molten globule state at basic pH (Dave et al., 2010b). Although considerable

progress have been made in studying the behavior of SB at different state, as well as

effect of various surfactants for aggregation induction, the effect of temperature on


37

SB at both (acidic and basic) conditions is still not fully deciphered. Due to different

structure and composition of these intermediates, they show different aggregation

behaviors at different pH during heat treatment.

Here, we attempted to find out effect of temperature on aggregation behavior of SB at

both pH 2.0 (net positive charge) and 10.0 (net negative charge) by employing various

spectroscopic techniques. The protein secondary structural changes were monitored

through far-UV circular dichroism spectroscopy. The nature of aggregated particles

were analyzed by turbidity measurements, ThT binding, fluorescence microscopy and

transmission electron microscopy. Particle size was measured by using dynamic light

scattering.

2.2. Materials and methods

2.2.1. Reagents

Stem bromelain (from Ananas comosus B 4882), 1-anilino-8-naphthalene sulfonate

(ANS) and Thioflavin T (ThT) were procured from Sigma–Aldrich Chemical Co., St.

Louis, MO, USA. All other reagents used were of analytical grade. Double deionized

water, free from all fluorescent contaminant, was used throughout the study. Glycine-

HCl buffer of pH 2.0 and Glycine-NaOH buffer of pH 10.0 were used. Prior to use

buffers and stock solutions were filtered using PVDF 0.45 µm syringe filters

(Millipore Milex-HV).

2.2.2. Sample preparation

Stem bromelain was dissolved in sodium phosphate buffer (20 mM) at pH 7.4. To

avoid complications due to autocatalysis, sodium tetrathionate (5 mM) was added to

buffer prior to protein addition, for inactivation of its proteolytic activity. Protein

solution was dialyzed extensively against the 20 mM sodium phosphate buffer and

subjected to size-exclusion chromatography (Qadeer et al., 2013). Protein

concentration was determined by Perkin-Elmer Lambda 25 double beam UV-Visible

spectrophotometer and extinction coefficient (ε %1

nm280 = 20.1) was used. Molecular

weight of protein was taken as 23,800 Dalton. Protein samples at 25ºC at both pH

served as control for all measurements.


38

2.2.3. pH measurements

pH measurements were carried out on a Mettler Toledo pH meter (seven easy S 20-K)

using an Expert „„Pro3 in 1‟‟ type electrode. pH was adjusted by 0.1 M HCl and 0.1

M NaOH for pH 2.0 and pH 10.0 respectively.

2.2.4. Protein charge determination

The charge on stem bromelain at various pH was calculated using the program protein

calculator v3.4 (Zaman et al., 2016a).

2.2.5. Turbidity measurements

Turbidity measurements of SB (5 µM &10 µM) at pH 2.0 and 10.0 were taken from 0

to 24 h at 65ºC. The measurement of aliquots was carried out at 350 nm on a Perkin-

Elmer Lambda 25 double beam UV–Vis spectrophotometer, in a cuvette of 1cm path

length.

2.2.6. Rayleigh light scattering measurements

Rayleigh scattering experiments were performed on a Shimadzu (RF-5301PC)

fluorescence spectrophotometer. Samples were incubated with specified pH (pH 2.0

and 10.0) at 65°C for 0 to 24h. To obtain reliable data the scattered intensity was

averaged for 3 samples for each concentration. Samples were excited at 350 nm and

emission spectra were recorded from 300 to 400 nm. Both the excitation and emission

slit widths were set at 1.5 nm.

2.2.7. Thioflavin T (ThT) binding assay

Thioflavin T (ThT) was dissolved in double distilled water and filtered through 0.45

µM syringe filter. Concentration of ThT was determined by using a molar extinction

coefficient (εM = 36,000 M -1

cm -1

) at 412 nm (Biancalana and Koide, 2010). Protein

samples were incubated for 0 to 12h at both pH conditions i.e., at pH 2.0 and 10.0. ThT

was added to aggregate samples of protein in a molar ratio of 2:1 followed by 30 min of

incubation. ThT fluorescence spectra were recorded on Shimadzu (RF-5301PC)

fluorescence spectrofluorometer. The intensity of the excitation was 440 nm and

emission intensity was in the range of 450 to 600 nm. Both excitation and emission slit

widths were fixed at 5 nm. Spectra were subtracted from appropriate blanks.


39

2.2.8. ANS fluorescence measurements

ANS is the extrinsic fluorescent dye with susceptibility towards exposed

hydrophobic patches and therefore a means for symbolizing the molten globule

states and certainly the surface hydrophobicity in proteins could be resolved

smoothly (Park et al., 2011; Honda et al., 2014). In order to scrutinize the exposed

hydrophobic patches upon heat induced aggregation of stem bromelain, ANS

binding assays were executed. ANS was properly dissolved in distilled water and

filtered with 0.45 µM syringe filter followed by measurement of concentration using

molar extinction coefficient, εM = 5,000 M-1

cm-1

at 350 nm. The ANS fluorescence

spectra of aliquots incubated for varying time (0-12 h) at different pH (pH 2.0 and

10.0) were observed on Shimadzu (RF-5301PC) fluorescence spectrophotometer

equipped with water circulator (Julabo Eyela). Prior to measurements, aliquots were

incubated with 20 fold molar excess of ANS for 30 min in dark. For ANS

fluorescence, emission spectra between 400-600 nm were collected with excitation

wavelength at 380 nm. Both excitation and emission slits were set at 5 nm. Spectra

were subtracted from appropriate blanks.

2.2.9. Circular dichroism measurements

Circular dichroic (CD) measurements were performed on JASCO spectropolarimeter

(J-815) equipped with a microcomputer to monitor secondary structural changes in

SB at different conditions. All CD measurements were taken in far UV range from

190 to 250 nm in a cuvette of 0.1 cm path length. Spectra were measured with 5 µM

& 10 µM of SB concentration at different pH. Each spectrum was an average of three

scans. Savitzky–Golay method with 23 convolution width was used for smoothening

of data. The MRE (Mean Residue Ellipticity) was calculated using the following

equation: (Xiao et al., 2008).

(2.1)

where, θobs is CD in millidegree, n is the number of amino acid residues, l is the path

length of the cell and Cp is the molar concentration of protein.


40

2.2.10. Dynamic light scattering (DLS) measurements

To measure the hydrodynamic radii dynamic light scattering measurements were

performed at 830 nm. DynaPro-TC-04 (Protein solutions, Wyatt Corporation, Santa

Barbara, CA) equipped with a temperature-controlled microsampler instrument was used

for DLS measurements. Samples at different pH (pH 2.0 & 10.0) were incubated at 65ºC

for overnight. Before the experiment, all samples were filtered through 0.22 µm pore

sized micro-filters. Aliquots of samples were loaded into a 12 µl quartz cuvette. For each

experiment 20 measurements were taken. Polydispersity and mean hydrodynamic radii

were analyzed at optimized resolution. The hydrodynamic radii (Rh) were predicted on

the basis of an autocorrelation investigation of scattered light intensity based on the

translational diffusion coefficient, using the Stokes–Einstein equation.

D

kTR

6h

(2.2)

where Rh is the hydrodynamic radius, k is the Boltzmann‟s constant, T is the absolute

temperature, η is the viscosity of water and D is the translational diffusion coefficient

(Qadeer et al., 2012).

2.2.11. Fluorescence microscopic measurements

The fluorescence microscopic system is used to observe individual amyloid fibrils.

Fibrils obtained by incubation of protein samples in different pH at 65ºC were assayed

by using ThT as an amyloid specific fluorescent probe. The aliquots as mentioned

above were supplemented with 1:2 molar ratio of ThT for 30 min in dark. The

samples were washed thoroughly and then placed on a glass slide and covered with a

cover slip. Samples were visualized using a carl zeiss imager equipped with 20x, 40x

or 63x (oil) objective magnification. The ThT molecule was excited and fluorescent

image was filtered with FITC channel. Finally images were visualized with American

digital camera attached with microscope.

2.2.12. Transmission electron microscopy measurements

To understand the morphology of amyloid, transmission electron microscopy is a

useful tool (Kaur et al., 2014). The morphology of aggregates for various samples was

examined on transmission electron microscope (JEOL-2100F) operating at an


41

accelerating voltage of 200 kV. Samples were prepared properly at pH 2.0 and pH

10.0 followed by 12h of incubation at 65ºC. The samples were diluted accurately and

applied to copper grids (200mesh), blotted and air-dried. Excess fluid was removed

after 2 min and the grids were then negatively stained with 2% (w/v) uranyl acetate.

Uranyl acetate is known to produce high electron density, image contrast, and impart

fine grained impression to the image (Qadeer et al., ; Ohi et al., 2004).

2.3. Results and discussion

2.3.1. Charge on stem bromelain at various pH

The total charge as well as isoelectric point (pI) on stem bromelain was calculated

using the program protein calculator v3.4. We had calculated the charge on stem

bromelain between pH 2.0 to 10.0. Total charge at pH 7.4 was found to be +3.1.

However total charge on SB at pH 2.0 and 10.0 was found to be +22.9 and -18.3

respectively. The positive charge developed at pH 2.0 and negative charge developed

at pH 10.0 agrees well with the result anticipated that SB acquires net negative charge

at above its pI and positive charge below its pI as all other proteins possess. By using

this program the isoelectric point (pI) was found to be 8.55.

2.3.2. Turbidity measurements to monitor aggregation behavior of SB

Turbidity gives an idea of the extent of aggregation in the protein solution. Thermally

induced aggregation in stem bromelain (at both conditions i.e pH 2.0 and 10.0) was

monitored at 350 nm and summarized in figure 2.1. Time zero corresponds to the time

at which the target pH was attained. Extent of aggregation in acid induced partially

folded intermediates of stem bromelain at pH 2.0 was insignificant as no turbidity was

observed at both concentrations (figure 2.1). This suggests that acid induced partially

folded intermediates of stem bromelain were unable to induce aggregation at 65ºC.

However, at pH 10.0, turbidity gradually increases with time and attained maximum

value at four hour of incubation followed by steady state after 12h incubation.

Further, it is notable that turbidity of samples increases with increasing concentration

of proteins as noticed in figure 2.1. Thermal aggregation behavior of stem bromelain

was observed with two different concentrations. This is because different aggregation

pathways occur (thermally) with increase in protein concentration. The acceleration

towards aggregate formation (thermally) with increasing protein concentration, may


42

be due to increased probability of association or precipitation due to the solubility

limit while crowding effect leads to decrease in thermal aggregation of proteins.

0 2 4 6 8 10 12

0.0

0.2

0.4

0.6

0.8 5 µM (pH 2.0)

10 µM (pH 2.0)

5 µM (pH 10.0)

10 µM (pH 10.0)

Tu

rbid

ity

at

35

0 n

m

Time (hrs)

Figure 2.1 Turbidity measurements of SB samples at pH 2.0 & pH 10.0 at two different

concentrations of SB with respect to time at 65°C.

Proteins acquire net positive charge (below pI) and net negative charge (above pI) as

verified by earlier report. These charges play a crucial role in aggregation pathway

due to charge-charge repulsion and attraction. Amino acid composition is also a

governing factor in aggregation process. Stem bromelain comprises more number of

basic amino acid residues rather than acidic amino acid residues. This leads to

generation of more number of positive charges in SB at pH 2.0. Insignificant turbidity

at pH 2.0 was observed, which may be due to more charge repulsion and less

hydrophobic exposure. This disfavors the aggregation process with compare to SB at

pH 10.0. However, at pH 10.0 due to minimization of charge repulsion (that favors

the aggregation) SB tends to aggregated as confirmed by enhanced turbidity. Most

likely, increase in turbidity for stem bromelain at pH 10.0 is concerned with the

enlargement of aggregates, depending on incubation at 65ºC. The high aggregation

propensity at pH 10.0 could be also due to higher molecular flexibility that would

facilitate favorable intermolecular interactions leading to aggregation. Further, to

confirm the protein aggregation, Rayleigh scattering measurements were performed.

2.3.3. Monitoring the aggregation behavior of SB by Rayleigh light scattering

method

Light scattering at 350 nm is another important parameter used to determine extent of

aggregation. Figure 2.2 show the intensity of scattered light as a function of time

dependent heating at 65ºC for SB (5 & 10 µM). The scattered intensity for stem


43

bromelain at elevated temperature (pH 2.0) stays virtually constant up to 12h, thus

showing no sign of aggregation. However, prominent increase in scattering intensity

was observed at pH 10.0, confirms formation of large amount of protein aggregates.

The dependence of the scattered intensity, where aggregation occurs (pH 10.0), is

very characteristic. Initially, the scattering intensity increases gradually and then

becomes constant. Larger intensity is observed for sample containing 10 µM stem

bromelain at pH 10.0 (figure 2.2).

0 2 4 6 8 10 120

250

500

750

1000

1250 5 µM (pH 2.0)

10 µM (pH 2.0)

5 µM (pH 10.0)

10 µM (pH 10.0)

Ray

leig

h s

catt

eri

ng

at

35

0 n

m

Time (hrs)

Figure 2.2 Rayleigh light scattering intensity at 350 nm of thermally induced SB samples at

pH 2.0 & pH 10.0 at two different concentrations of SB with respect to time.

Further, these findings confirm that stem bromelain undergoes thermal aggregation

and extent of aggregation increases with increasing the protein concentration at

alkaline pH.

2.3.4. Thioflavin-T binding with SB aggregates

Thioflavin-T, a benzothiazole dye, is widely used to visualize and quantify the

presence of amyloids that displays enhanced fluorescence upon binding to amyloids

(Krebs et al., 2005). In solutions, initially ThT has a low fluorescence quantum yield

which increases considerably after binding with amyloid fibrils (Morshedi et al.,

2007). Thus to characterize the heat induced SB aggregates, ThT binding assay was

performed. Figure 2.3 (A-D) show ThT fluorescence spectra of SB and ThT

fluorescence intensity (485 nm) at both conditions i.e pH 2.0 and pH 10.0. SB at pH

2.0 has negligible ThT fluorescence intensity, however at pH 10.0 an increase in

fluorescence intensity occurs till four hour of incubation. Further, no significant


44

increase in ThT binding was observed for overnight incubation. Fluorescence

intensity increases with increasing protein concentration (figure 2.3 C & D).

Figure 2.3 Time-dependent changes in ThT fluorescence spectra and fluorescence intensity at

485 nm of SB at pH 2.0 & pH 10.0. 5 μM (A & B) and 10 μM (C & D)

This confirms aggregate formation with fibrillar morphology. At pH 2.0, amyloid

fibril was not formed due to positive charge repulsion among the amino acid residue

of SB, and hence no significant ThT binding were observed. It can be also attributed

to fact that at acidic pH the positively charged N-atom of a benzothiazole group of

ThT repels the N-atom of arginine and lysine (basic amino acid residue) of stem

bromelain that may not allow binding due to charge-charge repulsion (Khurana et al.,

2005). However at pH 10.0, SB amyloid fibrils (generated at 65°C) provides some

sort of hydrophobic surfaces to thioflavin T, and as a consequence strong ThT binding

with amyloids were observed as confirmed by increase in fluorescence intensity. The

intensity of scattered light, turbidimetric measurements as well as ThT fluorescence

intensity (485 nm) at different conditions is summarized in table 2.1. In solution, polar

group and hydrophobic regions of ThT forms structures in such a way that the

positively charged nitrogen of benzothiazole pointing towards solvent with

hydrophobic interiors made up of dimethylaminophenyl group (Khurana et al., 2005).

Specific ThT binding is also due to hydrogen bond formation between charged

nitrogen of thiazole group to amyloids that were generated at pH 10.0 in SB.


45

Table 2.1 Spectroscopic Properties of SB at different conditions following heat

treatment.

Conditions RLS350 nm FI485 nm Turbidity350 nm

5 µM native SB 16.41 ± 2.24 4.25 ± 2.1 0.02 ± .002

5 µM SB + pH 2 (0.5h) 17.29 ± 3.24 6.95 ± 1.2 0.02 ± .001

5 µM SB + pH 2 (12 h) 37.89 ± 8.23 13.45 ± 2.2 0.04 ± .001

5 µM SB + pH 10 (0.5 h) 24.65 ± 2.32 5.36 ± 1.6 0.03 ± .002

5 µM SB + pH10 (12 h) 538.93 ± 21.67 232.17 ± 11.6 0.29 ± 0.01

10 µM native SB 24.93 ± 2.34 7.23 ± 2.1 0.04 ± 0.01

10 µM SB + pH 2 (0.5 h) 19.42 ± 2.44 9.23 ± 3.1 0.01 ± .001

10 µM SB + pH 2 (12 h) 88.98 ± 4.31 31.57 ± 3.9 0.03 ± 0.01

10 µM SB + pH 10 (0.5 h) 18.32 ± 2.31 10.45 ± 2.7 0.04 ± 0.02

10 µM SB + pH 10 (12 h) 1012.78 ± 26.97 455.55 ± 13.6 0.56 ± 0.04

It is generally suggested that ThT binds as excimers to large hydrophobic cavities of

fibrils (Biancalana and Koide, 2010). This binding will be also based on charged

interaction as positive charge (positive charge nitrogen in benzothiazole moiety of

hydrophobic phenyl ring linked to thiazole ring) of ThT can interact with negatively

charge molecules present in SB amyloid fibrils at pH 10.0. Due to presence of lesser

number of negative charges (due to acidic amino acid residue) at pH 10.0 on SB, it

collapses after heating, and turns into amyloid fibrils, However SB at pH 2.0 possess

more number of positive charges that resist heating and does not turn into amyloid

fibrils.

2.3.5. Aggregation behavior of SB studied by ANS fluorescence assay

1-Anilino-Naphtalene 8-sulfonic acid (ANS) is a fluorescent dye that is extensively

used as a probe for presence of exposed hydrophobic patches or cavities on proteins

(Naseem et al., 2004). To investigate the exposure of hydrophobic patches on heat

induced aggregation in SB at pH 2.0 and 10.0, ANS binding measurements were

performed. Presence of aggregates causes exceptionally more exposure of

hydrophobic patches where large number of ANS molecules bind and give high

emission intensity (McDuff et al., 2004). An enhanced fluorescence emission

accompanied with a blue-shift of spectral maximum is observed with preferential


46

binding of ANS. A comparison of ANS fluorescence spectra ans intensity of SB (pH

2.0 and 10.0) for 5 & 10 µM are shown in figure 2.4 A - C.

Figure 2.4 ANS fluorescence spectra of (A) 5 μM and (B) 10 μM thermally induced stem

bromelain at pH 2.0 and pH 10.0 at various time intervals. (C) ANS fluorescence intensity at

480 nm of SB (5 μM & 10 μM) at pH 10.0. (D) Shift in λmax of SB at pH 10.0 (5 μM & 10

μM).

In accordance to previous results, spectra of SB at pH 2.0 show insignificant ANS

fluorescence intensity for various time intervals (only two data are shown for clarity).

Although, blue shifted emission maxima were observed at pH 2.0, where protein

seems to be trapped in unfolded state. However, at pH 10.0, ten times more ANS

fluorescence intensity than native state clearly indicates enhanced exposure of

hydrophobic patches. Maximum fluorescence intensity was observed at four hour of

incubation followed by steady state on further incubation (12h). Time dependent

increase in fluorescence intensity revealed that size of particle increases with time, as

ANS fluorescence intensity increases with aggregated size increment (Hawe et al.,

2008). Increase in fluorescence is due to reduction in intermolecular charge transfer

rate constant between sulfonate group of ANS and Lys or Arg of stem bromelain

(Gasymov and Glasgow, 2007). Besides, binding to ANS to the SB resulted in

prominent blue shift at both conditions (figure 2.4D). Occurrence of blue shift is due

A B

C D

450 500 550 6000

50

100

150

200

250

300 SB (pH 7.4)

SB (pH2.0) 0.5 h

SB (pH2.0) 12 h

SB (pH10.0) 0.5 h

SB (pH10.0) 1h

SB (pH10.0) 2h

SB (pH10.0) 3h

SB (pH10.0) 4h

SB (pH10.0) 12h

AN

S F

luore

scen

ce i

nte

nsi

ty

Wavelength (nm)

450 500 550 6000

100

200

300

400

500 SB (pH 7.4)

SB (pH2.0) 0.5h

SB (pH2.0) 12h

SB (pH10.0) 0.5h

SB (pH10.0) 1h

SB (pH10.0) 2h

SB (pH10.0) 3h

SB (pH10.0) 4h

SB (pH10.0) 12h

AN

S F

luo

resc

ence

in

ten

sity

Wavelength (nm)

0 1 2 3 4 120

100

200

300

400

500 5 µM SB (pH 10.0)

10 µM SB (pH 10.0)

AN

S f

luo

resc

en

ce i

nte

nsi

ty a

t 4

80

nm

Time (hrs)

0 1 2 3 4 12468

470

472

474

476

478

480m

ax n

m

Time (hrs)

5 µm SB (pH 10.0)

10 µm SB (pH 10.0)


47

to change in intramolecular charge transfer rate by positive charge that is present near

the –NH group of ANS. The emission maxima were remarkably blue shifted up to 12

nm in SB aggregates, possibly due to the non- polar environment being provided to

the exposed Trp residues inside the aggregates. The ANS fluorescence intensity and

λmax (wavelength shift) of stem bromelain obtained under various conditions are

summarized in table 2.2.

Table 2.2 ANS fluorescence intensity and shift in λmax.

Conditions FI480 nm λmax (nm) Shift in λmax

ANS 15.07 ± 2.13 481 nm 0

5 µM SB + pH 2 (12 h) 47.71 ± 2.45 471 nm 10

5 µM SB + pH10 (12 h) 247.30 ± 8.69 469 nm 12

10 µM SB + pH 2 (12 h) 96.19 ± 4.32 471 nm 10

10 µM SB + pH 10 (12 h) 431.83 ± 11.2 470 nm 11

2.3.6. Far-UV circular dichroism to monitor secondary structural measurements

The experimental results from turbidity, light scattering and dye binding indicates a

pronounced thermally pH dependence aggregation, which is most likely related to

protein stability at that pH and temperature. Far UV-CD was used to investigate the

conformational alterations in secondary structure of stem bromelain, altered due to

temperature at different pH. The spectra of native SB (pH 7.4) exhibited a large

minimum at 208 nm and a shoulder at 222 nm that are characteristic of α + β

structure (Bhattacharya and Bhattacharyya, 2009; Rani and Venkatesu, 2015). The

spectra of SB (incubated at respective pH) revealed a decrease in MRE values as well

as peak shift at respective minima. This indicates loss of secondary structure under

both conditions i.e acidic as well as basic conditions. The MRE208nm at pH 7.4 was -

8430 deg cm2 dmol

-1 while at pH 2.0 and 10.0 it was reduced to -6083 deg cm

2dmol

-1

and -7289 deg cm2dmol

-1 respectively. The % α-helical content of SB were calculated

to be 16.23% for native protein (pH 7.4), 7.2% for MG state (pH 10.0) and 6.5% for

PFI (pH 2.0). This implies that SB retains about 44% and 41% of the native α-helical

structure at pH 10.0 and pH 2.0 respectively. These findings support the formation of

characteristic PFI as well as MG state of SB as reported earlier (Haq et al., 2002).

Thermal treated SB at pH 10.0 shows a single negative peak at 218 nm which point

towards prominent acquisition of β-sheet structure (figure 2.5A & B).


48

Figure 2.5 Far-UV CD spectra of SB at pH 7.4 (native SB), pH 2.0 (PFI state) & pH 10.0

(MG state) at two different concentration exposed at different temperature.

Beta-sheet content increases with increasing concentration of SB as ellipticity was

found to be maximum for 10 µM of SB. This indicates that the aggregates formed by

SB at pH 10.0 have characteristics of amyloid fibrils which might be due to

conformation rearrangement of inter and intramolecular associations lead to α-β

transition. In contrast, the spectra obtained at pH 2.0 after incubation (65ºC) were

very similar to the previous one that is obtained at pH 2.0 before heat treatment

(figure 2.5 A & B). From all the above results that are obtained during secondary

structural changes, we found that SB at pH 10.0 is more prone to form amyloids.

Moreover, at pH 2.0, it retains in PFI state and does not favor the thermally induced

amyloidogenesis. These findings were in agreement with the results obtained from

turbidity and ThT measurements.

2.3.7. Dynamic light scattering measurements to monitor the size of SB aggregates

Dynamic light scattering is a useful technique to characterize the size of particles

(Zaman et al., 2014). Presence of protein aggregates and size in the solution could be

detected through DLS. Changes in hydrodynamic radii of SB in native state, and heat

induced SB (5 µM & 10 µM) at pH 2.0 and pH 10.0 were measured. The Rh value of

native SB (5 µM) was 2.3 nm (figure 2.6 A), while at pH 2.0 it increases slightly to

3.8 nm and 5.4 nm for 5 µM and 10 µM respectively (figure 2.6 B&C). This increase

in Rh may be due to unfolding of proteins driven by structural loss at pH 2.0.

However, heat treatment to SB leads significant increase in Rh at pH 10.0, suggests

formation of aggregated species. The hydrodynamic radii for SB at pH 10.0 were

found to be 100.8 nm and 166.0 nm (figure 2.6 D&E).

A B

190 200 210 220 230 240 250-12000

-8000

-4000

0

4000

8000

12000 SB (pH 7.4)

SB (pH 2.0) 12h (25°C)

SB (pH 2.0) 12h (65°C)

SB (pH 10.0)12h (25°C)

SB (pH 10.0)12h (65°C)

MR

E (

deg

cm

2 d

mo

l-1)

Wavelength (nm)

190 200 210 220 230 240 250-12000

-8000

-4000

0

4000

8000 SB (pH 7.4)

SB (pH 2.0) 12h (25°C)

SB (pH 2.0) 12h (65°C)

SB (pH 10.0)12h (25°C)

SB (pH 10.0)12h (65°C)

MR

E (

deg

cm

2 d

mo

l-1)

Wavelength (nm)


49

Figure 2.6 DLS measurements to determine the hydrodynamic radii of stem bromelain at

65ºC. The hydrodynamic radii of stem bromelain (A) 5 μM (pH 7.4), (B) 5 μM (pH 2.0), (C)

10 μM (pH 2.0), (D) 5 μM (pH 10.0), (E) 10 μM (pH 10.0)

This increase in hydrodynamic radii upon heat treatment may be due to complex

formation between charged moieties in SB at pH 10.0. Here, we also observed at

higher concentration of protein (10 µM), the hydrodynamic radii is changed, and

found to be 166.0 nm, as compare with radii of 5 µM SB (100.8 nm). The lower value

of polydispersity (< 15%) in native SB (pH 7.4) is indicative of homogeneous species

and that protein behaved as monomer in the solution. However, greater value of

polydispersity (22.8% and 31.3%) was observed for aggregated species confers

heterogeneity of solution. All results are summarized in table 2.3.

Table 2.3 Hydrodynamic radii (Rh) and polydispersity (Pd) of SB at different pH

Conditions Rh (nm) Pd (%)

(A) SB (pH 7.4) 2.3 ± 0.1 10.6 ± 1.1

(B) SB (pH 2.0) (5 µM) 3.8 ± 0.3 11.1 ± 1.4

(C) SB (pH 2.0) (10 µM) 5.4 ± 0.4 11.4 ± 1.6

(D) SB (pH10.0) (5 µM) 100.8 ± 3.4 22.8 ± 2.1

(E) SB (pH10.0) (10 µM) 166.0 ± 4.2 31.3 ± 3.2

2.3.8. Morphology of SB aggregates examined by fluorescence microscopy and

transmission electron microscopy

Finally, thermally induced aggregation of stem bromelain was further elucidated by

microscopic imaging techniques (Hoyer et al., 2002; Ghosh et al., 2014). Figure 2.7

A&B shows fluorescence microscopic images of SB (5 &10 µM) at pH 2.0. Absence

of fibrils at pH 2.0 clearly indicates that SB is unable to form aggregate at these


50

conditions. However, at pH 10.0 large number of fibrils in microscopic imaging

confirmed aggregation property of SB (figure 2.7 C-D).

Figure 2.7 Fluorescence microscopy of thermally induced aggregation of SB (A) 5 μM (pH

2.0), (B) 10 μM (pH 2.0), (C) 5 μM (pH 10.0), (D) 5 μM (pH 10.0) and transmission electron

microscopic images of heat induced SB (E) 5 μM (pH 2.0), (F) 10 μM (pH 2.0), (G) 5 μM

(pH 10.0), (H)10 μM (pH 10.0).

The morphology of thermally induced aggregation of SB was further examined by

TEM. In parallel with above results amyloid fibrils of SB at pH 10.0 were clearly

visible in TEM images, however no such type of structure was observed at pH 2.0 as

shown in figure 2.7 E-H. In figure 2.8, we have proposed a hypothetical model to

explain how temperature affects different intermediate states of stem bromelain.

Figure 2.8 Schematic representation of thermal induced fibrillation of stem bromelain at

different intermediate states.


51

2.4. Conclusion

We studied the effect of pH (2.0 & 10.0) on SB aggregation propensity. During

adsorption it encounters in acidic conditions (as in gastrointestinal tract) as well in

basic conditions (in intestine). Native state of SB (pH 7.4) undergoes conformational

changes under both conditions, and acquires partially folded state at pH 2.0 and

molten globule state at pH 10.0. The charge developed at both conditions play a major

role during aggregation of SB. We found that at pH 2.0, net positive charge develop

on SB is large in number which resist towards thermal aggregation. However, the

lower number of negative charge develops in SB at pH 10.0 leading to assembly of

proteins into characteristics amyloid fibrils. Consequently it can be easily stated that

negative charges on SB is mainly responsible for aggregation of SB at pH 10.0.

However aggregation phenomenon at different pH provides to make prediction about

the stability during pharmaceutical formulations. Moreover, our findings reveal the

role of charge during aggregation phenomenon, further experimental work is

necessary to understand the mechanism and interactions involve in the fibrillation of

SB. Besides these care should be taken during pharmaceutical formulation of SB.

CHAPTER 3

Published in RSC Advances 6 (44), 37591-37599

Chapter 3 DNA induced aggregation

52

DNA induced aggregation of stem bromelain: a mechanistic

insight

3.1. Introduction

Proteins misfolding and subsequent aggregation are hallmark of a number of clinical

disorders including Alzheimer‟s, Parkinson‟s, Huntington‟s disease, Type II diabetes

and many others (Ono et al., 2014; Huang et al., 2015). Different proteins and

peptides generate morphologically similar amyloid fibrils under carefully chosen

conditions (Qadeer et al., 2014). Generally, higher aggregation propensity of protein

was observed when they are present in partially folded state produced due to harsh

conditions such as low pH, high temperature and moderate concentration of organic

solvents (Bhattacharya et al., 2011; Loveday et al., 2011). But, it is not yet cleared

whether native state of proteins, partially folded or totally folded state leads to

amyloid fibrils development. However, it is believed that exposure of hydrophobic

patches may promote the intermolecular interactions and consequently conformational

changes may be responsible for aggregation. Further, structurally different aggregates,

amorphous aggregates or amyloid fibrils, are formed as a result of different

aggregation pathway acquired under different conditions due to different

conformational changes (Militello et al., 2003; Vetri and Militello, 2005; Gazova et

al., 2013). In spite of the dissimilarity in conformation of the proteins, all amyloid

fibrils share common morphological features (Dobson, 2003; Vetri et al., 2007a). It

has been observed that a large number of intracellular and extracellular factors affect

the fibrillation property of proteins. Negatively charged nucleic acid has been found

associated with proteinaceous deposits in the tissue of patients of amyloid disease

(Jiang et al., 2007). Though enormous investigation and comprehensive details

describes the mechanism of protein aggregation, but still there is no real cure for such

type of devastating diseases.

Stem bromelain [EC 3.4.22.32], a proteolytic enzymes obtained from Ananas

comosus is a widely accepted phytotherapeutical drug member of the bromelain

family (Mahajan et al., 2012). Like other cysteine proteases stem bromelain belongs

to α+β protein class (Ahmad et al., 2007). It is widely accepted therapeutic drug due

to its broad medicinal application such as anti-inflammatory effects for the treatment

DNA induced aggregation Chapter 3

53

of angina pectoris, bronchitis and sinusitis. It also helps in adsorption of drugs

principally antibiotics, analgesic and anti-tuberculosis activity (Aichele et al., 2013).

It has its application in oncology, odontology and a wide range of sports injuries due

to rare chances of side effects. Anti-metastasis and anti-inflammatory activities of SB

are independent of its proteolytic activity, however pleiotrophic effects in stem

bromelain originate from its unusual ability to traverse membranes (Seifert et al.,

1979; Grabovac and Bernkop-Schnurch, 2006). It has been reported that stem

bromelain exhibits partially folded state at pH 2.0, characterized by disrupted tertiary

contacts and exposed hydrophobic surfaces (Haq et al., 2002). These partially folded

intermediates are more prone to aggregate as they have less ordered conformation and

more exposed hydrophobic surfaces (Qin et al., 2007).

DNA interacts with various compounds like metal ions, either by intercalation or

groove binding (Yamada et al., 2014). Besides, nucleic acids also interact with

different amyloid peptides (like prions, beta amyloid and tau protein) and thus

modulate their aggregation behavior (Cherny et al., 2004). In many cases they

generally stimulate fibrillation process and induce structural changes from alpha

helical to beta sheet isoform by forming stable complexes. Various studies revealed

that nucleic acid act as allosteric effectors of PrP amyloidogenesis or act as an

efficient polyanionic scaffold catalyst to misfold the cellular PrP into a scrapie-like

conformer (Lima et al., ; Cordeiro et al., 2001; Deleault et al., 2008; Macedo et al.,

2012; Noble et al., 2015). In addition, RNA molecules may have a stimulatory role in

the pathogenesis of prion proteins (Deleault et al., 2003). However, another finding

revealed conformational switch of engineered RepA-WH1, a bacterial protein that

show some analogies to nucleic acid promoted amyloidosis (Giraldo et al., 2011).

DNA also leads to aggregation of Superoxide dismutase that is responsible for

pathogenesis of amyotrophic lateral sclerosis under acidic conditions (Jiang et al.,

2007). Since, conditions that are required to induce aggregation vary with protein to

protein, we use an appropriate pH condition based on proteins pI. In the present study,

we attempted to monitor the effect of calf thymus DNA on SB at pH 2.0, by using a

combination of various spectroscopic and microscopic techniques. Moreover, we

demonstrated that CT-DNA can induce aggregation in SB with fibrillar morphology.


54


3.2.1. Materials

Stem bromelain (SB) EC 3.4.22.32 lot no. B4882, Calf thymus DNA (CT-DNA), 1-

anilino-8-naphthalene sulfonate (ANS) and thioflavin T (ThT) were purchased from

Sigma–Aldrich Chemical Co., St. Louis, MO, USA. All other reagents used were of

analytical grade.


To avoid complications due to autocatalysis, stem bromelain was dissolved in 20 mM

sodium phosphate buffer (pH 7.4) containing sodium tetrathionate (5 mM) and

subjected to size-exclusion chromatography followed by dialysis (Dave et al., 2010b).

Buffer (pH 2.0) was prepared with Gly-HCl and filtered with 0.45 µm Millipore

Millex-HV PDVF filter. The concentration of protein and CT-DNA were determined

spectrophotometrically using specific extinction coefficient (ε %1

nm280 = 20.1 on Perkin

Elmer Lambda 25 UV-Visible spectrophotometer (Murachi, 1970). The molecular

mass of protein was taken as 23,800 Dalton (Ritonja et al., 1989). CT-DNA stock

solution was prepared in 20 mM Gly–HCl buffer pH 2.0. To check the purity of CT-

DNA solution absorbance ratio A260/A280 was recorded. No further purification was

required since the attenuation ratio was between 1.8 and 1.9. DNA concentration was

determined on Perkin Elmer Lambda 25 UV-Visible spectrophotometer using average

molar extinction coefficient value of 6600 M-1

cm-1

of a single nucleotide at 260 nm

(Kumar et al., 2011). All solutions were filtered through 0.45 µm syringe filter.

3.2.3. pH determination

pH was measured by Mettler Toledo Seven Easy pH meter (model S20) using

Expert„„Pro3 in 1‟‟ type electrode. The least count of the pH meter was 0.01 pH unit.


The charge on stem bromelain at pH 2.0 was calculated using the program protein

calculator v3.4.


55


For turbidity measurements, stem bromelain (5 µM) in absence and presence of

varying concentration of CT-DNA (0-120 µM) were monitored by UV absorbance at

350 nm using JascoV-660 double beam UV–Vis spectrophotometer in 1 cm cuvette

path length. Prior to turbidity measurements, all samples were incubated for 24 h.


Rayleigh light scattering of SB (5 µM) in the absence and presence of varying

concentration of CT-DNA (0-120 µM) were carried out using a Shimadzu RF 5301

PC fluorescence spectrofluorometer equipped with a water circulator (Julabo Eyela) at

25°C. Protein samples were excited at 350 nm and spectra were recorded from 300 to

400 nm. The DNA samples without protein were considered as the control. All the

samples were incubated for 24 h prior to measurements.

3.2.7. ThT fluorescence spectroscopic measurements

Thioflavin T (ThT) was dissolved in distilled water, filtered and then its concentration

was determined using a molar extinction coefficient of ε412nm = 36,000 M-1

cm-1

at 412

nm (Biancalana and Koide, 2010). Protein samples (5 µM) in absence and presence of

CT-DNA (0-100 µM) were incubated in 1:2 molar ratio of protein to ThT for 30 min

at 25°C in dark. The bound ThT was excited at 440 nm and spectra were recorded

from 450 to 600 nm. The excitation and emission slit widths were fixed at 5 nm.

Proper controls were taken in consideration.

3.2.8. ANS fluorescence assay

A stock solution of ANS was prepared in distilled water and its concentration was

determined using molar extinction coefficient, εM = 5000 M-1

cm-1

at 350 nm (Qadeer

et al., 2012). For ANS binding experiment, SB (5 µM) in absence and presence of

varying concentration of CT-DNA (0-100 µM) were incubated at pH 2.0 for 24h.

Further these samples were incubated with 20 fold molar excess of ANS for 30 min in

dark. The excitation wavelength for ANS fluorescence was set at 380 nm and the

emission spectra were taken in the range of 400 to 600 nm. Both the excitation and

emission slits were set at 5 nm.


56

3.2.9. Circular dichroism spectroscopic measurements

Circular dichroic (CD) measurements were performed to monitor secondary structural

changes of SB in the presence of CT-DNA. JASCO spectropolarimeter (J-815) was

used for circular dichroic measurements using quartz cell with 0.1 cm path length.

The temperature was controlled using Peltier Thermostat with Multitech water

circulator and instrument was calibrated with d-10-camphorsulfonic acid. SB (5 µM)

was taken for all far UV CD measurements. Spectra were obtained in the range of

190-250 nm and each spectrum was average of two scans. Scan speed of 100 nm/min,

data pitch 1 nm and response time of 1 s were used for spectra collection. All spectra

were smoothed by the Savitzky–Golay method with 25 convolution width. Prior to the

measurements, all the samples were incubated for 24h. The results were expressed as

MRE (Mean Residual Ellipticity) in degcm2dmol

-1 and MRE was calculated using the

following equation (Xiao et al., 2008).

(3.1)

where, θobs is the observed ellipticity in millidegree, n is the number of amino acid

residues, l is the length of light path in cm, the cell in cm and Cp is the molar

concentration of protein.

3.2.10. Dynamic light scattering measurements

The aggregation behavior changes at different CT-DNA concentration were

determined by DLS measurements. DLS experiments were performed on a Dynamic

Pro-TC-04 dynamic light scattering instrument (Protein solutions, Wyatt technology,

Santa Barbara, CA) equipped with a temperature controlled microsampler that

monitors the scattered light at 90°C to the excitation. SB samples (40 µM) were

incubated with increasing concentration of CT-DNA over 24h at pH 2.0. The protein

to CT-DNA ratio (1:20) was unaltered throughout the DLS measurements. Prior to

DLS measurements, all samples were filtered through a 0.22 µm pore size microfilter

(Whatman International, Maidstone UK). At least 20 measurements were performed

for each sample. The data were analyzed by Dynamic 6.10.0.10 software at optimized

resolution. The hydrodynamic radii (Rh) and polydispersity (Pd) were determined

using Stokes- Einstein relationship.


57

D

kTR

6h

(3.2)

where Rh is the hydrodynamic radius, k is the Boltzmann constant, T is the absolute

temperature, η is solvent viscosity and D, is the diffusion coefficient (Chaturvedi et

al., 2015a).


Amyloid species obtained upon incubation of SB (5 µM) at pH 2.0 in presence of CT

DNA (100 µM) were assessed by using ThT as an amyloid specific fluorescence

probe. Samples were incubated in 1:2 molar ratio of protein to ThT for 30 min at 25°C

in dark and then placed on a glass slide and covered with a cover slip. For ThT

excitation and emission, Z stacking was done in the FITC channel. Images were

acquired using a Carl Zeiss imager equipped with 20x, 40x or 63x (oil) objective

magnification. Final images were captured with american digital camera attached with

microscope.

3.2.12. Transmission electron microscopic measurements

Transmission electron micrograph of SB (5 µM) in presence of CT-DNA (100 µM)

was collected on JEOL 2100F transmission electron microscope (TEM) operating at

an accelerating voltage of 200 kV. Protein sample incubated with different

concentrations of DNA was applied to copper grids (200 mesh) covered by carbon-

coated formvar film. Excess of fluid was removed after 2 min, and then negatively

stained with 2% (w/v) uranyl acetate. The samples on grids then were air-dried and

viewed under electron microscope.

3.2.13. Isothermal titration calorimetric measurements

The binding energetic of SB and CT-DNA was measured using VP-ITC titration

micro-calorimeter from Microcal (Northampton, MA). Before titration experiment, all

samples were degassed appropriately on a thermovac. The sample cell was loaded

with SB (25 µM) prepared in buffer of pH 2.0, and the reference cell contained

respective buffer. Further, multiple injections of 10 µL of CT-DNA (2.6 mM),

prepared in pH 2.0 buffer were injected into sample cell containing SB. Duration of


58

each injection was 20s with an interval of 180s between successive injections. Stirring

speed and reference power were set to 307 rpm and 16 µcal/s respectively. Heat of

dilutions for CT-DNA were determined in control experiments, and subtracted from

the integrated data before curve fitting. The heat signals obtained from ITC were

integrated using Origin 7.0 software supplied by Micro Cal Inc.


3.3.1. Net charge on stem bromelain

The total charge at pH 2.0 on stem bromelain was found to be +22.9 as determined by

Protein Calculator v3.4. The nature of charge agrees well with the result expected as

an outcome of the fact that below its pI, SB acquires positive charges as all other

protein possesses.


Turbidity of samples at 350 nm is a measure of insoluble aggregates and gives an idea

of the extent of aggregation in the solution. The turbidity at 350 nm was monitored in

SB samples in absence and presence of CT-DNA. SB at pH 2.0 in presence of

increasing concentration of CT-DNA exhibited an exponential increase in turbidity up

to 100 µM after which it leveled off (figure 3.1).

Figure 3.1 Turbidity at 350 nm of SB in absence and presence of varying concentration of

CT-DNA (0-120 µM) at pH 2.0.

However, when CT-DNA and SB were incubated individually at pH 2.0, no increase

in turbidity was observed. This indicates the formation of SB aggregates in presence

of CT-DNA at pH 2.0. The chances of aggregation of CT-DNA and SB alone at pH

0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8CT-DNA (pH 2.0)

SB (pH 2.0) + CT-DNA

Tu

rbid

ity

at

35

0 n

m

DNA (µM)


59

2.0 were negated by insignificant turbidity values (figure 3.1). Proteins acquire net

positive charge below its pI that results in electrostatic repulsion between charge

moieties, leading to partial unfolding of proteins with exposed hydrophobic patches.

In such system aggregation occurs due to neutralization of positive charges. The SB

aggregates in presence of CT-DNA at pH 2.0 is due to the interaction of negatively

charged phosphate groups of CT-DNA with positively charged partially folded

intermediates of SB, which possess exposed hydrophobic residue. Further, on

interaction with CT-DNA rate of aggregate formation accelerated which is due to

charge neutralization of SB followed by hydrophobic collapse. Aggregation of the

protein-CT-DNA complex at pH 2.0 has also been reported for the zinc superoxide

dismutase-DNA system (Jiang et al., 2007). However, to further confirm the presence

of protein aggregation, Rayleigh scattering measurements were performed.


Rayleigh light scattering at 350 nm is another parameter to measure the extent of

aggregation due to increase in size and number of aggregates. Thus, the change in

light scattering was monitored in SB at pH 2.0 in absence and presence of varying

CT-DNA concentration (figure 3.2).

Figure 3.2 Rayleigh light scattering at 350 nm of SB in the absence and presence of varying

concentration of CT-DNA (0-120 µM) at pH 2.0.

As can be observed in figure 3.2, steep rise in light scattering intensity was observed

up to 100 µM CT-DNA and no further increase was observed thereafter. However,

control samples (SB and CT-DNA incubated separately at pH 2.0), revealed

insignificant values of light scattering. This indicates that the CT-DNA induces the

0 20 40 60 80 100 1200

200

400

600

800

1000

1200

Ray

leig

h L

igh

t S

catt

erin

g a

t 3

50

nm

DNA (µM)

CT-DNA (pH 2.0)

SB (pH 2.0) + CT-DNA


60

aggregation of SB at pH 2.0 and thus results further support findings from turbidity

measurements.

3.3.4. ThT fluorescence spectroscopic measurements

ThT, a standard fluorescent dye, produces a marked enhancement in fluorescence

intensity upon binding to amyloid structures (Krebs et al., 2005). Thus, to predict the

nature of aggregates, ThT fluorescence intensity measurements of SB (pH 2.0) were

performed in the absence and presence of CT-DNA. Figure 3.3 A & B represents ThT

fluorescence intensity at 485 nm and ThT fluorescence spectra of SB in absence and

presence of CT-DNA respectively. The aggregation did not occur in SB at pH 2.0

(control samples). It may be due to the fact that at pH 2.0, protein exists in a partially

unfolded conformation with exposed hydrophobic patches and positively charged

residues (Haq et al., 2002). The charged residues may prevent aggregation because of

the electrostatic repulsion between positively charge residues of protein molecules.

Insignificant ThT binding was observed with CT-DNA (100 µM) at pH 2.0 (figure 3.3

B).

Figure 3.3 (A) ThT fluorescence intensity of SB at 485 nm at varying concentration of CT-

DNA (0-100 µM) at pH 2.0. (B) ThT fluorescence spectra of SB (pH 2.0) in the absence and

presence of 50 µM and 100 µM CT-DNA.

This observation is in accordance with the finding that ThT show binding with G-

quadruplexes structure of DNA but it has no significant binding with the other forms

of DNA i.e ssDNA, dsDNA and triplex DNA (Gabelica et al., 2013; Luo and Mu,

2015). Therefore, we have also checked the binding of ThT with CT DNA alone to

rule out the ThT fluorescence of CT-DNA and ThT complex. However, addition of

CT-DNA induces the fibril formation process in SB at pH 2.0. The fibril formation

increases with increase in CT-DNA concentration as can be observed from increase in

460 480 500 520 540 560 580 6000

60

120

180

240

SB (pH 2.0)

CT-DNA (pH 2.0)

SB (pH 2.0) + 50 µM CT-DNA


Th

T F

luo

resc

ence

in

ten

sity

Wavelength (nm)

A B

0 10 20 30 40 50 60 70 80 90 1000

50

100

150

200

250 SB (pH 2.0) + CT-DNA

Th

T F

luo

resc

en

ce I

nte

nsi

ty a

t 4

85

nm

DNA (µM)


61

ThT fluorescence intensity at 485 nm (figure 3.3 A&B). This suggests that protein

and DNA interact with each other and form amyloid fibrils. All spectroscopic results

were summarized in table 3.1.

Table 3.1 Spectroscopic properties of SB at different conditions.


SB (pH 2.0) 25.71 ± 3.20 4.26 ± 1.2 0.003 ± 0.01

CT-DNA (pH 2.0) 38.63 ± 2.61 27.9 ± 2.1 0.009 ± 0.01

SB (pH 2.0) + 515.2 ± 13.25 72.3 ± 5.32 0.424 ± 0.01

50 µM CT-DNA

SB (pH 2.0) + 991.3 ± 21.21 195.72 ± 6.95 0.617 ± 0.03

100 µM CT-DNA

3.3.5. ANS binding measurements



(Hawe et al., 2008). Presence of aggregates cause greater exposure of hydrophobic

patches where large number of ANS molecules could bind and yield significant

increase in fluorescence intensity (Khan et al., 2017).

Figure 3.4 (A) ANS fluorescence spectra of SB in the absence and presence of varying

concentrations of CT-DNA at pH 2.0. (B) Changes in emission maxima (λmax) and

fluorescence intensity of SB as a function of varying CT-DNA concentration.

The concentration-dependent effects of CT-DNA on SB at pH 2.0 were evident in the

ANS fluorescence assay as shown in figure 3.4 A. SB in presence of CT-DNA

produced an increase in intensity with concomitant blue shift of 14 nm that indicates

0 20 40 60 80 100470

472

474

476

478

480

482

484

486 max shift

Fluorescence Intensity

DNA (µM)

m

ax

nm

50

100

150

200

250

300

350

400

450

Flu

ore

scen

ce in

ten

sity

A

B

450 500 550 6000

150

300

450

600

AN

S F

luo

resc

ence

in

ten

sity

(a.

u)

Wavelength (nm)

SB (pH 2.0)

CT-DNA (pH 2.0)





62

formation of aggregates (figure 3.4 A&B). Besides, ANS fluorescence spectra

obtained for controls do not show any noticeable fluorescence intensity thus negating

the chances of aggregation.

3.3.6. Far-UV CD measurements to monitor secondary structural changes in stem

bromelain

The changes in the secondary structure of SB in presence of increasing concentration

of CT-DNA were monitored by far-UV CD spectroscopy as it is highly sensitive to

ascertain in secondary structure conformation. Native state (pH 7.4) of SB exhibited

two minima centered on 208 and 222 nm that are characteristic of alpha helical

structure (Bhattacharya and Bhattacharyya, 2009; Dave et al., 2010a) as shown in

figure 3.5.

Figure 3.5 Far-UV CD spectra of SB at pH 7.4, and in the absence and presence of CT-DNA

at pH 2.0.

The spectra of SB (pH 2.0) showed a decrease in MRE value at respective minima as

compared to native state of protein (pH 7.4). This indicates loss of secondary structure

at pH 2.0. The percent α-helical content value of SB at pH 7.4 is 14.5% that reduces

to 6.12% at pH 2.0. This confers retention of about 42% of the native like structure of

SB at pH 2.0 that supports the formation of characteristic PFI as reported earlier

(Qadeer et al., 2013). With increasing CT-DNA concentration, peak at 208 nm

became disappeared and a single minima at 218 nm was obtained, those points toward

prominent acquisition of β-sheet structure (figure 3.5). This clearly indicates that

these conformations with prominent beta-sheet structure are amyloids in nature as

reported in earlier literature (Kardos et al., 2011; Suzuki et al., 2011). Furthermore,

the β-sheet content increases with increasing concentration of CT-DNA were

summarized in table 3.2.

190 200 210 220 230 240 250-12000

-8000

-4000

0

4000

8000

12000

MR

E (

deg

cm

2 d

mo

l-1)

Wavelength (nm)

SB (pH 7.4)

SB (pH 2.0)



63

Table 3.2 Secondary structure properties of SB at different CT-DNA concentrations.

Properties pH7.4 10 µM CT-DNA 50 µM CT-DNA 100 µM CT-DNA

% α 14.52 11.62 8.94 6.01

% β 6.51 7.83 10.63 15.30

% RC 78.97 80.55 80.43 78.69


The change in hydrodynamic radii of the aggregated species was monitored by DLS

as it is capable of unbiased analysis of size distribution of particle (Zaman et al.,

2014). It is well known that during amyloid formation the size of protein molecules

increases due to complex formation, between additives and protein molecules (Liu et

al., 2010). The Rh of SB at pH 7.4 was found to be 2.3 nm (figure 3.6 A).

Figure 3.6 Dynamic light scattering measurement of SB in the absence and presence of

various concentration of CT-DNA (A) SB at (pH 7.4), (B) SB at pH 2.0, (C) SB (pH 2.0) + 50

µM CT-DNA, (D) SB (pH 2.0) + 100 µM CT-DNA.

However, slight increase in hydrodynamic radii (3.2 nm) was observed at pH 2.0

(figure 3.6 B). This increase in Rh is due to partial unfolding of SB at pH 2.0. When

SB was incubated with increasing concentration of CT-DNA at pH 2.0, the Rh was

found to increase. Further, smaller aggregates were observed at lower concentration

of CT-DNA after 24h of incubation. This indicates the formation of complex between

SB and CT-DNA at pH 2.0. In the presence of CT-DNA (50 µM), SB showed

existence of different species with hydrodynamic radii ranging from 2.7-175.8 nm,


64

whereas aggregates with hydrodynamic radii of 178.6 nm were observed at 100 µM

CT-DNA (figure 3.6 C&D). All results are summarized in table 3.3.

Table 3.3 Hydrodynamic radii and polydispersity of SB at different conditions.


(A) SB (pH 7.4) 2.3 ± 0.1 11.4 ± 1.2

(B) SB (pH 2.0) 3.2 ± 0.3 11.7 ± 1.4

(C) SB (pH 2.0) + CT-DNA (1:50) 2.7 ± 0.2 10.6 ± 1.1

9.6 ± 1.1 11.6 ± 1.2

175.8 ± 2.28 20.2 ± 1.8

(D) SB (pH 2.0) + CT-DNA (1:100) 178.6 ± 3.24 38.8 ± 2.2

Further, the broadening of DLS peak with polydispersity (a parameter of

homogeneity) > 40% in the presence of CT-DNA is attributed to the formation of

species of heterogeneous size (Chaturvedi et al., 2015a). However, native protein has

< 15% polydispersity that confirms the existence of protein in homogeneous

monomeric population.

3.3.8. Morphology of fibrils as examined by fluorescence microscopy and

transmission electron microscopy

Fluorescence microscopy is one of the most widely used methods to investigate the

complex formation process and to study the topology of protein aggregates (ordered

or unordered). To examine the CT-DNA induced aggregation in SB at pH 2.0,

fluorescence microscopic images were taken (figure 3.7).

Figure 3.7 Fluorescence microscopic images for (A) Native SB (pH 7.4) (B) SB (pH 2.0) (C)

SB (pH 2.0) + 100 µM CT-DNA, and transmission electron microscopic images for (D)

Native SB (pH 7.4) (E) SB (pH 2.0) (F) SB (pH 2.0) + 100 µM CT-DNA.


65

The SB at pH 7.4 and pH 2.0 did not form any aggregates (figure 3.7 A&B

respectively), while large number of fibrils was observed following incubation with

CT-DNA at pH 2.0 (figure 3.7 C). The morphology of fibrils was also determined by

transmission electron microscopy. In TEM images, no aggregated species could be

observed in sample containing SB alone at pH 7.4 and pH 2.0 (figure 3.7 D&E),

whereas fibrillar structure (similar to that observed in fluorescence microscopy) was

observed in protein samples at pH 2.0 incubated with CT-DNA (figure 3.7 F).

3.3.9. Mode of interaction between SB and CT-DNA as investigated by isothermal

titration calorimetric (ITC) measurements

ITC measurements were performed to gain an insight into the type of interactions

between SB and CT-DNA. The binding isotherms obtained were best fitted by single

binding site model and obtained thermodynamic parameters are compiled in table 3.4.

Table 3.4 Thermodynamic parameters obtained by isothermal titration calorimetric

measurements of SB with CT-DNA.

Ligand Thermodynamic parameters Values

CT-DNA K (M-1

) 2.18 x 104 ± 5.18 x10

3

∆H (Kcal mol-1

) -1.74 ± .05

T∆S (Kcal mol-1

) 3.99

∆G (Kcal mol-1

) -5.78

CT-DNA + NaCl K (M-1

) 3.48 x 103 ± 2.05 x10

3

∆H (Kcal mol-1

) -1.28 ± .03

T∆S (Kcal mol-1

) 3.6

∆G (Kcal mol-1

) -4.81

The binding constant (K) was found to be 2.18 x 104

M-1

which indicates the moderate

binding between CT-DNA and SB. Furthermore, from the sign of enthalpy changes

(∆Hº) and entropy changes (∆Sº), the forces involved in binding of CT-DNA to SB

can be predicted (Ross and Subramanian, 1981). Negative ∆Hº and positive ∆Sº are

indicative of involvement of electrostatic interaction in the binding of CT-DNA to SB

at pH 2.0. However, due to positive ∆Sº hydrophobic interaction cannot be negated.

Moreover, the negative ∆Gº also suggests the interaction to be spontaneous. To

further confirm the involvement of electrostatic interaction, the binding constant of

SB to CT-DNA was calculated in the presence of equimolar NaCl to CT-DNA. The

decrease observed in value of binding constants confirms the involvement of


66

electrostatic interaction at pH 2.0 because the decrease may occur due to competitive

interaction of NaCl ions to SB (Thoppil et al., 2008; Zaidi et al., 2014). It is

established that CT-DNA irreversibly unwinds at pH 2.0 and exposes more phosphate

groups. SB is positively charged at pH 2.0 and thus the chances of its interaction with

negatively charged accessible phosphate groups of unwind CT-DNA are fair and is

probable mechanism that leads to aggregation. Thus, the negatively charged groups is

likely to contribute significantly the formation of aggregates in vitro as reported for

the heparin and mAcP interaction (Khan et al., 2012; Qadeer et al., 2014).

3.3.10. Aggregated assemblies are DNase resistant

The fibrillar assembly of SB was analyzed to ascertain whether they survive on

digestion with DNase. It is easily explored by multiple approaches like Rayleigh

scattering, ThT fluorescence spectroscopy, far UV-CD spectroscopy, and

transmission electron microscopy. The aggregated assembly of SB and CT-DNA (0-

100 µM) was treated with DNase and the obtained results are shown in figure 3.8.

Figure 3.8 Aggregated assembly of SB treated with DNase (A) Rayleigh light scattering (B)

ThT fluorescence intensity at 485 nm (C) Far-UV CD spectra and (D) Transmission electron

microscopic image.

The scattering and ThT fluorescence intensity of DNase treated aggregated assembly

shows similar trend as of untreated CT-DNA (0-100 µM) with SB; however, the

values are lower for former. Similarly, far UV-CD experiment revealed that DNase


67

treated aggregated assembly shows similar secondary structure as of untreated CT-

DNA (0-100 µM) with SB as shown in figure 3.8 C. Furthermore, figure 3.8 D shows

unaltered amyloid fibers after digestion with DNase as visualized in TEM image.

Survival of aggregates even after treatment with DNase confirms that aggregates are

DNase resistant. A schematic representation of CT-DNA induced aggregation is

shown in figure 3.9.

Figure 3.9 Schematic representation of DNA mediated fibrillation of stem bromelain.

3.4. Conclusion

Negatively charged species like DNA is found associated with the proteinaceous

deposits in the tissues of patients suffering from amyloid diseases. Thus in presence of

CT-DNA, we examined the aggregation behavior of SB, a model protein that exists as

partially folded state at pH 2.0. Results conclude that at pH 2.0, SB undergoes

conformational changes and acquires PFI state, which converts into fibrillar structure

upon incubation with CT-DNA. However, initially fibrils are smaller in size that

further progressed into mature fibrils at higher concentration of CT-DNA. It is due to

electrostatic interaction between negatively charged phosphate groups of CT-DNA to

the positively charged residues in SB. Thus, negatively charged groups generally

enhance the protein aggregation and aggregates thus formed are DNase resistant. The

implication of this work is the formation of DNA accelerated protein inclusions that

can be utilized as therapeutic treatment to avoid accumulation of toxic protein

oligomers.

CHAPTER 4

Published in J Biomol Struct Dyn. 2016 Jul 14:1-13.

Chapter 4 Surfactant induced fibrillation in SB

68

Surfactant-mediated amyloidogenesis behavior of stem

bromelain: a biophysical insight

4.1. Introduction

Amyloid plaques are the dominant pathological authentication for neurodegenerative

diseases like Alzheimer‟s, Huntington‟s disease, Parkinson‟s disease etc (Diamant et

al., 2006; Berhanu and Masunov, 2015; Chaari et al., 2015; Kumar et al., 2015).

Amyloid generation is usually pathogenic, where proteins are subjected to

conformational alteration and self association that gives rise to non-functional but

toxic aggregates (Zerovnik, 2002; Ghosh et al., 2015; Sohail et al., 2015). Unfolding

of proteins results in formation of intermediate state that reorganize themselves to

form aggregates which are oligomeric in nature, and finally transform into ordered

fibrils (Chiti and Dobson, 2006; Park et al., 2015). However it is still uncertain

whether amyloid formation is induced by native, partial unfolded or totally unfolded

state of proteins. Partially unfolded state of proteins are believed to have a greater

susceptibility towards aggregation and such states can be formed in vitro under

stressful conditions like extremes of pH, temperature, pressure, additives including

organic solvents and surfactants (Tougu et al., 2009; Khan et al., 2012; Giugliarelli et

al., 2015). Numerous proteins have been recognised to form amyloid fibrils in vivo,

apart from their variation in the amino acid sequence, demonstrating an intrinsic

property of polypeptides to form amyloid fibrils. Some of the common indications for

amyloid structures are enhanced ThT fluorescence intensity, red shift in absorbance

upon binding with Congo red, high β-sheet content and fibrillar morphology (Dobson,

2004). Based upon their significant applications in chemical, cosmetic and

pharmaceutical industries, a large number of compounds have been explored to

induce amyloid fibril formation (Wang, 2005; Li et al., 2006; Wang et al., 2011). In

this framework protein-surfactant interactions have been broadly studied due to their

capability towards amyloid induction based upon their charge and hydrophobicity (Li

et al., 2006; Qadeer et al., 2013).

Stem bromelain (SB) [EC 3.4.22.32], a proteolytic enzyme is obtained from Ananas

comosus. SB is a widely accepted phytotherapeutic drug by virtue of its enormous

medicinal applications including inhibition of angina pectoris, platelet aggregation

Surfactant induced fibrillation in SB Chapter 4

69

and enhanced absorption of drugs particularly antibiotics, antituberculosis activity,

analgesic and anti-inflammatory (Maurer, 2001; Baez et al., 2007; Mahajan et al.,

2012). It has been suggested that such properties of SB emerge from its remarkable

efficiency to span the membranes (Seifert et al., 1979). SB exists in molten globule

state with unorganized tertiary conformation and exposed hydrophobic patches, at pH

10.0 as stated in earlier reports (Dave et al., 2010b). SB encounters alkaline pH in the

intestine, the main site of its adsorption (Dave et al., 2010b). The unfolded and molten

globule states are admitted to hold a greater susceptibility for aggregate formation due

to its less ordered conformation and exposed hydrophobic patches. Molten globule are

oftenly accompanied for better understanding of protein folding pathways (Moosavi-

Movahedi et al., 2003; Chamani and Moosavi-Movahedi, 2006). As SB is destined to

pass the intestinal epithelium membrane during its assimilation in the blood, it may

turn into fibrillar and toxic species upon interaction with membrane components such

as lipids similar to other protein (Dave et al., 2010b; Dave et al., 2011). Thus, the

study of SB interaction with membrane alike compounds has become more relevant at

extreme pH (generally not significant physiologically), but at moderate conditions

(pH 7 to 10) they are significant, as proteins forms stable folding intermediates under

these conditions. Cationic surfactants like CTAB (cetyl trimethylammonium bromide)

and DTAB (dodecyl trimethylammonium bromide) which possess antiseptic

properties are known to be in amyloid induction at low concentration. The structure of

both surfactants is shown in figure 4.1.

Figure 4.1 Chemical structures of CTAB and DTAB.

Here we examined the amyloid induction behavior by both the surfactants (CTAB &

DTAB) at a concentration (0-500 µM) on SB at pH 10.0, using spectroscopic

techniques like turbidity measurements, Rayleigh scattering, circular dichroism, ANS


70

fluorescence assay, Congo red binding and ThT fluorescence measurements. Since

protein surfactant complexes are greatly used as a carrier for drugs and nanoparticles,

it is necessary to know the nature of surfactants towards proteins. This study is

significant in highlighting the role of charge on stem bromelain above its pI on the

structure and function. Further this study may be helpful in understanding the role of

surfactants in amyloid induction and inhibition in proteins.


4.2.1. Materials

Stem bromelain (SB) [EC 3.4.22.32], Thioflavin T (ThT), Congo red, ANS were

obtained from Sigma Chemical Co.,St. Louis, and USA. Hexadecyltrimethyl-

ammonium bromide (CTAB), Dodecyltrimethylammonium bromide (DTAB) and all

other chemicals used were of best analytical grade.


The stock solution of protein was prepared in 20 mM sodium phosphate buffer (pH

7.4) containing sodium-tetrathionate (5 mM). Sodium tetrathionate was used to

inactivate the preteolytic property of SB. Protein concentration of dialyzed stock was

determined using specific extinction coefficient of ε %1

nm280 = 20.1 (Murachi, 1970) on

Perkin Elmer Lambda 25 UV-Visible spectrophotometer. The molecular weight of

stem bromelain was taken as 23,800 Dalton (Gupta et al., 2003). The stock solution of

surfactants were prepared in Gly-NaOH buffer (pH 10.0) and filtered through 0.45 μm

syringe filter. Protein samples were incubated (overnight) with desired range of

surfactant concentration (0-500 µM) before all spectroscopic measurements.

4.2.3. pH determination

pH measurements were made using Mettler Toledo Seven Easy pH meter (model

S20), which is routinely calibrated by standard buffer.


The charge on stem bromelain at pH 10.0 was calculated using the program protein

calculator v3.4 (Zaman et al., 2016a).

http://www.sigmaaldrich.com/catalog/product/sigma/d8638?lang=en&region=US


71


Turbidity of SB (5 µM) samples in the absence and presence of CTAB and DTAB (0-

500 µM) at pH 10.0 were measured at 350 nm. The measurements were carried out on

a JASCO V-660 spectrophotometer. The measurements were carried out in a cuvette

of 1 cm path length at 25°C. The spectra were corrected by subtracting the controls

samples.


Rayleigh scattering measurements was performed at 25°C in a 1 cm path-length cell

on a Shimadzu (RF-5301PC) fluorescence spectrophotometer. Stem bromelain (pH

10.0) in the absence and presence of surfactants were incubated overnight and their

extent of light scattering was measured. Further, spectra of incubate samples were

recorded between 300 to 400 nm at 350 nm excitation wavelength. Excitation and

emission slits were fixed at 1.5 nm. The spectra were corrected by subtracting the

controls samples.


ANS is an extrinsic fluorescent dye with susceptibility towards exposedhydrophobic

patches and therefore a means for symbolizing the molten globule states, certainly

with the help of this dye the surface hydrophobicity in proteins could be examined

(Honda et al., 2014). ANS fluorescence was performed on Shimadzu (RF-5301PC)

fluorescence spectrophotometer equipped with water circulator (Julabo Eyela). For

ANS binding measurements protein samples (incubated with desired concentration of

surfactant) were incubated with 20 fold molar excess of ANS for 30 min in dark. The

ANS fluorescence was measured at 25°C by exciting the samples at 380 nm and

recorded the emission spectra between 400 to 600 nm. Both the excitations an

emission slits widths were set at 3 nm. The spectra were corrected by subtracting the

controls samples.


Thioflavin T (ThT) was dissolved in distilled water and filtered with 0.45 µm Millex

PDVF filter. Further concentration of ThT was determined by using extinction

coefficient of 36,000 M-1

cm-1

at 412 nm (Biancalana and Koide, 2010). Protein


72

samples containing desired range of CTAB and DTAB (0-500 µM) concentration

were incubated in 1:3 molar ratio of protein to ThT for 30 minutes at 25°C in dark.

Samples were excited at 440 nm. The spectra were recorded in the range of 450 nm to

600 nm and plotted at 485 nm. The excitation and emission slit width were both fixed

at 5 nm. The samples prepared in the absence of protein served as blanks. The spectra

were corrected by subtracting the controls samples.

4.2.9. Congo red binding assay

A stock solution of CR was prepared in double distilled water and filtered for further

use. The concentration was determined using εM = 45,000 M-1

cm-1

at 498 nm (Khan et

al., 2014a). The protein samples were incubated with desired range of CTAB and

DTAB (0-500 µM). Further, aliquots of CR were mixed with incubated protein

samples in the absence and presence of CR at a molar ratio of 1:1 and kept for 20

minutes at 25°C in dark. The absorbance spectra (200 to 700 nm) of the resulting

samples were recorded on a UV-Visible spectrophotometer (Perkin Elmer Lambda

25) in a 1 cm path length cuvette and results were analyzed.

4.2.10. Circular dichroic (CD) measurements

Circular dichroic measurements were performed on a Jasco spectropolarimeter (J-

815). The instrument was calibrated with D-10 camphor sulfonic acid. All

measurements were made at 25°C with a thermostatically controlled cell holder

attached to a peltier with Multitech water circulator. Spectra were obtained with a

scan speed of 100 nm/min with a response time of 2s. Each spectrum was average of

five scans. Far-UV CD spectra were taken in the wavelength range of 190-250 nm

range in a cell of 0.1 cm path length. For near-UV CD (250-320 nm) spectral

measurements, the protein concentration was 30 µM and the path length of the cuvette

was 1 cm. All spectra were smoothed by the Savitzky–Golay method with 19

convolution width. The MRE (Mean Residue Ellipticity) in degcm2dmol

-1 was

calculated using the following equation:

lCn

m

10

deg)(MRE

obs

(4.1)

where θ is the CD in milli-degree, n is the number of amino acid residues, l is the path

length of the cell and the C is the molar concentration of SB (Zaidi et al., 2014).


73

4.2.11. Intrinsic fluorescence measurements

Intrinsic fluorescence measurements were performed on Shimadzu (RF-5301PC)

fluorescence spectrophotometer at 25°C in 1 cm path length cuvette. An excitation

wavelength of 295 nm was used to monitor Trp fluorescence. The emission spectra

were recorded over a wavelength range of 300 to 400 nm. Both the excitation and

emission slit widths were fixed at 5 nm.


To measure the size of the aggregated particles in solution dynamic light scattering is

a useful technique. All measurements on DLS were carried out at 830 nm by using

DynaPro-TC04 (Protein solutions, Wyatt Technology, Santa Barbara, CA). Stem

bromelain with surfactants (DTAB and CTAB) was incubated at room temperature

for overnight. The samples were then filtered with 0.22 µm Millipore filter directly

into a 12 µl quartz cuvette. Ten measurements were taken for each experiment. The

polydispersity and mean hydrodynamic radius (Rh) were analyzed using Dynamics

6.10.0.10 software at optimized solution. On the basis of autocorrelation analysis

using stokes equation the Rh value was estimated.

D

kTR

6h

(4.2)

Where Rh is hydrodynamic radius, k is Boltzmann‟s constant, T is temperature and ƞ

is viscosity of the water and D is diffusion coefficient (Qadeer et al., 2012).

4.2.13. Fluorescence microscopy measurements

To observe fibrils, fluorescence microscopic technique is a very reliable technique.

Amyloids obtained by overnight incubation of SB samples with CTAB and DTAB

were assayed by using ThT as an amyloid specific fluorescent probe. The protein

samples were supplemented with 1:3 molar ratio of ThT for 30 min in dark. Further,

samples were washed carefully and then positioned on a glass slide and covered with

a cover slip. Samples were visualized using a carl zeiss imager equipped with 20x,

40x or 63x (oil) objective magnification. FITC channel were used to filter the

fluorescent image. Finally images were visualized with American digital camera

attached with microscope.


74

4.2.14. Transmission electron microscopic measurements

Morphology of protein samples incubated under different conditions was examined on

JEOL 2100F transmission electron microscopy operated with 200 kV voltages. The

aggregates were examined by placing aliquots of aggregated samples on 200 mesh

copper grids covered by carbon-coated formvar film. They were subsequently

negatively stained with 2% uranyl acetate. Further, the grids were air-dried and

viewed under electron microscopy.



Turbidity gives an idea of the extent of aggregation in the solution. Surfactant-

induced aggregation of SB was monitored by measuring turbidity at 350 nm. Figure

4.2 shows the change in turbidity of SB at pH 10.0 incubated with different

concentration of DTAB and CTAB (0-500 µM). In control samples (SB incubated at

pH 10.0), observed turbidity were negligible. The aggregation was insignificant in the

presence of DTAB up to 500 µM. However in the presence of CTAB, the turbidity

gradually increased and attained at maximum value at 200 µM of CTAB as observed

in figure 4.2.

Figure 4.2 Turbidity measurements of SB samples at 350 nm in the presence of 0-500 µM

CTAB and DTAB at pH 10.0. Prior to measurements all samples were incubated overnight at

25°C.

Further increase in CTAB concentration disfavored the aggregation process as evident

from decrease in turbidity in samples containing (250-500) µM CTAB. This suggests

0 100 200 300 400 5000.0

0.3

0.6

0.9

1.2

Tu

rbid

ity

at

35

0 n

m

Surfactant (µM)

SB (pH 10.0) + CTAB

SB (pH 10.0) + DTAB


75

that SB at pH 10.0 favors aggregation upon incubation with CTAB (0-200 µM). It is

known that proteins acquire net negative charge above its isoelectric point (Shaw et

al., 2001; Qadeer et al., 2012). Unfolding as well as exposure of hydrophobic patches

takes place as a result of electrostatic repulsion among charge moieties of proteins.

Aggregation induction in such kind of system takes place due to neutralization of

charges together with increment in hydrophobic interactions. Both necessities are

satisfied with cationic detergents such as CTAB and DTAB. Increased turbidity in

presence of CTAB may be due to higher hydrophobicity as it has longer carbon chains

(14C) than DTAB (12C). At pH 10.0 native conformation of SB exists in unfolded

state and also acquires some negative charge that leads to aggregation upon

interaction of cationic surfactants due to neutralization of charges. Cationic surfactant

(CTAB) neutralizes this negative charge and favors aggregation process. This may be

due to the interaction between negatively charged head group of SB with positively

charged head group of CTAB. Solvent-solvent interaction as a consequence of

repulsion between water molecules (wrapped around the proteins) leads to protein

aggregation (Pertinhez et al., 2002). The higher hydrophobicity of CTAB may be

responsible for the aggregation of SB.


Another parameter used to determine the aggregation is light scattering at 350 nm

(Qadeer et al., 2014). Increase in light scattering at this wavelength is attributed to the

aggregate formation or new complex formation (Sarzehi and Chamani, 2010). No

significant light scattering was observed in protein samples incubated with 0-500 µM

DTAB as shown in figure 4.3. However, significant increase in light scattering at 350

nm was observed in samples incubated with CTAB (0-200 µM). Beyond this

concentration there was a remarkable decrease light scattering which can be attributed

to suppression of aggregation (figure 4.3). It should be noted that the micelles

formation by surfactant and large particles may also contribute to increase in light

scattering. Therefore, control experiments were conducted to rule out such possibility.

No significant increase in light scattering was observed in protein samples as well as

for surfactant samples that are incubated with buffer (pH 10.0) suggesting that no

aggregate formation. CTAB is more hydrophobic in nature. From all these

observation, we suggest that hydrophobicity of surfactant plays a significant role in


76

aggregation process as aggregation was observed only in the presence of CTAB, as it

is more hydrophobic in nature as compared to DTAB.

Figure 4.3 Rayleigh light scattering of SB at 350 nm in the absence and presence of CTAB

and DTAB (0-500 µM) at pH 10.0. Before measurements all samples are incubated overnight

at 25°C.

4.3.3. Charge on stem bromelain

The total charge at pH 10.0 on stem bromelain was found to be -18.3 as determined

by Protein Calculator v3.4. The negative charge developed at pH 10.0 agrees well

with the result anticipated that above its isoelectric point SB acquires net negative

charge as all other protein possess.




(Qadeer et al., 2012). Further, to assets aggregation of protein samples, ANS was used

as an extrinsic fluorophore. An enhanced ANS fluorescence emission together with

blue shift in emission maxima occurred due to preferential binding of ANS to exposed

hydrophobic patches. Native SB (pH 7.4) shows negligible intensity at 480 nm

confirmed that hydrophobic patches were not exposed. On the other hand, samples

incubated with CTAB (0-200 µM), showed a remarkable increase in ANS

fluorescence intensity due to exposure of buried hydrophobic patches figure 4.4 A.

Since all the spectra for 0-500 µM DTAB were observed, so for clarity in figure 4.4

only final 500 µM spectra of DTAB were shown.

0 100 200 300 400 5000

200

400

600

800

1000

Surfactant (µM)

Ray

leig

h s

catt

erin

g a

t 3

50

nm

SB (pH 10.0) + CTAB

SB (pH 10.0) + DTAB


77

Figure 4.4 (A) ANS fluorescence spectra of SB in the absence and presence of 0-500 µM

CTAB and DTAB at pH 10.0. (B) ANS fluorescence intensity of SB at 480 nm in the

presence of different concentration of CTAB.

Further increase in CTAB concentration results in a significant decrease in ANS

fluorescence which indicates reduction in exposed hydrophobic patches. Apart from

this, at lower concentration of CTAB, the emission maxima was also remarkably blue

shifted by 9-11 nm, possibly due to the non- polar environment being provided to the

exposed Trp residues inside the aggregates. However, when CTAB concentration was

enhanced further, the aggregation was diminished significantly and the tryptophan

was again exposed to solvent (figure 4.4 B). A significant ANS fluorescence intensity

was also observed in the presence of DTAB (500 µM) due to exposure of

hydrophobic patches of SB at pH 10.0. However, the F.I was lower than that of 200

µM CTAB where maximum amyloid formation was observed (figure 4.4 A).

4.3.5. ThT binding studies

ThT has been most widely used dye to differentiate between amorphous aggregation

and amyloid fibrils (Zaidi et al., 2014). In general, ThT does not binds to non-

amyloid structures and amorphous aggregates (Khurana et al., 2005). Therefore to

illustrate the nature of aggregates, we performed ThT binding assay of SB samples

that were overnight incubated with CTAB & DTAB (figure 4.5). Degree of

aggregation was increased in SB samples incubated with increasing CTAB

concentration as confirmed by increase in ThT fluorescence intensity. Samples that

were incubated with CTAB (0-200 µM) showed enhanced ThT fluorescence intensity

(figure 4.5 A). Further, increase in CTAB concentration (beyond 200 µM) shows

A B

440 480 520 560 6000

200

400

600

800

1000

1200

AN

S F

luo

resc

en

ce i

nte

nsi

ty

Wavelength (nm)

SB (pH 7.4)

SB (pH 10.0) + 50 µM CTAB

SB (pH 10.0) +100 µM CTAB




SB (pH 10.0) +500 µM DTAB

0 100 200 300 400 5000

200

400

600

800

1000

1200

AN

S f

luo

resc

en

ce i

nte

nsi

ty a

t 4

80

nm

CTAB (µM)

SB (pH 10.0) CTAB


78

reduction in amyloid formation as ThT fluorescence intensity decreases (figure 4.5

B).

Figure 4.5 (A) ThT fluorescence spectra of SB in the absence and presence of 0-500 µM

CTAB and DTAB at pH 10.0. (B) ThT fluorescence intensity at 485 nm of SB under various

conditions.

Amyloid fibrils generated in the presence of CTAB may have provided exposed

hydrophobic surfaces for ThT binding resulting in significant increase in fluorescence

intensity. The binding of ThT to amyloid fibrils may be due to hydrogen bond

formation between charged nitrogen that is present in the thiazole group of amyloid

fibrils and ThT (Khurana et al., 2005). All spectroscopic results are summarized in

table 4.1.

Table 4.1 Spectroscopic properties of SB at different conditions.


SB (pH 7.4) 10.1 ± 2.13 12.77 ± 1.20 0.002 ± 0.01

SB (pH 10.0) 15.4 ± 3.61 27.91 ± 2.10 0.040 ± 0.01

SB (pH 10.0) + 320.2 ± 12.12 136.68 ± 10.2 0.463 ± 0.01

50 µM CTAB

SB (pH 10.0) + 804.4 ± 10.71 213.12 ± 16.3 1.011 ± 0.01

150 µM CTAB

SB (pH 10.0) + 987.3 ± 34.25 224.61 ± 25.3 1.121 ± 0.05

200 µM CTAB

SB (pH 10.0) + 240.6 ± 8.54 195.72 ± 6.95 0.317 ± 0.03

500 µM CTAB

SB (pH 10.0) + 42.8 ± 4.24 15.27 ± 2.30 0.102 ± 0.01

50 µM DTAB

SB (pH 10.0) + 120.5 ± 12.67 35.47 ± 4.52 0.152 ± 0.02

500 µM DTAB

A B

460 480 500 520 540 560 580 6000

50

100

150

200

250

300

Th

T F

luo

rese

cnec

in

ten

sity

Wavelength (nm)

SB (pH 7.4)

SB (pH10.0)

SB (pH10.0) + 50 µM CTAB

SB (pH10.0) +100 µM CTAB





SB (pH10.0) +500 µM DTAB

0 100 200 300 400 5000

50

100

150

200

250

Th

T F

.I a

t 4

85

nm

Surfactant concentration (µM)

SB (pH 10.0) + DTAB

SB (pH 10.0) + CTAB


79

4.3.6. Congo red binding assay

Since ThT binding is also observed for protein oligomers therefore we performed

another dye binding assay using Congo Red, a well-known amyloid specific dye

(Nilsson, 2004; Khan et al., 2014b). Congo red binding exhibits increase in

absorbance with a prominent red shift (Kim et al., 2003; Srinivasan et al., 2003). The

CR binding assay was performed with the SB samples incubated in the absence and

presence of surfactants and results are summarized in table 4.2.

Table 4.2 Shift in λmax of SB at pH 10.0 upon binding with CTAB and DTAB

Conditions λmax (nm) Shift in λmax

Congo red (pH 10.0) 485 nm 0

Congo red + SB ( pH 10.0) 485 nm 0

Congo red + SB ( pH 10.0) + 50 µM CTAB 505 nm 20




Insignificant red shift was observed for samples incubated with 0-500 µM DTAB

(data not shown). This indicates that DTAB was incapable of inducing amyloid

formation in SB. Congo red binding was observed in protein samples incubated with

CTAB (0-200 µM). Samples containing 200 µM CTAB concentrations showed

maximum absorption with significant red shift (32 nm). These results confirmed the

formation of characteristics fibrillar species and ruled out possibility of amorphous

aggregation. During fibril formation process, nucleation is the preliminary step in

which hydrophobic patches of proteins come together and form nucleus. Nucleation,

which is directly proportional to the surfactant hydrophobicity increased due to

availability of additional sites presented by the hydrophobic tails of CTAB. The

reason for this shift is that stem bromelain becomes more compact and this may have

allowed some electrostatic and hydrophobic interactions to take place under such

conditions.


80

4.3.7. Intrinsic fluorescence spectroscopic measurements

Fluorescence spectroscopy is widely used for the study of proteins-ligand interaction.

Among the three aromatic amino acid residue Tyr, Phe and Trp, Trp has the highest

fluorescence yield. This may be due to influence of microenvironment due to presence

of indole group (Otzen, 2002). Thus to get the information on the effect of surfactant

(CTAB & DTAB), Trp was excited. SB contains five Trp residues. As shown in figure

4.6, native state of SB (pH 7.4) exhibited maximum fluorescence at 345 nm suggesting

that most of the Trp residues are exposed to the solvent. At pH 10.0 the λmax was blue

shifted to 3 nm accompanied by an increase in fluorescence intensity, thus suggests the

formation of molten globule state as reported previously (Dave et al., 2010b). The

concentration dependent effects of surfactants (CTAB & DTAB) were also evident in

the intrinsic fluorescence parameters. A sharp blue shift of 8-13 nm accompanied by

pronounced decrease in intrinsic fluorescence intensity was observed up to 200 µM

CTAB concentration followed by a gradual increase till 500 µM CTAB (figure 4.6). A

sharp dip in emission maxima indicates that Trp residue is considerably shielded from

solvent due to strong aggregation of SB. This may be due to Trp residue that were

relatively exposed in molten globule state might have got buried in the non-polar

environment inside the aggregates. The λmax began to rise with increase in CTAB

concentration and became more or less constant with concurrent increase in

fluorescence intensity. This suggests suppression of fibril formation at higher

concentration due to re-exposure of Trp residue in the microenvironment.

Figure 4.6 Intrinsic fluorescence measurements (A) Changes in Trp emission maxima of SB

in native state (pH 7.4) and MG state (pH 10.0) as a function of CTAB concentration. (B)

Changes in fluorescence intensity of SB at pH 7.4 and pH 10.0 as a function of CTAB

concentration.

A B

0 100 200 300 400 50090

105

120

135

150

165 pH 7.4

pH 10.0

RF

I at

34

5 n

m

CTAB (µM)

0 100 200 300 400 500

335

340

345

350 pH 7.4

pH 10.0

m

ax (

nm

)

CTAB (µM)


81

Further it is worth noticing that emission maxima in presence of higher concentration

of CTAB (300-500 µM) was slightly lower (340-344 nm) than molten globule state

(348 nm).This indicates that some of the Trp residue might have been sequestered in

non-polar environment around the newly formed secondary structures that were

formed with higher concentration of CTAB (as noticed in far UV-CD spectra). This

further supports that SB in native state and incubated with higher concentration of

CTAB had different tertiary environment. However, DTAB at all concentration does

not induce fibrillation in SB at pH 10.0, that‟s why we do not show structural changes

that occurred due to intrinsic fluorescence measurements.

4.3.8. Secondary structure determination

Far UV-CD was used to study the changes in secondary structure of stem bromelain

as a result of interaction with different surfactants. The interaction with different

ligand and drugs have been reported to induce secondary structure alterations in

proteins due to disruption of electrostatic interactions and hydrogen bonds

(Rashidipour et al., 2016). Far-UV CD spectra of native SB (pH 7.4) showed

characteristic α-helical signatures and exhibited minima at 208 and 222 nm as

reported by many others (Bhattacharya and Bhattacharyya, 2009; Dave et al., 2010a).

Samples at pH 10.0 (overnight incubated) acquired a characteristic molten globule

conformation due to the loss of secondary structure as demonstrated by decrease in

ellipticity around 208 nm (figure 4.7 A).

Figure 4.7 (A) Far UV-CD spectra of SB at pH 7.4, pH 10.0 and in the presence of different

concentration of CTAB and DTAB. (B) Near UV-CD spectra of SB under different

conditions.

A B

190 200 210 220 230 240 250

-20

-15

-10

-5

0

5

10

15

20

Ell

ipti

cit

y (

md

eg

)

Wavelength (nm)

SB (pH 7.4)

SB (pH 10.0)




SB (pH10.0) +500 µM DTAB

250 260 270 280 290 300 310 320

-6

-3

0

3

6

9

12

15

18

Ell

ipti

cit

y (

md

eg

)

Wavelength (nm)

SB (pH 7.4)

SB (pH 10.0)

SB (pH 10.0) + 200 µM CTAB


82

However, SB acquired the single negative peak around 218 nm in presence of CTAB

(150 & 200 µM), which gave an indication for prominent increase in β-sheet content

of protein that is indicative of the presence of fibrillar structure (Kardos et al., 2011;

Suzuki et al., 2011). However, when CTAB concentration was increased further, the

protein adopted the conformation similar to the native state as observed in figure 4.7

A. The transition of SB α-helix to β-sheet is hierarchical process. It is well known fact

that the folding mechanism with a molten globule intermediate is a hierarchical

model, in which protein folding proceeds according to the established hierarchy of the

native structure i.e, involvement of secondary and tertiary structures (Shiraki et al.,

1995). In present study, SB first acquired a molten globule state at pH 10.0, and then

it formed aggregates in the presence of CTAB. The aggregation process was

suppressed at higher CTAB concentration (200 µM onwards) and the protein acquired

a native like secondary structures. This could probably be due to the hydrophobic

forces gaining predominance at high concentration of surfactant. As a result, the

protein acquired a conformation more like that of native state. No such type of

transition was observed for samples that were incubated with 0-500 µM DTAB

(figure 4.7 A). For clarity only 500 µM DTAB + SB spectra is being shown. Near UV

CD spectral analysis showed that at pH 10.0, SB had fewer tertiary contacts to that of

native one as shown in figure 4.7 B. However, tertiary structure was lost in

aggregated species (figure 4.7 B). Further, tertiary structure of protein (200 µM

onwards CTAB concentration), was found to be significantly disrupted compared to

the native state as inferred from the significantly reduced ellipticity in the near UV-

CD spectra (data not shown).


Dynamic light scattering is a widely used technique to monitor the changes in size of

aggregates (Francis Simpanya et al., 2008; Zaman et al., 2014). This technique was

used to determine the size of SB that was being altered due to presence of surfactants

at pH 10.0. Results obtained from DLS experiments in absence and presence of

surfactants are shown in figure 4.8. The Rh value obtained for SB at native state (pH

7.4) was 2.3 nm, while at pH 10.0 due to unfolding it was slightly increases from 2.3

nm and was found to be 4.5 nm (figure 4.8 A & B). The Rh was slightly altered in

presence of maximum DTAB concentration that was being used (figure 4.8 D).


83

Figure 4.8 Dynamic Light Scattering of SB under different conditions. (A) Native state (pH

7.4), (B) SB (pH 10.0), (C) SB (pH 10.0) + 200 µM CTAB, (D) SB (pH 10.0) + 500 µM

DTAB.

However, in presence of CTAB Rh slightly increased and maximum Rh was found to

be 120.6 nm for protein samples that were incubated with 200 µM of CTAB (figure

4.8 C). Conformational changes in protein were governed under solvent motion as

protein behaves like a dynamic molecular system in aqueous environment.

Interaction of surfactant with the hydrophobic tail and charged head groups leads to

increase in hydrodynamic radii. Since CTAB contains more long hydrophobic tails

than DTAB that maximizes the chance of binding sites. Hydrodynamic radii and

polydispersity index was summarized in table 4.3. Hydrophobic interaction play

important role in complex formation (Khorsand Ahmadi et al., 2015). Polydispersity

for native protein (pH 7.4) was found to be 10.8 which was < 15%, thus signifies

homogeneity of protein samples.

Table 4.3 Hydrodynamic radii (Rh) and polydispersity (Pd) of SB at different

conditions.


(A) SB (pH 7.4) 2.3 ± 0.1 10.8 ± 1.2

(B) SB (pH 10.0) 4.5 ± 0.4 11.1 ± 1.4

(C) SB (pH 10.0) + 200 µM CTAB 120.6 ± 3.6 32.4 ± 2.3

(D) SB (pH 10.0) + 500 µM DTAB 13.8 ± 1.1 12.2 ± 1.1

However, this parameter was found maximum (32.4%) for protein samples that were

incubated with 200 µM CTAB signifies that samples are heterogeneous in nature.


84

4.3.10. Fluorescence and transmission electron microscopic measurements

During protein characterization and optimization of pharmaceutical formulations,

detection of protein aggregates is needed and for this we analyzed protein aggregates

by fluorescence microscopy (staining with ThT) including transmission electron

microscopy (Demeule et al., 2007). No fibrils were observed under microscopy for

native protein (SB at pH 7.4) as well as for SB samples incubated in 500 µM DTAB

(pH 10.0) as shown in figure 4.9 A & B. Fluorescence microscopic images showed a

network of SB fibrils when incubated with 200 µM CTAB at pH 10.0 (figure 4.9 C).

Figure 4.9 Fluorescence microscopic images of SB (A) SB at pH 7.4, (B) SB at pH 10.0, (C)

SB + 200 µM CTAB, (D) SB + 500 µM DTAB. Transmission electron microscopic images of

(E) SB at pH 7.4, (F) SB + 200 µM CTAB, (G) SB + 500 µM DTAB.

At higher concentration of CTAB (500 µM) negligible amount of fibrils were

observed that clearly indicates CTAB at higher concentration is unable to induce

fibrillation in SB at pH 10.0 (data not shown). Figure 4.9 D shows sample incubated

with 500 µM DTAB. Further amyloids were characterized by another microscopic

technique i.e, transmission electron microscopy. SB at native state (pH 7.4) and in

presence of 500 µM DTAB did not show any type of amyloids (figure 4.9 E & G

respectively). However in presence of 200 µM CTAB, SB (pH 10.0) showed fibrillar

type of species as shown in figure 4.9 F. This all results suggest that SB acquire

fibrillar structure in presence of CTAB which is more hydrophobic in nature. A

schematic illustration has been shown in figure 4.10.


85

Figure 4.10 Schematic representation of effect of CTAB and DTAB on MG state of SB at pH

10.0.

4.4. Conclusion

Stem bromelain undergoes conformational changes under basic conditions (pH 10.0)

and acquires molten globule state as confirmed by spectroscopic and circular dichroic

measurements. We studied the effect of cationic surfactants (CTAB & DTAB) on MG

state of SB, a phototherapeutic protein. It was found that CTAB acts as an amyloid

inducer in SB at a concentration range of (50- 200 µM) at pH 10.0 while DTAB was

unable to induce fibrillation. In view of above results, we can conclude that

hydrophobicity triggers aggregation. Larger hydrophobicity of CTAB and its strong

potential towards amyloid fibril formation suggests that aggregation process is

majorly driven by hydrophobicity. Considering all these conditions, stem bromelain

should be extensively examined for fibrillation propensity under in vivo and in vitro

condition during processing of pharmaceutical formulations.

CHAPTER 5

Chapter 5 Cysteine as anti-amyloidogenic molecule

86

Cysteine act as a potential anti-amyloidogenic agent with

protective ability against amyloid induced cytotoxicity for

stem bromelain

5.1. Introduction

Protein misfolding and their subsequent aggregation under various conditions has

been concerned in various neurodegenerative diseases such as Prion, Parkinson‟s,

Alzheimer‟s and systemic amyloidosis diseases (Hashimoto et al., 2003; Ross and

Poirier, 2004; Zaman et al., 2016a). Although, fibrillation is associated with the

progress of a variety of debilitating neurological diseases, cytotoxicity appears to be

linked through pre-fibrillar aggregated states. It is due to their ability to permeabilize

cell membranes and diffusion of hydrophobic surfaces that may catalyze redundant

reactions (Holm et al., 2007). In spite of the variation in the biochemical properties

and amino acid sequence, almost every protein show propensity towards aggregation

under various altered situation like extreme pH, temperature, ionic strength and

presence of denaturants (Vernaglia et al., 2004; Wang, 2005; Wang et al., 2010b;

Zaman et al., 2016c). Among these, temperature and pH are most persistent aspect

that affects the succession of protein aggregation process (Rondeau et al., 2010; Tutar

et al., 2010). This is due to unfolding (at altered pH) and exposure of hydrophobic

patches of proteins at elevated temperature (Yan et al., 2004). Conversely, different

protein possess different mechanism for misfolding and aggregation (Morris et al.,

2009). Using different groups of compounds and molecules, researcher develops

several potential treatment strategies to avert and/or reverse the aggregation

processes. Plant derivatives, drug molecules, polyphenols, amino acids, metal ions are

some structurally unrelated compounds that have been proved to weaken the

intermolecular interactions during inhibition of self assembly progression of proteins

(Lorenzi et al., 2004; Qadeer et al., 2013; Churches et al., 2014; McKoy et al., 2014).

These compounds or additives may control the proteins stability and solubility in the

native and unfolded states respectively (Shiraki et al., 2002). The folding rates are

also altered by these additives or compounds to avert or accelerate the development of

unambiguous aggregates. Amyloid aggregation induction or inhibition by additives

Cysteine as anti-amyloidogenic molecule Chapter 5

87

depends upon their specific group binding with different amino acid residue of

proteins as reported by Shah et el for arginine (Shah et al., 2011).

Stem bromelain (SB), a proteolytic enzyme obtained from Ananas comosus is non-

toxic with great remedial values. It is extensively notable phytotherapeutic drug by

virtue of its massive medicinal applications including fibrinolytic agent (both in vitro

and in vivo), prevention of platelet aggregation, anti-inflammatory and anti-

tuberculosis activity, preventing cancer, cytokine induction in mononuclear cells as

well as enhanced absorption of drugs particularly antibiotics, analgesic (Maurer,

2001; Baez et al., 2007; Bhattacharyya, 2008; Mahajan et al., 2012). It has been

suggested that diverse effects of stem bromelain comes from its astonishing ability to

span the membranes (Seifert et al., 1979). The protein may turn into fibrillar and toxic

species upon interaction with membrane components such as lipids similar to other

protein (Dave et al., 2010b; Dave et al., 2011). Because of its exceptional properties

and therapeutic efficacy, it is crucial to understand its aggregation behavior under

such conditions to which it is exposed during oral administration. Stem bromelain,

when taken orally encountered low pH in the stomach followed by alkaline pH in the

intestine, the main site of its absorption (Dave et al., 2010b). At alkaline pH it adopts

a molten globule state that has a greater susceptibility towards aggregation.

Thermally, under these condition stem bromelain acquires fibrillar structure (Zaman

et al., 2016b). Various studies revealed the aggregation inhibition of different model

proteins under in vitro and in vivo conditions that can serve as a model for

formulating anti-amyloidogenic drug (Ignatova and Gierasch, 2006; Bhattacharya et

al., 2014). They might stabilize the native state of proteins, slow down the fibrillation

progression or repeal the misfolding process.

Cysteine (2-Amino-3-sulfhydrylpropanoic acid) is a semi-essential amino acid

possesses a thiol side chain that participates in enzymatic reactions. The thiol group is

prone to oxidization or reduction, chelation of transition metals that serves a

significant structural role in several proteins (Lipton et al., 2002). Cysteine is found

mostly in high protein diets i.e, meat, eggs, dairy products, wheat germ, and garlic and

also synthesized in the animals with the amino acid serine (Hell, 1997). In humans in

can be synthesized from methionine under physiological conditions. Further, side

chains of cysteine has been shown to stabilize hydrophobic interactions and also tend

to associate with hydrophobic regions of proteins (Heitmann, 1968). Cysteine and its


88

analogue has several applications in pharmaceutical and personal care industries and

also have been reported for inhibition of platelet aggregation, anti-amyloidogenic and

stabilization of proteins against aggregation (Jia et al., 2000; Samuel et al., 2000;

Baynes et al., 2005).

In order to get further insight into cysteine mediated aggregation inhibition of stem

bromelain we evaluated the effect of cysteine by using various spectroscopic and

microscopic techniques. The inhibitory potential of this amino acid was investigated

by employing fluorescence spectroscopy, UV-vis spectroscopy and dye binding

assays (ThT and ANS). The changes in secondary structures were monitored by far-

UV circular dichroism spectroscopy. Morphological changes were analyzed by

fluorescence and transmission electron microscopic techniques.


5.2.1 Reagents

Stem bromelain (B 4882), Cysteine, Thioflavin T (ThT) and 1-anilino-8-naphthalene

sulfonate (ANS) were purchased from Sigma–Aldrich Chemical Co., St. Louis, MO,

USA. All other reagents used were of analytical grade. Double deionized water, free

from all fluorescent contaminant, was used throughout the study. Glycine-NaOH

buffer of pH 10.0 were used. Buffers and stock solutions were filtered using

(Millipore Milex-HV) PVDF 0.45 µm syringe filters.


Stem bromelain was dissolved in sodium phosphate buffer (20 mM) of pH 7.4.

Autocatalysis complications were avoid by adding sodium tetrathionate (5 mM) prior

to protein addition in buffers for proteolytic activity inactivation. Stem bromelain was

dialyzed in 20 mM sodium phosphate buffer (pH 7.4) and subjected to size-exclusion

chromatography (Qadeer et al., 2013). Finally using extinction coefficient (ε %1

nm280 =

20.1), the concentration of stem bromelain was determined by Perkin-Elmer Lambda

25 double beam UV-Visible spectrophotometer. Molecular weight of stem bromelain

was taken as 23,800 Dalton.


89

5.2.3. pH measurements

pH measurements were carried out on a Eutech instruments pH tutor (cyber scan)

using an thermo-scientific type electrode. pH 10.0 was adjusted by using NaOH.


Turbidity measurements of SB samples (10 µM) incubated in absence and presence of

cysteine (0-2 mM) at pH 10.0 were carried out on a Perkin-Elmer Lambda 25 double

beam UV–Vis spectrophotometer, in a cuvette of 1cm path length. We also measured

turbidity with time intervals from 0-12h.


Rayleigh scattering experiments of SB in absence and presence of cysteine were

performed on a Shimadzu (RF-5301PC) fluorescence spectrophotometer at pH 10.0.

Aliquots were excited at 350 nm and emission spectra were recorded from 300 to 400

nm. Both the excitation and emission slit widths were set at 1.5 nm. Spectra were

substracted from their appropriate blanks.


A stock solution of Thioflavin-T was prepared in distilled water and filtered with 0.45

µm Millex Millipore filter. A molar extinction coefficient (εM = 36,000 M-1

cm-1

) was

used to determine the concentration of ThT at 412 nm (Biancalana and Koide, 2010).

Aliquots (10 µM) of SB solutions in absence and presence of desired concentration of

0-2 mM cysteine (incubated overnight) were mixed with ThT (20 µM) at pH 10.0

followed by 30 min of incubation in dark. The resulting fluorescence spectra were

measured on Shimadzu (RF-5301PC) fluorescence spectrofluorometer at excitation

and emission wavelengths of 440 and 450 to 600 nm respectively. Excitation and

emission slit widths were fixed at 3 to 5 nm respectively. Spectra were subtracted

from their appropriate blanks.


A stock solution of ANS (hydrophobic dye) was dissolved in double distilled water,

filtered and further its concentration was determined at 350 nm using a molar

extinction coefficient, εM = 5000 M-1

cm-1

(Qadeer et al., 2014). For ANS fluorescence

experiments, aliquots of SB (10 µM) in absence and presence of 0-2 mM cysteine

(overnight incubation) were further incubated with 50 fold molar excess of ANS for


90

30 min in dark. All ANS fluorescence spectra were recorded on Shimadzu (RF-

5301PC) fluorescence spectrophotometer equipped with water circulator (Julabo

Eyela) with a slit width of 3 to 5 nm. Excitation wavelength (380 nm) was taken with

an emission wavelength in the range of 400 to 600 nm. All spectra were subtracted

from their appropriate blanks.

5.2.8. Circular dichroism spectroscopy measurements

Far-UV CD spectra (190-250 nm) were collected on a JASCO spectropolarimeter (J-

815) using a 0.1 cm path length. Spectra were measured of SB aliquots (10 µM) in the

absence and presence of cysteine (0 - 2mM). For all CD spectral analysis an average

of three scans after subtracting the appropriate ligand (cysteine) concentration was

obtained. For spectra smoothening, we used Savitzky–Golay method with 17

convolution width. The MRE (Mean Residue Ellipticity) in deg cm was calculated

using the following equation (Xiao et al., 2008).

(5.1)

where, θobs is CD in millidegree, n is the number of amino acid residues, l is the path

length of the cell and Cp is the molar concentration of protein.


Dynamic light scattering were performed with a DynaPro-TC-04 (Protein solutions,

Wyatt Corporation, Santa Barbara, CA) equipped with a temperature-controlled at

830 nm. Before the experiment, aliquots were filtered through 0.22 µm pore sized

micro-filters directly into a 12 µl quartz cuvette. For each analysis 15 measurements

were averaged with measurement duration of 20 s. Mean hydrodynamic radii and

polydispersity were analyzed at optimized resolution. The Rh (hydrodynamic radii)

were predicted on the basis of an autocorrelation examination of scattered light

intensity based on the translational diffusion coefficient, with the Stokes–Einstein

equation.

D

kTR

6h

(5.2)


91

where k is the Boltzmann‟s constant, η is solvent viscosity (which was assumed to

that of water), T is the absolute temperature, and D is the translational diffusion

coefficient and Rh is the hydrodynamic radius (Qadeer et al., 2012).

5.2.10. Fluorescence microscopic (FM) measurements

The fluorescence microscopic system (based on inverted microscope) was used to

observe individual fibrils in the solution. Aliquots obtained by incubation of SB (10

µM) in absence and presence of various concentration of cysteine (2 mM) were

assayed through a specific amyloid fluorescent probe i.e, thioflavin T. ThT (20 µM)

were added to protein samples (mentioned above) and incubated for 30 minutes in

dark. Further, aliquots were washed carefully, positioned on a glass slide and roofed

with a cover slip. Using a carl zeiss imager (equipped with 20x, 40x or 63x objective

magnification) samples were visualized. The fluorescent image was filtered using

FITC channel and visualized using american digital camera attached with microscope.

5.2.11. Transmission electron microscopy (TEM) measurements:

Transmission electron microscopy is a functional tool to identify the morphology of

amyloid (Kaur et al., 2014). The morphology of aggregates for different aliquots was

viewed on JEOL-2100F electron microscope operating at an accelerating voltage of

200 kV. SB samples in absence and presence of cysteine (2 mM) were prepared and

placed on 400-mesh copper grids, covered with carbon-stabilized Formvar film,

blotted and air-dried. After 2 minutes excess fluid were removed, and the grids were

negatively stained with uranyl acetate (2%). Excess fluid was further detached and

images were taken. To produce high image contrast, electron density, and impart fine

grained impression to the image we use uranyl acetate (Ohi et al., 2004).


Isothermal titration is an influential technique to determine the thermodynamic

parameters of protein-protein and protein-ligand interactions in solutions. To gain

insight into the binding energetics of stem bromelain and cysteine, we performed

isothermal titration calorimetry using VP-ITC titration micro-calorimeter (Microcal,

Northampton, MA). All samples (protein, cysteine) were degassed properly on a

thermovac before titration. The sample and reference cell (1.44 mL) was loaded with

SB (20 µM) and 20 mM Glycine-NaOH buffer (pH 10.0) respectively. Using a 288


92

µL injection syringe (307 stirring speed), SB was titrated with multiple injections of

10 µL of cysteine (4 mM), prepared in same buffer (pH 10.0). Between successive

injections, the time duration of each injection was 20 s with an interval of 180 s. The

reference power was set to 16 µcal/s. Before curve fitting, we determined heat of

dilutions for cysteine (in control experiments) and subtracted from the integrated data.

The heat signals obtained from ITC were integrated using Origin 7.0 software

supplied by Micro Cal Inc. Standard enthalpy change (∆H°) and association constant

(Kb) were directly obtained by fitting and ∆G° and ∆S° were calculated using

following equation.

∆G° = ∆H° - T∆S° (5.3)

∆G° = - RT ln Kb (5.4)

5.2.13. Cell culture

Breast cancer cells (MDA-MB-231) were obtained from American Type Culture

Collection (ATCC, Manassas, VA, USA). These cells were cultured and maintained

in RPMI-1640 medium in humidified 5% (v/v) CO2/air at 37°C, supplemented with

10% FBS and 1% penicillin-streptomycin solution. Further, these cells were

maintained in M-200 medium (Invitrogen, Carlsbad, CA) supplemented with LSGS

(Invitrogen) with 1% penicillin-streptomycin solution.

5.2.14. Cell viability (MTT) assay

To measure cell viability we performed MTT (3, (4, 5-dimethylthiazol-2-yl) 2, 5-

diphenyltetrazolium bromide) reduction assay on human breast cancer cell line MDA-

MB-231. For MTT reduction assay, approximately 5 × 103 breast cancer MDA-MB-

231 were seeded in 96-well plates and treated with different aliquots (SB in absence

and presence of cysteine) for 24 h. At the end of the incubation time, MTT solution

(0.5 mg/mL) was added to each well and incubated for 4h at 37°C in CO2 incubator.

The media containing MTT solution was aspirated from the wells and the MTT-

formazan crystals were dissolved in DMSO. Absorbance was recorded at 540 nm

wavelength. Cell viability was compared with control samples without prior exposure

to the fibrils solutions.


93


5.3.1. Inhibitory effect of cysteine on aggregation of SB as monitored by turbidity

and Rayleigh scattering measurements

The value of turbidity increases and becomes constant after 4h of incubation of SB

samples (pH 10.0) at 65°C as shown in figure 5.1 A. This is due to increment in size

and number of aggregates at 350 nm as shown in our previous study (Zaman et al.,

2016b). However, in presence of varying concentration of cysteine (0-2 mM)

significant decrease in turbidity indicates inhibition of aggregation (figure 5.2 B).

This fall in turbidity is due to decrease in size and number of aggregated particles as

reported earlier (Qadeer et al., 2013).

Figure 5.1 (A) Turbidity measurements of SB at 350 nm with increasing time (0-12 h) and

(B) in the presence of various concentrations of cysteine (0-2 mM). (C) Rayleigh light

scattering measurement of SB in the absence and presence of various concentration of

cysteine (0-2 mM).

0 2 4 6 8 10 120.00

0.15

0.30

0.45

0.60

Tu

rbid

ity

at

35

0 n

m

Time (hrs)

0.0 0.5 1.0 1.5 2.0

0.2

0.3

0.4

0.5

0.6

Tu

rbid

ity

at

35

0 n

m

Cysteine (mM)

A B

0.0 0.5 1.0 1.5 2.00

200

400

600

800

1000

Ray

leig

h s

cate

eri

ng

(3

50

nm

)

Cystiene (mM)

C


94

Additionally, in figure 5.1 C, the scattering intensity follows similar pattern to that of

obtained in turbidity method. Inhibition may be due to decrease the protein-protein

association or accelerate dissociation of amyloids by cysteine molecules as observed

earlier in insulin aggregation by arginine molecules (Baynes et al., 2005). The most

likely interaction between SB and cysteine might be hydrogen bonding. Thiophilic

interaction takes place between aromatic amino acid residue of protein and sulphur

compound of ligand (Berna et al., 1998). So, thiophilic interaction could also occur

between aromatic amino acid residues of SB with sulphur compound of cysteine.

From all these, we inferred that thiophilic interaction between aromatic amino acid

residue of SB and sulphur atom of cysteine along with hydrogen bonding may play a

major role in deceleration and reduction of SB aggregation.

5.3.2. ThT binding assay to monitor nature of aggregates

The light scattering and turbidometric method are known to detect the presence of

both fibrillar and amorphous material, whereas ThT fluorescence is selective for

detection of cross β-sheet structure of amyloids (Krebs et al., 2005). Thus, ThT

(Thioflavin T) fluorescence assay was performed to inspect and enumerate the impact

of cysteine on amyloid fibril formation by SB aggregates. The characterization and

growth of SB amyloids were monitored through ThT flour. SB samples incubated at

65°C (pH 10.0) in absence and presence of varying concentration of cysteine (0-

2mM) were shown in figure 5.2 A & B. For clarity only selected spectra were shown.

A significant reduction in ThT fluorescence intensity was observed for samples

incubated in presence of cysteine (figure 5.2 A). Further, a marked reduction in ThT

fluorescence intensity at 485 nm (figure 5.2 B) in presence of cysteine clearly

indicates inhibition of SB amyloid fibril formation. The observed decrease in ThT

fluorescence intensity might be due to presence of thiol group in cysteine and its

derivative as they prevent amyloid aggregation. Figure 5.2 C shows kinetics of SB

fibril formation in absence and presence of different concentration of cysteine. The

aggregation kinetics of SB display an emblematic sigmoidal facade containing a lag

phase (associated with nucleation), exponential phase (fast growing phase) and a

stationary phase. Conversely, concurrent incubation of cysteine with SB considerably

attenuated the ThT fluorescence intensity during the same time frame of the

experiment (figure 5.2 C).


95

Figure 5.2 (A) ThT fluorescence spectra of SB incubated at 65°C in the absence and presence

of cysteine (0-2 mM). (B) ThT fluorescence intensity of SB at 485 nm incubated in the

absence and presence of cysteine (0-2mM) at 65°C, (C) Effect of cysteine on SB ThT

fluorescence kinetics in the absence and presence of different concentration of cysteine.

It can be clearly observed from figure 5.2 B that ThT fluorescence intensity of SB was

maximally reduced in presence of 2 mM cysteine. Further, insignificant decrease in

ThT fluorescence intensity, after addition of cysteine on performed fibrils of SB

undoubtedly suggests that it can only inhibit the amyloid formation instead of reversal

of amyloidogenesis (data not shown). All these results revealed that cysteine inhibits

fibrillation of SB form the very beginning as observed in case of inhibition of chicken

cystatin (Wang et al., 2015).

5.3.3. ANS fluorescence measurements to modulate surface hydrophobicity

The presence of hydrophobic patches on surfaces of amyloids or proteins is widely

characterized by ANS (a hydrophobic dye) binding assay (Zaman et al., 2016b).

Based on this, we examined the impact of cysteine on surface hydrophobicity of SB.

480 510 540 570 600

0

100

200

300

400

Th

T f

luo

resc

en

ce i

nte

nsi

ty

Wavelength (nm)

ThT

SB (65 °C)

SB (65 °C) + 0.1 mM Cys

SB (65 °C) + 0.5 mM Cys

SB (65 °C) + 1.0 mM Cys

SB (65 °C) + 1.5 mM Cys

SB (65 °C) + 2.0 mM Cys

0.0 0.5 1.0 1.5 2.00

100

200

300

400

Th

T f

luo

resc

en

ce i

nte

nsi

ty a

t 4

85 n

m

Cysteine (mM)

A B

0 2 4 6 8 10 120

100

200

300

400

SB

SB + 0.5 mM cys

SB + 1.0 mM cys

SB + 2.0 mM cys

Th

T F

luo

resc

en

ce I

nte

nsi

ty a

t 4

85

nm

Time (hrs)

C


96

A significant ANS fluorescence intensity suggests interaction of ANS with exposed

hydrophobic patches of proteins. The ANS fluorescence intensity in absence and

presence of cysteine at 25°C was found to be insignificant (data not shown). In

contrast, in figure 5.3 (A&B), a marked increase in ANS fluorescence intensity was

observed in SB (incubated at 65°C, 12h). However, low ANS fluorescence intensity

was observed for those samples that were co-incubated with cysteine (0.5 mM & 2

mM) as shown in figure 5.3 A&B. For clarity only selected spectra were shown.

Figure 5.3 (A) ANS fluorescence spectra of SB incubated at 65ºC in the absence and

presence of different concentration of cysteine. (B) ANS fluorescence intensity of SB at 480

nm incubated at 65ºC in the absence and presence of different concentration of cysteine.

This suggests that in presence of cysteine exposure of hydrophobic patches were

considerably reduced. The decreased ANS fluorescence intensity with lesser number

of exposed hydrophobic patches suggests that cysteine might alleviate the entire SB

conformation although it was subjected to higher temperature. Cysteine might have

interacted via non-covalent interaction with amino acid residue and interfere with the

formation of SB fibrils as non-covalent bonds stabilize the core structure of amyloids.

5.3.4. Effect of cysteine of conformational transition of SB studied by far-UV

circular dichroism

Changes in secondary structural of proteins are well characterized by CD

spectroscopic techniques. The transformation of alpha helix or intermediate state of

proteins to beta sheet is characteristic of amyloid formation (Alam et al., 2016).

Native SB at pH 7.4 (non-incubated with cysteine) shows double minimum bands at

208 and 222 nm which indicates that it is α-helical in nature (figure 5.4). However,

450 500 550 600

0

100

200

300

AN

S f

luo

resc

en

ce i

nte

nsi

ty (

a.u

)

Wavelength (nm)

ANS Only

SB (65 °C )

SB (65 °C) + 0.5 mM Cys

SB (65 °C) + 2.0 mM Cys

0.0 0.5 1.0 1.5 2.00

100

200

300

AN

S F

.I a

t 4

80

nm

Cysteine (mM)

A B


97

SB spectrum obtained at pH 10.0 incubated at 25°C (MG state) revealed a decrease in

MRE value along with shift at respective minima as reported earlier (Zaman et al.,

2016b). The far UV-CD spectrum of SB incubated at 65°C in absence of cysteine

shows a single peak near 218 nm, which is indicative of β-sheet structure (figure 5.4).

Figure 5.4 Far-UV CD spectra of SB in native state (pH 7.4), MG state (pH 10.0) and

aggregated state in the absence and presence of cysteine (2 mM).

Conversely, SB incubated in presence of cysteine (2 mM) at same experimental

conditions regains its native like structure as observed in far UV-CD spectrum (figure

5.4). These outcomes indicate that structural transitions in SB (from β-sheets to native

like structure) were mitigated upon incubation with different concentration of cysteine

(0-2 mM). For clarity of results only one spectrum is shown.

5.3.5. Dynamic light scattering to measurements

Dynamic light scattering were performed in absence and presence of cysteine to

determine the size distribution of SB. It is quite evident from figure 5.5 (A&B) that

hydrodynamic radii increase from 2.3 nm (native state) to 4.5 nm (MG state) at pH

10.0. This increase in Rh might be due to acquisition of MG state of SB in which it

unfolds at basic pH. Further, low polydispersity index <15% corresponding to the

monomeric state of SB. Conversely, SB incubated in absence of cysteine at 65°C

leads significant increase in Rh (157.3 nm) that indicates formation of aggregated

species (figure 5.5 C). This observed increment in Rh at these conditions is due to

complex formation between charged moieties in SB. However, in presence of 2 mM

cysteine value of size of aggregates decreases and found to be 2.4 and 138.9 nm

(figure 5.5 D).

190 200 210 220 230 240 250

-8000

-4000

0

4000

MR

E (

deg

cm

2 d

mo

l-1)

Wavelength (nm)

SB (pH 7.4 + 25 °C)

SB (pH 10.0 + 25 °C)

SB (pH 10.0 + 65 °C)

SB (pH 10.0 + 65 °C) + 2 mM Cysteine


98

Figure 5.5 Dynamic light scattering measurement of SB in the absence and presence of

cysteine (A) SB in native state (pH 7.4, 25°C), (B) SB in MG state (pH 10.0, 25°C), (C) SB at

pH 10.0, 65°C, (D) SB at pH 10.0, 65°C + 2 mM cysteine.

This decrease in size of aggregates and distribution of two species, one nearly

identical to the native one attributed to the stabilization of SB in presence of cysteine.

All results are summarized in table 5.1.

Table 5.1 Hydrodynamic radii (Rh) and polydispersity (Pd) index of stem bromelain

(SB) in absence and presence of cysteine.


(A) SB at 25°C (pH 7.4) 2.3 ± 0.02 10.4

(B) SB at 25°C (pH 10.0) 4.5 ± 0.17 12.6

(C) SB at 65°C (pH 10.0) 157.3 ± 4.2 1 30.1

(D) SB at 65°C (pH 10.0) 2.4 ± 0.04, 138.2 ± 3.9 11.1, 17.3

+ 2 mM cysteine

5.3.6. Aggregation inhibition visualized by fluorescence and transmission electron

microscopy

Additionally, to explore the efficacy of cysteine beside SB fibril formation, FM and

TEM analysis were performed. SB incubated at 65°C for 12h upon binding with ThT

fluorescence green that imply the presence of amyloid fibrils unlike control SB (pH

10.0, 25°C) as shown in figure 5.6 A&B. Further, SB aliquots incubated with 2 mM

cysteine showed reduced ThT fluorescence that clearly indicates inhibition of amyloid


99

fibrils (figure 5.6 C). In addition, transmission electron microscopy also revealed

inhibition of SB amyloids in presence of cysteine under similar experimental

conditions. SB (pH 10.0) at 25°C shows negligible fibers (figure 5.6 D), while at

65°C a large number of amyloid fibrils were observed (figure 5.6 E).

Figure 5.6 Fluorescence microscopic images of (A) SB at 25°C, (B) SB incubated at 65°C in

the absence of cysteine, (C) SB incubated at 65°C in the presence of 2 mM cysteine.

Transmission Electron Microscopic images of (D) SB at 25°C, (E) SB incubated at 65°C in

the absence of cysteine (F) SB incubated at 65°C in the presence of 2 mM cysteine.

However, SB samples incubated with cysteine showed lesser number of fibrillar

aggregate as shown in figure 5.6 F. All these results clearly revealed cysteine inhibits

fibrillation of SB.


Isothermal titration calorimetric along with controls was performed properly to

measure the heat evolved or absorbed at the time of protein-ligand interaction. The

thermodynamic and binding parameters of cysteine with SB that were obtained after

fitting were summarized in table 5.2. The results show that cysteine binds with

moderate affinity (104) to SB at pH 10.0. Negative values of ∆H° and ∆S° clearly

indicate that reaction was accompanied by favorable enthalpic change (∆H < 0) and

unfavorable entropic changes (∆S < 0). This is a usual observation in protein-ligand

interaction as a favorable binding enthalpy essentially results in greater entropic

constraint leading to more unfavorable contribution to binding free energy (Ross and

Subramanian, 1981).


100

Table 5.2 Thermodynamic parameters obtained by isothermal titration calorimetric

measurements of SB with cysteine.

Ligand Thermodynamic

parameters

Values

Cysteine

K (M-1

)

∆H (Kcal mol-1

)

T∆S (Kcal mol-1

)

∆G (Kcal mol-1

)

1.74 x 104 ± 2.28 x10

3

- 12.74 ± 0.71

- 6.85

- 5.89

In addition, the type of forces responsible for cysteine association was determined by

thermodynamic parameters obtained from cysteine-SB interaction. It is well described

that net entropy contribution is sum up of hydrophobic interaction, restriction of degree

of freedom of main and side chain of polypeptide, changes in vibration content, burial

of water-accessible area and the uptake/release of ion and water molecules. Besides

these, free energy of association (net enthalpy) is sum up of van der Waals interactions

and hydrogen bond formation (Ross and Subramanian, 1981; Ahmad et al., 2011).

Based on these result, we inferred that hydrogen bonding plays a significant role, since

the interaction results the involvement of negative sign of both i.e, enthalpy change

(∆H) and entropy change (∆S). Furthermore, negative value of ∆G° signifies that

interactions between cysteine and SB were spontaneous in nature at given conditions.

From all these results we inferred that hydrogen bonding play an important role in

aggregation inhibition along with thiophilic interaction between aromatic amino acid

residues of stem bromelain with thiol group of cysteine to some extent.


The amyloid fibrils are known to be harmful for cells; however pre-amyloid fibrils or

oligomers are thought to be more cytotoxic. To examine the capability of cysteine to

slow down cell death caused by SB fibrils, we performed MTT assay using cancer

cell lines (MDA-MB-231). No cytotoxic effect was found when cysteine alone was

added to MDA-MB-231 cell line (data not shown). SB amyloids and their effect on

cell viability were examined by treating MDA-MB-231 cell line with these fibrils and

data were shown in figure 5.7. In presence of SB amyloids cell viability decreases up

to 68% as inferred from figure 5.7. Conversely, cysteine increases cell viability even

in presence of SB amyloids. Cell viability was rescued to 81% and 83% in presence of

1 mM and 2 mM cysteine respectively.


101

Figure 5.7 MDA-MB-231 cell viability after being exposed to SB fibrils formed in the

absence and presence of cysteine (1 mM and 2 mM). *Statistically significant from the

control group, p ≤ 0.01 and # statistically significant from the SB, p ≤ 0.01.

It may be due to the decrease in fibril formation (in presence of cysteine) which

disrupts the cell membrane. This confirmed that regained cell viability was due to

anti-amyloidogenic property and ability of cysteine against amyloid fibrils of SB. All

these results suggest that non-fibrillar aggregates formed in presence of cysteine are

less toxic to cancer cells and therefore related compounds may have therapeutic

intervention against amyloid diseases (Hayden et al., 2015).

5.4. Conclusion

By exploration of various biophysical techniques, the current study established that an

amino acid (cysteine) have anti-amyloidogenic property as it inhibits amyloid fibril

formation of stem bromelain. Microscopic as well as dynamic light scattering studies

revealed formation of smaller size aggregates with sparingly population. Additionally,

cysteine which seems to be remedial through conventional biophysical and imaging

approaches too imparts favorable possessions in decreasing cytotoxicity of human

breast cancer cell line. Hydrogen bonding and conformational changes play an

important role in aggregation inhibition of SB as conferred by isothermal titration

calorimetry. The thiophilic interaction between aromatic amino acid residue of SB

and sulphur atom of cysteine may also contribute in deceleration and reduction of SB

amyloid fibril formation. Cysteine is a compound that is found in blood and cytosol

plentifully, so that it or its derivative (e.g peptides containing cysteine,) may be a

potential candidate for the structural design against amyloidogenesis in future

Control

SB amyloids

SB amyloids + 1 mM Cys

SB amyloids + 2 mM Cys

0

20

40

60

80

100

#

#

*

% C

ell

Via

bil

ity


102

projections. The results of the present study may provide a new insight of protein

fibrillation inhibition by cysteine; pave the way for breakthrough of novel anti-

amyloidogenic agents and other small molecules that may exert similar effect against

amyloidogenesis and its related neurodegenerative diseases.

CHAPTER 6

Anti-amyloidogenic behavior of salicylic acid derivatives Chapter 6

103

Amino group of salicylic acid exhibits enhanced inhibitory

potential against insulin amyloid fibrillation with protective

aptitude towards amyloid induced cytotoxicity

6.1. Introduction

Protein misfolding and aggregation into amyloidal fibrils play a noteworthy role in the

pathogenesis of various neurological disorders due to excessive accumulation of it

into organs and tissues (Kelly, 1998; Sipe and Cohen, 2000; Hong et al., 2012). Such

kind of aggregated species might append to cell membranes and cause membrane

permeabilization, which induces cell dysfunction that results into pathogenic

symptoms (Gong et al., 2014). These diseases possess different biochemical and

pathological properties with distinct functions of their corresponding precursor

proteins (Wang et al., 2010a). Under various conditions like extreme pH, elevated

temperature, interaction with surfactants and in presence of nucleic acids these

proteins acquire fibrillar structure (Malisauskas et al., 2003; Necula et al., 2003;

Zaman et al., 2016c). Undoubtedly, extensive efforts have been developed to

understand the molecular detail of amyloid deposition to screen out of anti-

aggregating or anti- amyloidogenic molecules as a means for therapeuting

implications. Under both conditions (in vitro and in vivo), several synthetic and

natural compounds/molecules have been reported to inhibit or retard the fibril

formation process (Wang et al., 2005; Porat et al., 2006). A potential approach to

tackle such type of diseases is to inhibit or capturing these species during their

production into pathogenic fibrillar structures.

Bovine insulin is a 51 amino acid residue peptide hormone that regulates glucose

metabolism and used in treatment of diabetes. It is composed of two polypeptide

chains (A & B) linked together by a pair of inter chain disulphide bonds (Nielsen et

al., 2001). At pH 2.0, insulin exists as a mixture of oligomeric states that include

monomer, dimer and hexamer in solutions with helical conformation that includes

almost 44 % α-helical structure (Hua and Weiss, 2004). Bovine insulin is an

extensively investigated protein, structurally homologous to human insulin (differ by

only three amino acid residues) which has been associated with the clinical syndrome

Chapter 6 Anti-amyloidogenic behavior of salicylic acid derivatices

104

injection localized amyloidogenesis (Wang et al., 2010a). Therefore, bovine insulin

serves as an excellent model system under provided suitable conditions such as low

pH and high temperature at which it forms typical amyloid fibrils (Kitagawa et al.,

2015). At these conditions hexamer dissociation, monomers unfolding (partial) as

well as hydrophobic surfaces contact lead to aggregation and subsequent amyloid

firbril formation (Gibson and Murphy, 2006). Many aggregate inhibitors have been

identified to affect aggregation process such as small molecules, natural and synthetic

compounds, osmolytes, peptides or amyloid specific antibodies (Cheng et al., 2013;

Choudhary et al., 2015). Conversely, their associated groups and intermolecular

interactions with associated energies are still very much lacking during inhibition

progression.

Acetylsalicylic acid (ASA) and 5-aminosalicylic acid (5-ASA) are inflammatory

drugs that has been used widely in medical sciences (Lindsay and Shall, 1971; Abdu-

Allah et al., 2016). ASA is a common analgesic and antipyretic that is metabolized

and transferred to salicylic acid which is essential for metabolism in humans (Zhang

et al., 2014). It also inhibits the formation of prostaglandin G2 (an intermediate in

human platelet biosynthesis) that causes platelet aggregation in its isolated form (Roth

and Majerus, 1975). Various studies revealed that ASA acylates a large number of

cytosolic proteins, suggesting an important role for acetylation in the function of

cellular proteins (Nagaraj et al., 2012). Generally, it transfers its acetyl moiety to N-

terminal residue and to the free amino group of lysine in both conditions i.e in vitro as

well as in vivo. However, 5-ASA is unique among salicylates and has a broad

spectrum of biological activities including analgesic, antitumor, neuroprotective and

anti-inflammatory with least induction of gastrointestinal side effects (Abdu-Allah et

al., 2016). It acts as a scavenger of superoxide radicals that are released by

neutrophils at the inflammatory sites by neutralizing the toxic hypochlorite.

Here, we investigated a detailed understanding of inhibition mechanism of fibrillation

in bovine insulin by these two drugs (ASA & 5-ASA) that contain two different

functional groups i.e acetyl and amino group respectively. The kinetics of heat

induced amyloid formation in bovine insulin and then its attenuation by ASA and 5-

ASA was investigated by numerous spectroscopic and imaging techniques followed

by computational approaches.


105


6.2.1. Materials

Bovine insulin (BI), Acetylsalicylic acid (ASA), 5-aminosalicyclic acid (5-ASA),

Thiofalvin T (ThT), 1-anilino-8-naphthalene sulfonate (ANS), penicillin and

streptomycin were purchased from Sigma–Aldrich Chemical Co., St. Louis, MO,

USA. Breast cancer cells that were used for cell cytotoxicity assay were procured

from ATCC, Manassas, VA (USA). All other reagents were of highest analytical

grade. Double deionized water free from all fluorescent contaminations was used.

Experiments were performed in 20 mM glycine-HCl buffer (pH 2.0) filtered with

PVDF 0.45 µm syringe filters (Millipore Milex-HV) throughout the study.

6.2.2. In vitro insulin amyloid fibrils formation

The bovine insulin stock solution was prepared in 20 mM glycine-HCl buffer (pH

2.0). The protein concentration was estimated on double beam Jasco-V660

spectrophotometer, using an extinction coefficient of A %1

1cm = 1.0 at 276 nm (Porter,

1953). The stock solution of bovine insulin was diluted by same buffer to a final

protein concentration of 10 µM. The resultant solution was heated in a heating block

at 65°C for desired time.

6.2.3. Turbidity and Rayleigh light scattering measurements

Turbidity of bovine insulin aliquots in absence and presence of ASA & 5-ASA (0-

1000 µM) were studied by using double beam UV-visible spectrophotometer at 350

nm. We also measured turbidity of both drugs (ASA & 5-ASA) incubated at 65°C in

pH 2.0, that serve as blank for the turbidometric analysis. Rayleigh scattering

experiments of bovine insulin in absence and presence of ASA & 5-ASA (0-1000

µM) were performed on a Shimadzu (RF-5301PC) fluorescence spectrophotometer at

pH 2.0. All samples of bovine insulin were excited at 350 nm and emission spectra

were recorded from 300 to 400 nm with fixed excitation and emission slit widths (1.5

nm). Samples were subtracted from their appropriate blanks (various concentrations

of drugs incubated in pH 2.0 at 65°C serve as blank).


106


Thioflavin-T fluorescence assay was performed on RF-5301PC Shimadzu fluorescence

spectrophotometer. A stock solution of ThT was prepared in distilled water and its

concentration was determined by using an extinction coefficient (εM = 36,000 M -1

cm -1

)

at 412 nm (Biancalana and Koide, 2010). Aliquots of bovine insulin samples (incubated

in absence and presence of both drugs) were further incubated for 30 minutes with ThT

in a molar ratio of 1:1 to that of protein. The ThT molecule was excited at 440 nm and

emission spectra were monitored between 450 to 600 nm. The excitation and emission

slit widths were fixed at 5 nm. Spectra were subtracted from their respective blanks.


A hydrophobic dye, 1-anilino-8-naphthalene sulfonate (ANS) was dissolved in double

distilled water, filtered and further its concentration was determined at 350 nm using a

molar extinction coefficient, εM = 5000 M-1

cm-1

(Qadeer et al., 2014). Bovine insulin

samples in absence and presence of ASA and 5-ASA (0-500 µM) were mixed with 50

fold molar excess of ANS and kept for 30 min in dark. ANS fluorescence spectra were

measured with an excitation of 380 nm and emission of 400 to 600 nm on Shimadzu

(RF-5301PC) fluorescence spectrophotometer with a slit width of 3 to 5 nm. All

spectra were subtracted from their appropriate blanks.

6.2.6. Circular dichroic measurements

Far-ultraviolet CD spectra were acquired on a J-815 JASCO spectropolarimeter

(thermostatically controlled cell holder attached to a peltier with multitech water

circulator) in the range of 200-250 nm. The concentration and path length was 10 µM

and 0.1 cm, respectively, for far UV-CD experiments. Each spectrum was base line

corrected and an average of three scans were taken at a scan rate of 100 nm/min with

a response time of 1sec. Savitzky–Golay method with 21 convolution width were

used for spectra smoothening.


Dynamic light scattering measurements were performed to measure the size of

different aliquots of bovine insulin. The hydrodynamic radii of bovine insulin at

different conditions (in absence and presence of both drugs) were obtained on a

DynaPro-TC-04 dynamic light scattering instrument (Protein Solutions, Wyatt


107

Technology, Santa Barbara, CA). Prior to measurements all samples were filtered

through a 0.22 µM Whatman international pore sized microfilter. The measured

hydrodynamic radii (Rh) were an average of 20 measurements. The polydispersity (Pd)

and hydrodynamic radii (Rh) were anticipated on the basis of autocorrelation study of

scattered light intensity, based on transitional diffusion coefficient, from the Stokes–

Einstein equation (Edward, 1970).

D

kTR

6h

(6.1)

Where, k is the Boltzmann‟s constant, Rh is the hydrodynamic radius, T is the

absolute temperature, D is the translational diffusion coefficient and η is the viscosity

of water.


Bovine insulin incubated in absence and presence of ASA and 5-ASA (500 µM) at

65ºC were assayed by using ThT as an amyloid specific fluorescent probe. The

aliquots as mentioned above were supplemented with ThT in a molar ratio of 1:1 to

that of protein and incubated for 30 minutes in dark. The samples were washed

carefully and kept on a glass slide and covered with a cover slip. A carl zeiss imager

equipped with 20x, 40x or 63x (oil) objective magnification were used to visualize the

samples. FITC channel were used to filter the fluorescent image. Images were

visualized with American digital camera attached with microscope.

6.2.9. Transmission electron microscopy measurements

To understand the morphology of amyloid, transmission electron microscopy is a

useful tool (Kaur et al., 2014). In order to visualize morphology of bovine insulin

amyloid fibrils under various conditions, the images were acquired on JEOL-2100F

transmission electron microscope, which operates at an accelerating voltage of 200

kV. For this purpose aliquots under different conditions (in absence and presence of

500 µM ASA and 5-ASA) were incubated properly at pH 2.0 and placed on the

Formvar-coated 200 mesh copper grids, blotted and air-dried. After 2 minutes, excess

fluid was removed and samples containing copper grids were negative stained with

2% (w/v) aqueous uranyl acetate solution. Uranyl acetate is known to produce high


108

electron density, image contrast, and impart fine grained impression to the image (Ohi

et al., 2004; Zaman et al., 2016b).

6.2.10. Cell culture

Breast cancer cells (MDA-MB-231) were obtained from American Type Culture

Collection (ATCC, Manassas, VA, USA). These cells were cultured and maintained

in RPMI-1640 medium in humidified 5% (v/v) CO2/air at 37°C, supplemented with

10% FBS and 1% penicillin-streptomycin solution. Further, these cells were

maintained in M-200 medium (Invitrogen, Carlsbad, CA) supplemented with LSGS

(Invitrogen) with 1% penicillin-streptomycin solution.


Cell viabilities of MDA-MB-231 cells were measured by MTT (3, (4, 5-

dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide) reduction assay. For MTT

reduction assay, approximately 5 × 103 breast cancer MDA-MB-231 were seeded in

96-well plates and treated with different aliquots (bovine insulin in absence and

presence of both drugs) for 24h and 48h. At the end of the incubation time, MTT

solution (0.5 mg/mL) was added to each well and incubated for 4 h at 37°C in CO2

incubator. The media containing MTT solution was aspirated from the wells and the

MTT-formazan crystals were dissolved in DMSO. Absorbance was recorded at

540 nm wavelength. The untreated cells were used as controls and results were

represented as percent proliferation over control.

6.2.12. Molecular docking study of interaction between insulin and drugs

The molecular docking study was performed with Autodock 4.2 and Autodock tools

(ADT) using Lamarckian genetic algorithm. The crystal structure of insulin and three

dimensional structures of ASA and 5-ASA were retrieved from protein data bank and

PubChem respectively, on which water molecules and ions were removed followed by

addition of hydrogen bonds. The grid size was generated so as to cover all active sites

residues and their size was set at 126, 126 and 126 along X, Y and Z axes with 0.431

A° spacing. In addition, partial Kollman charges were assigned to protein molecule.

During docking process solvent molecules were not considered and out of ten

generated binding modes, only the minimum energy conformation state of ligand

bound protein complex were considered. The GA population size was selected as 150


109

and 2,500,000 was set as maximum number of energy evolutions. To visualize and

identify the residue that was involved in binding, we used Discovery studio 3.5.


6.3.1. Inhibitory effect of acetylsalicylic acid (ASA) and 5-aminosalicylic acid (5-

ASA) on aggregation of bovine insulin observed by turbidity and Rayleigh

scattering measurements

The value of turbidity of bovine insulin samples (pH 2.0) at 65°C in absence and

presence of ASA and 5-ASA (0-1000 µM) are shown in figure 6.1 A. In presence of

varying concentration of ASA and 5-ASA (0 –1000 µM), significant drop in turbidity

was observed and became static after 500 µM. Conversely, much more significant

decrease in turbidity was observed for 5-ASA in comparison to ASA (Figure 6.1 A).

Decrease in size and number of aggregated particles are attributed to reduced

turbidity.

Figure 6.1 (A) Turbidity measurements of bovine insulin samples incubated at 65°C over a

period of 24h in the absence and presence of ASA and 5-ASA (0-1000 µM). (B) Rayleigh

scattering measurements of bovine insulin samples incubated at 65°C over a period of 24h in

the absence and presence of ASA and 5-ASA (0-500 µM).

Further, aggregation induction and inhibition were confirmed by Rayleigh scattering

measurements under similar conditions. Figure 6.1 B shows RLS profile of aliquots in

absence and presence of both drugs (ASA & 5-ASA) in a concentration range of 0-

500 µM. A prominent decrease in Rayleigh scattering in presence of 500 µM ASA

and 5-ASA clearly indicates inhibition of insulin aggregation. Moreover, maximum

reduction in light scattering intensity was observed in presence of 500 µM 5-ASA.

0 100 200 300 400 5000

200

400

600

800

1000

Ray

leig

h s

catt

eri

ng (

350 n

m)

Drug ( µM)

ASA

5-ASA

A B

0 200 400 600 800 10000.0

0.1

0.2

0.3

0.4

0.5

0.6

Tu

rbid

ity

at

35

0 n

m

Drug ( µM)

ASA

5-ASA


110

6.3.2. Thioflavin-T binding assay to monitor the anti-amyloidogenic property of

ASA and 5-ASA

Thioflavin-T (ThT), a cationic benzothiazole dye, which interacts mostly with

amyloid fibrils and gives enhanced fluorescence emission intensity after binding to

amyloid fibrils (Khurana et al., 2005). The inhibitory effect of two drugs, ASA and 5-

ASA, on insulin fibrillation was examined via ThT fluorescence measurements.

Figure 6.2 Effects of ASA and 5-ASA on ThT fluorescence of bovine insulin. (A) ThT

fluorescence spectra of bovine insulin in the absence and presence of ASA at different

concentrations. (B) ThT fluorescence spectra of bovine insulin in the absence and presence of

5-ASA at different concentrations. (C) ThT fluorescence intensity at 485 nm of bovine insulin

in the absence and presence of ASA and 5-ASA at different concentrations. (D) Kinetics of

bovine insulin fibrillogenesis in the presence of different concentrations of (ASA & 5-ASA).

Results represent means ± s.d (n=3).

A significant reduction in ThT fluorescent intensity in presence of ASA and 5-ASA at

different concentrations suggests that amount of amyloid fibrils formed were

substantially decreased (figure 6.2 A & B). However, maximum reduction in presence

of 500 µM 5-ASA clearly indicates that amino group of salicylic acid is more potent

inhibitor than acetyl group (figure 6.2 B). Further, in presence of 5-ASA, fluorescence

emission intensity at 485 nm was significantly more reduced to that of ASA with

equal concentration i.e, 250 & 500 µM (figure 6.2 C). These results suggest that 5-

450 480 510 540 570 6000

150

300

450

600 ThT only

BI + ThT

BI + 250 µM ASA+ ThT

BI + 500 µM ASA+ ThT

Th

T f

luo

resc

ence

in

ten

sity

(a.

u)

Wavelength (nm)

450 480 510 540 570 6000

150

300

450

600

Th

T f

luo

resc

ence

in

ten

sity

(a.

u)

Wavelength (nm)

ThT only

BI + ThT

BI + 250 µM 5-ASA + ThT

BI + 500 µM 5-ASA + ThT

0 250 5000

100

200

300

400

500

600

Drug ( µM)

Th

T f

luo

resc

ence

in

ten

sity

at

48

5 n

m ASA

5-ASA

0 8 16 24 320

150

300

450

600

Th

T F

.I a

t 4

85

nm

Time (hrs)

BI

BI + 250µM ASA

BI + 500µM ASA

BI + 250µM 5-ASA

BI + 500µM 5-ASA

A B

C D


111

ASA in comparison to ASA, is more effective drug to suppress amyloid fibril

formation of bovine insulin. As shown in figure 6.2 D, bovine insulin fibrillation

kinetics possesses nucleation dependent polymerization mechanism, comprising of an

initial lag phase, elongation phase followed by a final saturation phase. ThT

fluorescence intensity (at 485 nm) of bovine insulin gradually increases as the

amyloid formation progressed until reached to a plateau phase with said experimental

conditions. It was apparent from figure 6.2 D that presence of both drugs (ASA & 5-

ASA) resulted in a slower growth and a longer lag phase periods as compare to the

control. The observed prolonged lag phase of bovine insulin aggregation indicates that

ASA and 5-ASA may influences nucleation, an early step in amyloid formation

cascade, during which seed of amyloid formation takes place as reported earlier for

apoE (Harper and Lansbury Jr, 1997). However, in presence of 500 µM (5-ASA) the

inhibition occurs maximally as it reduces maximum fluorescence intensity as well as

longer lag phase were observed. All spectroscopic results are summarized in table 6.1.

Table 6.1 Spectroscopic properties of bovine insulin (BI) in absence and presence of

ASA and 5-ASA at various conditions.


BI 25°C 13.2 ± 2.21 8.26 ± 1.41 0.019 ± 0.01

BI 65°C 868.48 ± 26.67 531.74 ± 20.1 0.509 ± 0.03

BI 65°C 333.56 ± 7.32 247.83 ± 4.2 0.291 ± 0.02

+ 500 µM ASA

BI 65°C 83.06 ± 2.31 56. 21 ± 1.23 0.121 ± 0.01

+ 500 µM 5-ASA

6.3.3. Effect of ASA and 5-ASA on the microenvironment of bovine insulin fibril

formation

Aggregation occurs as a result of hydrophobic collapse during nucleation stage and

thioflavin-T binding confirms that both drugs were capable of delaying the nucleation

process. This phenomenon may occur due to reduction in surface hydrophobicity or

stabilization of native state of protein. To further explore the inhibition mechanism,

we investigated the alteration of microenvironment upon binding of both drugs (ASA

& 5-ASA) with bovine insulin fibril formation. ANS shows insignificant fluorescence


112

intensity in aqueous solutions but upon binding with hydrophobic patches the

observed fluorescence intensity was noteworthy. Hence ANS provides a significant

tool for proteins‟ conformational changes in solutions (Schwabe et al., 2016). In

presence of both drugs, ANS fluorescence intensity decreased in comparison to the

native one (bovine insulin alone) as shown in figure 6.3 (A & B).

Figure 6.3 (A) ANS fluorescence spectra of bovine insulin incubated at 65ºC in the absence

and presence of ASA. (B) ANS fluorescence spectra of bovine insulin incubated at 65ºC in

the absence and presence of 5-ASA. (C) (A) ANS fluorescence intensity at 480 nm of bovine

insulin incubated at 65ºC in the absence and presence of ASA and 5-ASA. Experimental data

represent the average ± s.d (n=3).

This decrease in fluorescence intensity in presence of various concentration of ASA

suggests that lesser number of aggregates were formed (figure 6.3 A). For clarity of

results only selected spectra were shown. However, in presence of 500 µM of 5-ASA,

utmost reduction in ANS fluorescence intensity clearly indicates that lesser number of

hydrophobic patches (propensity to form aggregates) were exposed to ANS dye as

show in figure 6.3 B (Ranade et al., 2015). In addition, reduction of ANS

fluorescence intensity was concentration dependent as observed in figure 6.3 C. From

these results, we can infer that 5-ASA due to less hydrophobic nature inhibits

0 250 5000

150

300

450

AN

S F

.I a

t 4

80

nm

Drug ( µM)

ASA

5-ASA

A B

C

450 500 550 6000

100

200

300

400

500ANS only

BI + ANS

BI + 250 µM ASA

BI + 500 µM ASA

AN

S f

luore

scence s

pectr

a

Wavelength (nm)

450 500 550 6000

100

200

300

400

500 ANS only

BI + ANS

BI + 250 µM 5-ASA

BI + 500 µM 5-ASA

AN

S f

luo

resc

en

ce s

pectr

a

Wavelength (nm)


113

maximum aggregation as hydrophobic residues and hydrophobic environment play a

key role during amyloid fibril formation (Marshall et al., 2011).

6.3.4. Influence of ASA and 5-ASA on bovine insulin secondary structure

Far UV-CD (200-250 nm) spectroscopy was utilized to explore protein secondary

structural changes in absence and presence of both drugs (ASA & 5-ASA) under

different conditions. Bovine insulin, a helical protein, at native state (pH 7.4, 25 °C)

shows a characteristic α-helical structure with two minima at 208 and 222 nm (Zako

et al., 2009). However, it exhibited a single pronounced minima at around 218 nm

with increasing incubation time (24h) at 65°C that clearly demonstrates a

characteristic pattern of β-sheet structure as shown in figure 6.4 A&B (Bouchard et

al., 2000; Zako et al., 2009).

Figure 6.4 (A) Far UV-CD spectra of bovine insulin in the absence and presence of 500 µM

ASA at different conditions. (B) Far UV-CD spectra of bovine insulin in the absence and

presence of 500 µM 5-ASA at different conditions.

Whereas, insulin samples co-incubated with ASA and 5-ASA (500 µM) shows

secondary structural transitions of amyloid fibrils peaks towards the native like

structure (figure 6.4 A & B). In addition, maximum retention towards control were

observed in presence of 5-ASA (500 µM) as shown in figure 6.4 B. Reduction in β-

sheet with concomitant increment in α-helicity in presence of ASA and 5-ASA

demonstrates that both drugs hinder in the conversion of native like α-helical structure

to β-sheet rich fibrillar species. It also suggests that 5-ASA possesses the strongest

suppressing activity against insulin fibrillation/aggregation in comparison to ASA

under the set experimental conditions. Further, our findings were in accordance with

our preceding turbidometric, Rayleigh scattering as well as dye binding assays (ThT

and ANS) measurements. Additionally, our outcomes were also in accord with prior

200 210 220 230 240 250

-45

-30

-15

0

15

30 BI (25°C)

BI (65°C)

BI (65°C) + 500 µM ASA

Ell

ipti

cit

y (

md

eg

)

Wavelength (nm)

200 210 220 230 240 250

-45

-30

-15

0

15

30 BI (25°C)

BI (65°C)

BI (65°C) + 500 µM 5-ASA

Ell

ipti

cit

y (

md

eg

)

Wavelength (nm)

A B


114

findings by Choudhary et al (Choudhary et al., 2015), anti- aggregation properties of

osmolytes against insulin fibrillation as well as inhibition of Aβ – 42 peptide by

vitamin K3 (Alam et al., 2016).

6.3.5. Dynamic light scattering to measure the size of aggregates

In order to analyze the size distribution of aggregates, we performed dynamic light

scattering measurements, because it can provide qualitative size of proteins as well as

their aggregated samples (Zaman et al., 2014). Hydrodynamic radii of different

aliquots (bovine insulin in absence and presence of 500 µM ASA and 5-ASA) was

measured and shown in figure 6.5. The hydrodynamic radii (Rh) of freshly prepared

bovine insulin was found to be 2.1 nm (figure 6.5 A) with slight increase Rh in

presence of both drugs and i.e, 2.7 nm for ASA and 2.5 nm for 5-ASA (figure 6.5 B &

C).

Figure 6.5 DLS pattern of bovine insulin in the absence and presence of ASA and 5-ASA.

(A) Bovine insulin alone (pH 7.4, 25°C) (B) Bovine insulin in the presence of 500 µM ASA

(pH 7.4, 25°C), (C) Bovine insulin in the presence of 500 µM 5-ASA (pH 7.4, 25°C), (D)

Bovine insulin alone (pH 2.0, 65°C), (E) Bovine insulin in the presence of 500 µM ASA (pH

2.0, 65°C) (F) Bovine insulin in the presence of 500 µM 5-ASA (pH 2.0, 65°C).

This increase in hydrodynamic radii and polydispersity revealed complex formation

between drug and proteins as a result of increase in water solvent shell around protein


115

molecules without affecting the monomeric population of bovine insulin (Armstrong

et al., 2004). However, bovine insulin incubated at 65°C (pH 2.0) for 24h showed a

prominent increase in hydrodynamic radii to 339.2 nm that clearly indicates formation

of aggregates (figure 6.5 D). Moreover, in presence of ASA and 5-ASA, appearance

of native like (monomeric insulin) peaks (with high intensity) as well as lower

polydispersity suggests that both drugs interfere with aggregation/fibrillation of

insulin (figure 6.5 E & F). In addition, maximum interference observed for 500 µM 5-

ASA, shows the strongest effects in reducing the average particle size of bovine

insulin.These results were consistent with ThT and spectroscopy results as derivatives

of salicylic acid showed inhibitory effects and the highest was found to be for 5-ASA

(500 µM). All results are summarized in table 6.2.

Table 6.2 Hydrodynamic radii (Rh) and polydispersity (Pd) index of bovine insulin

(BI) in absence and presence of varying concentration of ASA and 5-ASA.


(A) BI at 25 °C 2.1 ± 0.02 10.4

(B) BI at 25°C + 500 µM ASA 2.7 ± 0.04 11.7

(C) BI at 25°C + 500 µM 5-ASA 2.5 ± 0.04 13.9

(D) BI at 65°C 339.2 ± 9.17 32.6

(E) BI at 65°C + 500 µM ASA 3.7 ± 0.04, 285.2 ± 3.4 10.1, 21.5

(F) BI at 65°C + 500 µM 5-ASA 2.2 ± 0.04, 140.2 ± 4.9 11.1, 27.3

Interaction of ASA and 5-ASA with insulin may bring conformational changes that

either folds proteins which may be arrested in non-native state or both drugs interact

with unfolded protein or collapse it. As a consequence, reduction in water solvent

shell may occur with reduced hydrodynamic radii that come closer to the native state

of insulin.

6.3.6. Fluorescence microscopic and transmission electron microscopic measurements

to visualize the effect of ASA and 5-ASA on aggregation inhibition and morphology of

bovine insulin

To characterize the morphology of amyloid fibrils, TEM (transmission electron

microscopy) is a useful technique (Gras et al., 2011). We performed TEM analysis to

examine the morphology of different aliquots (in absence and presence of 500 µM

ASA & 5-ASA) of bovine insulin. Figure 6.6 (A-D) display representative


116

micrograph of fresh bovine insulin alone (25 °C, pH 2.0), bovine insulin (65°C, pH

2.0, 24h), bovine insulin in presence of 500 µM ASA (65°C, pH 2.0, 24h) and bovine

insulin in presence of 500 µM 5-ASA (65°C, pH 2.0, 24h, respectively). As shown in

figure 6.6 A, we did not observe any fibrillar structure of fresh bovine insulin at

native conditions (pH 7.4, 25°C). However samples incubated at 65°C (24h) at pH 2.0

show high density of needle like species reminiscent of typical amyloid fibrils (figure

6.6 B). Conversely, aliquots of bovine insulin co-incubated with ASA (500 µM)

showed decreased quantity of fibrils (figure 6.6 C). In addition, fibrils almost

disappeared in presence of 500 µM 5-ASA suggesting that it exhibited optimal

inhibitory activity as compared to other conditions examined (figure 6.6 D).

Figure 6.6 TEM and FM images of bovine insulin amyloid formation in the absence and

presence of salicylic acid derivatives. (A) Native bovine insulin (25°C) (B) Bovine insulin

incubated at 65°C. (C) Bovine insulin co-incubated with 500 µM ASA at 65°C, (D) Bovine

insulin co-incubated with 500 µM 5-ASA at 65°C, (E) FM image of native bovine insulin

(25°C), (F) FM image of bovine insulin incubated at 65°C, (G) FM image of bovine insulin

co-incubated with 500 µM ASA at 65°C, (H) FM image of bovine insulin co incubated with

500 µM 5-ASA at 65°C.

In addition, fluorescence microscopic technique is another useful method to visualize

and characterize the amyloid fibrils of protein aggregates (Demeule et al., 2007).

Bovine insulin aggregation inhibition by ASA and 5-ASA (500 µM) was further

monitored by fluorescence microscopic measurements under the set experimental


117

conditions using ThT as a fluorescent dye. ThT do not fluoresce when native bovine

insulin (25°C, pH 7.4) was examined (figure 6.6 E). However, 24h aged insulin

samples (65°C, pH 2.0) upon binding with ThT, fluoresces green suggesting the

presence of amyloid fibrils (figure 6.6 F). Conversely, bovine insulin co-incubated

with ASA (500 µM) and 5-ASA (500 µM) shows reduced and insignificant

fluorescence intensity respectively (figure 6.6 G, H).From all these results, it was

notable that 5-ASA was more pronounced as compared to ASA towards aggregation

inhibition as complete loss of fibrils were observed in presence of 5-ASA (figure 6.6

H).

6.3.7. Effects of ASA and 5-ASA on amyloid induced cytotoxicity

It is widely not universally believed that fibrillar or aggregated species are toxic to

neuronal like cells (Pike et al., 1991; Zako et al., 2009). So, we checked the ability of

both drugs to attenuate the cytotoxicity induced by insulin amyloid fibrils to confirm

whether ASA and 5-ASA would affect the function of insulin fibrils or not. We

investigated the cellular toxicity of insulin amyloids on human breast cancer cell lines

(MDA-MB-231) by using MTT reduction assay. The cell viability was measured by

their ability towards reduction of metabolic dye (MTT) to a blue formazan product.

The data were represented as the percentage of MTT reduced by cells treated with

insulin samples relative to MTT reduced by untreated cells. No significant decrease in

cell viability was observed, when cells were exposed to ASA (250 & 500 µM) and 5-

ASA (250 & 500 µM) as compared to control (data not shown). However, a

substantial loss in cell survival with insulin fibrils was observed when cells were

incubated with 24 h aged insulin fibrils for 24h and 48h as shown in figure 6.7 A & B.

Further, insulin samples pre incubated with ASA (250 & 500 µM) and 5-ASA (250 &

500 µM) showed significant increase in cell viability (figure 6.7). It is clear from

figure 6.7 that cell viability of MDA-MB-231 cells was increased from 40 ± 1% to 50

± 2% (250 µM ASA), 54 ± 2% (500 µM ASA), 81 ± 4% (250 µM 5-ASA) and 84 ±

3% (500 µM 5-ASA) in presence of various concentration of drugs after 24h

incubation (figure 6.7 A). In addition, we also investigated cell viability assay of 24h

aged insulin amyloids with increasing incubation time (48h) of fibrils to cells. We

observed that increasing incubation time insignificantly alters the results to that of 24h

incubated one, as cells viability was found to be 40 ± 2%, 50 ± 1% (250 µM ASA), 52


118

± 2% (500 µM ASA), 79 ± 3% (250 µM 5-ASA) and 82 ± 4% (500 µM ASA) at

various conditions (figure 6.7 B).

Figure 6.7 MTT reduction assay for cell cytotoxicity of 24h aged bovine insulin amyloid

fibrils in MDA-MB-231 breast cancer cell lines in the absence and presence of different

concentration of ASA and 5-ASA. (A) Incubation time 24h (B) incubation time 48h. Control

represents cell lines without prior exposure to bovine insulin fibrils. *Statistically significant

from the control group, p ≤ 0.01 and # statistically significant from the bovine insulin, p ≤

0.05 for ASA and 5-ASA.

Closer examination of our results revealed that both ASA and 5-ASA markedly

protect cells from amyloid induced toxicity but amino group of salicylic acid (5-ASA)

was evidently more potent or effective than acetyl derivative of salicylic acid (figure

6.7).Though the precise mechanisms implicated in amyloid induced cytotoxicity

remain unknown where the pro-inflammatory cytokines, overproduction of pro-

inflammatory cytokines, destabilization of cellular membrane and increased formation

of reactive oxygen species are some of the factors that play a significant role in this

process (Sayre et al., 2007; Jellinger, 2010). However, protective effect of both drugs

(increased cell viability due to pretreatment with both drugs) against bovine insulin

mediated cytotoxicity in human breast cancer cells (MDA-MB-231) might be

attributed to its strong anti inflammatory and analgesic properties.

6.3.8. Binding mode of ASA and 5-ASA to bovine insulin

Molecular docking was performed to determine the mode of interaction and putative

binding site between bovine insulin and drugs (ASA & 5-ASA). It is clear from the

results that bovine insulin interacts with both drugs in a different manner (figure 6.8 A

- D). It is generally assumed that hydrophobic interactions, aromaticity and hydrogen

A B

Control

Insulin amyloids (IA)

IA + 250 µM ASA

IA + 500 µM ASA

IA + 250 µM 5-ASA

IA + 500 µM 5-ASA

0

20

40

60

80

100

*

#

#

##

% C

ell

Via

bil

ity

A

Control

Insulin Amyloids (IA)

IA + 250 µM ASA

IA + 500 µM ASA

IA + 250 µM 5-ASA

IA + 500 µM 5-ASA

0

20

40

60

80

100

% C

ell

Via

bil

ity

A

*

##

##


119

bonding play a significant role in aggregation process and disruption of such forces in

presence of ASA and 5-ASA might be responsible for amyloid inhibition (Wu et al.,

2006).

Figure 6.8 Molecular docking results of drugs (ASA & 5-ASA) + bovine insulin complex.

(A) Acetylsalicylic acid is shown in a stick representation, and bovine insulin represented

with ribbon model. (B) Detailed view of the docking poses of acetylsalicylic acid + bovine

insulin complex. (C) 5-amino salicylic acid is shown in a stick representation, and bovine

insulin represented with ribbon model. (D) Detailed view of the docking poses of 5-amino

salicylic acid + bovine insulin complex.

The main interacting forces between drugs (both) with bovine insulin were found to

be hydrophobic interactions as well as hydrogen bonding as confirmed by docking

method. 5-amino salicylic acid (5-ASA) after interacting with protein was found to

possess more hydrogen bonding along with hydrophobic interactions that might be

responsible for enhanced anti-amyloidogenic behavior in comparison to ASA. In

addition, the value of Gibbs free energy for the excellent poses for 5-ASA was found

to be -5.23 (Kcal M-1

), which also suggests that it forms a more stable complex than

ASA which has a lower (-4.85 Kcal M-1

) Gibbs free energy. Our results suggest that

the inhibition of bovine insulin amyloid formation can be attributed mainly to the

binding of ASA and 5-ASA to protein resistant region. Results obtained through

docking are summarized in table 6.3.


120

Table 6.3 Molecular docking parameters for drugs (ASA and 5-ASA) - bovine insulin

interaction.

Drug

Amino acid

residue residues

Interactions involved

Binding energy

(Kcal M-1

)

Acetylsalicylic

acid

Ala 30

Lys 29

Pro 28

Thr 27

Glu4

Gly1

Tyr 19

Hydrophobic interaction





Hydrogen bonding


-4.85

5-Amino

salicylic acid

Leu 6

Gln 4

Ala 14

Val 2

Phe 1

Leu 13


Hydrogen bonding


Hydrogen bonding



-5.23

In figure 6.9, we have projected a hypothetical sculpt to enlighten the effect of ASA

and 5-ASA on bovine insulin fibrillation.

Figure 6.9 Schematic representation of bovine insulin aggregation and its inhibition by

acetylsalicylic acid (ASA) and 5-aminosalicylic acid (5-ASA).


121

6.4. Conclusion

The present study demonstrates that acetylsalicylic acid (ASA) and 5-aminosalicylic

acid (5-ASA) inhibit fibrillation of insulin and amino group of salicylic acid was

found to be more effective in inhibition over acetyl group. We established this by

using multiple approaches including spectroscopic, microscopic and cell viability

assays. The observed β-sheet retardation by circular dichroism that clearly indicates

restoration of native like secondary structure in presence of both drugs and maximum

was achieved in presence of 5-ASA. In addition, transmission and fluorescence

microscopic images in presence of 5-ASA confer inhibition of amyloid fibrils,

whereas in presence of ASA few fibrils were still observed. In addition, this may be

due to presence of amino group on aromatic ring of 5-ASA, which makes it more

efficient in prolonging the nucleation stage of insulin aggregation through

comparatively strong binding with amyloid prone region of proteins. This in turn

disrupts the hydrophobic interaction as well as aromatic stacking which are involved

in amyloid progression. Significant survival of breast cancer cells in presence of ASA

and 5-ASA against insulin amyloid mediated toxicity revealed that both drugs could

be potential therapeutic candidates during aggregation inhibition. Such study may

give clue to enlarge various pharmaceutical formulations and other drug derivatives

that can be turned to avert amyloid fibrillation. In addition, the native state

stabilization of protein can serve as a model system for development of potential

therapeutic strategies in combating to neurodegenerative diseases.

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ir.amu.ac.inir.amu.ac.in/11781/1/T10303.pdf · 2018. 6. 12. · i Acknowledgements All praise is almighty Allah who bestowed me with ample and perseverance that paved the path to