The effect of Berberine on the secondary structure

of human serum albumin

Ying Li, WenYing He, Jianniao Tian, Jianghong Tang, Zhide Hu*, Xingguo Chen

Department of chemistry, LanZhou University, LanZhou, GanSu province 730000, China

Received 10 November 2004; revised 21 February 2005; accepted 24 February 2005

Available online 5 April 2005

Abstract

The presence of several high affinity binding sites on human serum albumin (HSA) makes it a possible target for many drugs. This study is

designed to examine the effect of Berberine (an ancient Chinese drug used for antimicrobial, antiplasmodial, antidiarrheal and

cardiovascular) on the solution structure of HSA using fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD)

spectroscopic methods. The fluorescence spectroscopic results show that the fluorescence intensity of HSA was significantly decreased in the

presence of Berberine. The Scatchard’s plots indicated that the binding of Berberine to HSA at 296, 303, 318 K is characterized by one

binding site with the binding constant is 4.071(G0.128)!104, 3.741(G0.089)!104, 3.454(G0.110)!104 MK1, respectively. The protein

conformation is altered (FT-IR and CD data) with reductions of a-helices from 54 to 47% for free HSA to 45–32% and with increases of turn

structure5% for free HSA to 18% in the presence of Berberine. The binding process was exothermic, enthalpy driven and spontaneous, as

indicated by the thermodynamic analyses, Berberine bound to HSA was mainly based on hydrophobic interaction and electrostatic

interaction cannot be excluded from the binding. Furthermore, the displace experiments indicate that Berberine can bind to the subdomain

IIA, that is, high affinity site (site II).

q 2005 Elsevier B.V. All rights reserved.

Keywords: Human serum albumin; Fourier transform infrared (FT-IR); Circular dichroism (CD); Fluorescence; Berberine

1. Introduction

Human serum albumin (HSA) is a principal extracellular

protein with a high concentration in blood plasma

(40 mg/mL or 0.6 mM) [1,2] HSA is a globular protein

composed of three structurally similar domains (I–III), each

containing two subdomains (A and B) and stabilized by 17

disulfide bridges [1–4]. Aromatic and heterocyclic ligands

were found to bind within two hydrophobic pockets in

subdomains IIA and IIIA, which are site I and site II [1–4].

Seven binding sites are localized for fatty acids in

subdomains IB, IIIA, and IIIB and on the subdomain

interfaces [5]. The multiple binding sites underlie the

exceptional ability of HSA to interact with many organic

and inorganic molecules and make this protein an important

0022-2860/$ - see front matter q 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.molstruc.2005.02.032

* Corresponding author. Tel.: C86 931 891 2540; fax: C86 931 891

2582.

E-mail address: huzd@lzu.edu.cn (Z. Hu).

regulator of intercellular fluxes and the pharmacokinetic

behavior of many drugs [1–5]. Traditional Chinese

medicine prescriptions have been used for over 1000

years, and in recent years concentrated dosage form have

been widely adopted for clinical use. Berberine (structure

shown in Fig. 1) is quaternary protoberberine alkaloids.

Their salts are typically yellow and are widely distributed

in many species of the Berberidaceae, Fumariaceae,

Papaveraceae, and other plant families. Berberine displays

a great variety of biological and pharmacological activities

(e.g. antimicrobial, antiplasmodial, antidiarrheal and cardi-

ovascular). Both alkaloids contribute to the chemical

defense of plants by complex actions with several molecular

targets [6]. However, the interactions of Berberine with

proteins are poorly understood. Therefore, we have been

interested in examining the effects of Berberine on the

solution structure of HSA that can be used as a model for

Berberine–protein interaction.

We now report the fluorescence, Fourier transform

infrared (FT-IR), Circular dichroism (CD) spectroscopic

results of the interaction of HSA with Berberine took place

in aqueous solution in physiological conditions.

Journal of Molecular Structure 743 (2005) 79–84

www.elsevier.com/locate/molstruc

N

H3CO

OCH3

O

O

Fig. 1. The chemical structure of Berberine.

Y. Li et al. / Journal of Molecular Structure 743 (2005) 79–8480

Furthermore, the binding parameter including binding

constant (K), the number of binding sites (n) and

thermodynamic function for the reaction have been

calculated according to the Scatchard’s plot and Van’t

Hoff equation at different temperatures. The spectroscopic

results on Berberine–HSA interactions can elucidate the

nature of Berberine–protein complexation in vitro and in

vivo.

2. Materials and methods

2.1. Materials

Human serum albumin (HSA) was purchased from the

Sino-American Biotechnology Company (China). All

HSA solutions were prepared in a pH 7.40 buffer solution,

and the HSA stock solution was kept in the dark at 4 8C.

Berberine chloride was purchased from the National

Institute for Control of Pharmaceutical and Bioproducts

(China) and the Berberine chloride stock solution was

prepared in absolute ethanol. NaCl (analytical grade,

1.0 M) solution was used to maintain the ionic strength at

0.1. The buffer (pH 7.40) consisted of Tris (0.2 M) and

HCl (0.1 M). The pH was checked with a suitably

standardized pH meter.

2.2. Apparatus and methods

2.2.1. Fluorescence measurements

Fluorescence spectra were measured with RF-5301PC

spectrofluorophotometer (Shimadzu), using 5 nm/5 nm slit

widths. The excitation wavelength was 295 nm and the

emission wavelength was read at 300–500 nm.

2.2.2. Fluorometric titration experiments

Three milliliter of a solution containing an appropriate

concentration of HSA was titrated by successive additions

of a 3.33 mM ethanol stock solution of Berberine (to give

a final concentration of 26.67 mM). Titrations were done

manually by using trace syringes. The fluorescence

intensity was then measured (excitation at 295 nm and

emission at 331 nm). All experiments were measured at

three temperatures (296, 303 and 318 K). The temperature

of the sample was kept constant throughout the exper-

iment. The data obtained were analyzed by using the

Scatchard’s equation [7] to calculate the binding par-

ameters

r=Df Z nK KrK (1)

where r is the moles of drug bound per mole of protein,

Df is the molar concentration of free drug, n is binding

site multiplicity per class of binding site and K is the

association binding constant.

2.2.3. CD measurements

CD was measured with Jasco-20c automatic recording

spetropolarimeter (Japan), using a 2 mm cell at 296 K. CD

spectra (200–350) were taken at a HSA concentration of

1.5 mM, and the results are expressed as molar ellipticity

([q]) in deg cm2 dmolK1. The a-helical content of HSA

was calculated from the q½ � value at 208 nm using the

equation %helixZ{(K[q]208K4000)/(33,000K4000)}100

as described by Lu et al. [8].

2.2.4. FT-IR measurements

FT-IR measurements were carried out at room

temperature on a Nicolet Nexus 670 FT-IR spectrometer

(USA) equipped with a Germanium attenuated total

reflection (ATR) accessory, a DTGS KBr detector and a

KBr beam splitter. All spectra were taken via the

Attenuated Total Reflection (ATR) method with a

resolution of 4 cmK1 and using 60 scans. The spectra

processing procedure involved collecting spectra of buffer

solution under the same conditions. Next, the absorbance

of the buffer solution was subtracted from the spectra of

the sample solution to obtain the FT-IR spectra of the

protein. The subtraction criterion was that the original

spectrum of the protein solution between 2200 and

1800 cmK1 was featureless [9,10].

The protein secondary structure is determined from the

shape of the amide I band located at 1650–1600 cmK1.

Fourier self-deconvolution and second derivative resolution

enhancement were applied to increase the spectral resol-

ution in the region of 1700–1600 cmK1. The second

derivatives were produced using a point convolution of

11 or 13. The resolution enhancement resulting from self-

deconvolution and the second derivative was such that the

number and the position of the bands to be fitted were

determined. In order to quantify the area of the different

components of the amide I contour as revealed by the

second derivative, least-squares iterative curve fitting was

used to fit the Gaussian-shaped curves to the spectra

Table 1

Binding parameters and thermodynamic parameters of Berberine–HSA

T (K) K (!104 MK1) n DG (KJ molK1) DS (J molK1 KK1) DH (KJ molK1)

296 4.071G0.128 1.22 K26.093G0.004

303 3.741G0.089 1.23 K26.578G0.001 69.157G0.467 K5.619G0.143

318 3.454G0.110 1.24 K27.615G0.006

Y. Li et al. / Journal of Molecular Structure 743 (2005) 79–84 81

between 1700 and 1600 cmK1. Before curve fitting was

done, a straight baseline passing through the ordinates at

1700 and 1600 cmK1 was subtracted. The baseline was then

modified again by least-squares curve fitting, which allowed

for a horizontal baseline to be adjusted as an additional

parameter in order to obtain the beat fit. It is known that no

meaningful curve fitting can be performed by simple

examination of the original IR spectra, which is why the

self-deconvolution procedure has to be carried out first.

The curve fitting was analyzed as follows. Each band is

assigned to a secondary structure according to the frequency

of its maximum: a helix (1650K1658 cmK1), b sheet

(1610K1640 cmK1), turn (1660K1700 cmK1), random

coil (1640K1650 cmK1) [9]. The area of all the component

bands assigned to a given conformation are then summed up

and divided by the total area. The number obtained is taken

as the proportion of the polypeptide chain in that

conformation.

2.2.5. The displacement experiment

The displacement experiments were performed using the

site probes keeping the HSA and the probes concentration at

1.67 mM. The fluorescence titration was used as before to

determine the binding constants of Berberine with HSA in

presence of the site probes. Phenylbutazone (PB), fluofe-

namic acid (FA) and digitoxin (Dig) are used as site

probes of sites I, II and III, respectively, according Sudlow

et al. [11].

300 350 400 450 5000

5

10

15

20

25

30

35

g

a

Fluo

resc

ence

Int

ensi

ty

Wavelength (nm)

Fig. 2. The fluorescence Emission Spectra of Berberine–HSA system (a)

3.0 mM HSA; (b)–(g) 3.0 mM HSA in the presence of 1.67, 3.33, 5.0, 6.67,

8.33, 10.0 mM, Berberine; TZ296 K; pHZ7.4.

2.2.6. Thermodynamic parameters

In this section, the association constants obtain by the

Scatchard equation was used to calculation the thermo-

dynamic parameters. Thermodynamic parameters were

calculated based on the temperature dependence of

the binding constant for Berberine–HSA binding. The

temperatures were used 296, 303 and 318 K. The

enthalpy change (H0) is calculated from the slope of

the Van’t Hoff relationship:

ln KT ZKDH0=RT CDS0=R (2)

KT is the binding constant at temperature T and R is gas

constant. The value of DH0 and DS0 were obtained from

linear Van’t Hoff plot and are presented in Table 1.

The value of DG0 as calculated from the relation:

DG0 Z DH0 KTDS0 (3)

3. Results and discussion

3.1. Analysis of fluorescence quenching of HSA

by Berberine

The fluorescence spectra of HSA in the absence and

presence of Berberine in the pH 7.40 Tris buffer were

measured with the excitation wavelength at 295 nm and

their representative spectra are shown in Fig. 2. HSA shows

a strong fluorescence emission with a peak at 331 nm on

excitation at 295 nm due to its single tryptophan residue,

and Berberine was almost non-fluorescence under the

presence experiment conditions. The addition of a solution

of Berberine to HSA leads to a significant reduction in the

fluorescence signal. The results indicate that the binding to

HSA quenches the intrinsic fluorescence of the single

trypotophan in HSA.

The binding parameters for the Berberine–HSA inter-

action have been carried out using Scatchard equation at

various temperatures as shown in Fig. 3 and Table 1. It can

be seen in Table 1, Berberine can strongly binds to HSA

0.1 0.2 0.3 0.4 0.5 0.6 0.7

2.5x10–2

3.0x10–2

3.5x10–2

4.0x10–2

4.5x10–2

r / D

f (10

6 )

r

Fig. 3. The Scatchard curves of quenching of HSA with Berberine 3.0 mM

HSA; (&) 296 K, (C) 303 K, (:) 318 K; PHZ7.4; lexZ295 nm, lemZ331 nm.

Y. Li et al. / Journal of Molecular Structure 743 (2005) 79–8482

and the association constant decreased with the increasing

of temperature.

Fig. 5. FT-IR spectra of free HSA (A) and Berberine–HSA complexes (B)

in buffer solution in the region of 1700–1600 cmK1; (a) second derivative

spectra; (b) difference spectra; (c) self-deconvolution spectra (30 mM HSA;

60 mM Berberine); pHZ7.4.

3.2. FT-IR and CD spectra of Berberine–HSA complexes

Addition evidence regarding the Berberine–HSA com-

plexation comes from infrared spectroscopic results

obtained for Berberine–protein complexes. Since there

was no major spectral shifting for the protein amide I

band at 1600–1700 cmK1 (mainly CaO stretch) and the

amide II band z1548 cmK1 (C–N stretching coupled with

N–H bending modes) [12,13]. Upon Berberine interaction,

the difference spectra [(protein solutionCBerberine solu-

tion)K(Berberine solution)] were obtained in order to

monitor the intensity variations of these vibrations and the

results are shown in Fig. 4. Similarly, the infrared self-

deconvolution with second derivative resolution enhance-

ment and curve fitting procedures [14] were used to

1800 1600 1400

0.0

2.0x10–3

4.0x10–3

6.0x10–3

b

a

amide II

amide I

1543

1645

1547

1647

Abs

orba

nce

Wavenumbers (cm–1)

Fig. 4. FT-IR spectra and difference spectra of HSA in aqueous solution.

(a) FT-IR spectrum of HSA;(b) FT-IR difference spectra of HSA obtained

by subtracting the spectrum of the Berberine-free form from that of the

Berberine-bound form in the region of 1800–1350 cmK1 (30 mM HSA;

60 mM Berberine); pHZ7.4.

determine the protein secondary structure in the presence

of Berberine and the results are presented in Figs. 5 and 6.

CD spectroscopy was also used to analyze the protein

secondary structure in the presence of Berberine and results

are shown in Fig. 7.

At Berberine concentration (60 mM), a strong positive

feature at 1645 and 1543 cmK1 were observed in the

difference spectra of the Berberine–HSA complexes and

these are attributed to a decease in the intensity of the amide

I band and amide II band, upon Berberine–protein

complexation (60 mM, Fig. 4). The interaction of Berberine

with the protein C–N group is also evident from the shift of

the amide A band at 3303 cmK1 (peptide N–H stretching

mode) [14,15], towards a lower frequency at 3290

(spectrum not shown). It is important to note that the

decrease in the intensity of amide I band is due to the

decrease of the proportion of protein a-helix structure, upon

Berberine complexation, which is quantitatively determined

and will be discussed further on.

It can be seen from Fig. 7 that the CD spectra of HSA

exhibit two negative bands in the ultraviolet region at 208

and 218 nm characteristic of a-helical structure of protein

Fig. 6. The curve fitting amide I region with secondary structure

determination of the free HSA (A) and Berberine–HSA complexes (B) in

buffer solution in the region of 1700–1600 cmK1 (30 mM HSA; 60 mM

Berberine); pHZ7.4. *Average percent areas obtained by curve fitting

(nZ4); estimated error G5%.

Y. Li et al. / Journal of Molecular Structure 743 (2005) 79–84 83

when Berberine was added to the solution of HSA, the

intensity of negative Cotton effect of HSA at 208 and

218 nm decreased which clearly indicates the considerable

changes in the protein secondary structure, with the loss of

helical stability and it may be the result of the formation

200 220 240–2.0x104

–1.6x104

–1.2x104

–8.0x103

–4.0x103

0.0 a

b

[θ]

(deg

cm

2 dm

ole–1

)

Wavelength (nm)

Fig. 7. CD Spectra of the HSA–Berberine System. (a) 3.0 mM HSA; (b)

3.0 mM HSA in the presence of 6.0 mM Berberine; pHZ7.4; estimated error

G5%.

of complex between HSA and Berberine. The binding of

Berberine complex to HSA changes negative band at 208

and 218 nm, indicating the increase of the disorder structure

content in the protein.

A quantitative analysis of the protein secondary structure

for the free HSA and its Berberine complexes in H2O is

given in Fig. 6. The free protein contained major amounts of

a-helix (54%), b-sheet (39%) and turn structure (5%). Upon

Berberine complexation, the a-helix structures were

reduced from 54 to 45%, the b-sheet decreased from 39 to

38%, and the turn structure increased from 5 to17% (Fig. 6).

The CD spectra of the free HSA and Berberine complexes

also exhibited a reduction of a-helix from that of 47% (free

HSA) to 32%, which is consistent with the IR results.

However, X-ray structural analysis of the HSA in the solid

state shows a-helix 66%, which is much higher than 55%

obtained in aqueous solution in this work and other solution

studies [16,17]. The differences in a-helix contents are due

to the different structural arrangements of protein in the

solid state and in aqueous solution. Structural differences

between the solid state and aqueous solution were also

observed for other proteins [18,19]. The reduction of

a-helices and b-sheet in favor of turn structure is indicative

of a partial unfolding of protein in the presence of Berberine

at high concentrations.

3.3. Thermodynamic analysis

The thermodynamic parameters, enthalpy (DH) and

entropy (DS) of reaction, are important for confirming

binding mode. For this purpose, the temperature-depen-

dence of the binding constant was studied. The temperatures

chosen were 296, 303, and 318 K at which HSA does not

undergo any structural degradation. By plotting the binding

constants according to Van’t Hoff equation, the thermo-

dynamic parameters were determined from a linear Van’t

Hoff plot (spectrum not shown) and listed in Table 1. It is

clear from the values of standard entropy changes (DS0) and

standard enthalpy changes (DH0) that the binding of

Berberine to HSA is an exothermic process accompanied

by a positive values of DS0 and a negative values of DG0.

The binding process was always spontaneous as evidenced

by the negative sign of DG0 values. For typical hydrophobic

interactions, both DH0 and DS0 are positive, while negative

enthalpy and entropy changes arise from van der Waals

force and hydrogen bonding formation in low dielectric

media [20]. However, negative enthalpy might play a role in

electrostatic interactions. As shown in Fig. 1, Berberine

carries a positive charge in aqueous solution. Therefore, the

binding of Berberine to HSA might involve electrostatic

interactions. It can be considered that the Berberine bound

to HSA was mainly based on the hydrophobic interaction by

the positive values of DS0 and the electrostatic interactions

can also not be excluded.

0.2 0.3 0.4 0.5 0.6 0.7 0.8

2.4x10–2

2.8x10–2

3.2x10–2

3.6x10–2

4.0x10–2

4.4x10–2

r / D

f (1

0 6)

r

Fig. 8. Effect of site marker probe on the fluorescence of Berberine–HSA

(3.0 mM HSA; 1.67 mM PB; 1.67 mM FA; 1.67 mM Dig); (&) PB, (C) FA,

(:) Dig, (+) Berberine; TZ296 pHZ7.4; lexZ295 nm, lemZ331 nm.

Y. Li et al. / Journal of Molecular Structure 743 (2005) 79–8484

3.4. Location and nature of binding site

Human serum albumin, a protein of Mr 65KD, consists of

585 amino acids. Crystallographic studies have shown that

the protein is composed of three structurally homologous

domains (I–III): I (residues 1–195), II (196–383), III (384–

585), which have similar 3D structures and form a heart-

shaped tertiary structure. Drug-binding sites I and II of HSA

are located in hydrophobic cavities in subdomain IIA and

IIIA, respectively, which exhibit similar chemistry [1]. This

is despite a structure which is very susceptible to

environmental factors such as pH, ionic strength, etc. [21].

There is a large hydrophobic cavity present in subdomain

IIA that many drugs can bind.

Sudlow et al. [11] have suggested two main distinct

binding sites on HSA, sites I and II. Site I of HSA showed

affinity for warfarin, Phenylbutazone (PB, anti-inflamma-

tory drug in the treatment of arthritis), etc. and site II for

ibuprofen, fluofenamic acid (FA, non-steroidal anti-inflam-

matory drug in the treatment of osteoarthritis, rheumatoid

arthritis and other painful musculosketal illnesses [22]), etc.

Later studies indicated that digitoxin (Dig, a common

cardiovascular drug used for the treatment of individuals

with congestive heart failure or atrial fibrillation [23].)

binding is independent of sites I and II [24], and binds to

what was nominated as site III. To determine the specificity

of the drug binding, the displacement experiments were

used to study the binding site of Berberine to HSA. As

shown from Fig. 8, the binding constant of Berberine

with HSA remarkably decreased after the additions of FA,

while the addition of PB and Dig did not change the

binding constants. These results indicate that FA can

displace the Berberine but PB and Dig have no effect

on the binding of Berberine to HSA. The binding

constants are 3.276(G0.035)!104, 2.662(G0.069)!104,

3.821(G0.084)!104 MK1 in the presence of PB, FA and

Dig at 296 K, respectively. From these data, we demon-

strated FA displaces Berberine from its site of HSA.

This probably indicated that Berberine has one reactive site

of HSA, that is, high affinity site (site II).

4. Conclusions

It was demonstrated that the binding properties of the

Berberine and the HSA could be characterized by

fluorescence and FT-IR, CD method. From above exper-

iments we can conclude that Berberine binds tightly

but reversibly to HSA with an affinity constant of

4.071(G0.128)!104 MK1 (296 K). The drug induces

protein conformational changes with a reduction of a-

helix and an increase of turn structure. Displacement

experiments show that Berberine interacts with the site II

of HSA. Because there is a large hydrophobic cavity present

in subdomain IIA that Berberine can bind, the main

interaction in this process is hydrophobic force. However,

electrostatic force cannot be excluded.

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