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International Journal of Biological Macromolecules 54 (2013) 90– 98

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules

jo u rn al hom epa ge: www.elsev ier .com/ locate / i jb iomac

−)-Epicatechin gallate prevents alkali-salt mediated fibrillogenesisf hen egg white lysozyme

udeshna Ghosh, Nitin K. Pandey, Swagata Dasgupta ∗

epartment of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India

r t i c l e i n f o

rticle history:eceived 11 October 2012eceived in revised form8 November 2012ccepted 28 November 2012

a b s t r a c t

Green tea polyphenols (GTPs) are found to be potent inhibitors of amyloid fibril formation. We report theeffective inhibitory property of (−)-epicatechin gallate (ECG) during the alkali-salt induced fibrillogenesisof hen egg white lysozyme (HEWL) at 37 ◦C. Spectroscopic techniques such as fluorescence, circulardichroism and microscopic images show that (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG),and (−)-epigallocatechin gallate (EGCG) show moderate inhibition of fibrillation with ECG as the most

vailable online 5 December 2012

eywords:ibrillationen egg white lysozyme

−)-Epicatechin gallate (ECG)

potent polyphenol. Aromatic interactions, hydrophobic interactions, the radical scavenging activity andautoxidation of polyphenols are likely to be the major reasons for ECG being the most effective inhibitor.

© 2012 Elsevier B.V. All rights reserved.

nhibition

. Introduction

Green tea polyphenols (GTPs) have drawn considerable atten-ion due to their potential role in neurodegenerative andardiovascular diseases as well as for their anticarcinogenic, antiox-dative, anti-inflammatory properties that has increased theirpplication for medicinal and pharmaceutical purposes [1–4]. Theajor polyphenol constituents of green tea are (−)-epicatechin

EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG) withhe most abundant being (−)-epigallocatechin gallate (EGCG). GTPsre known to interact with several proteins like human serum albu-in (HSA), bovine serum albumin (BSA), hen egg white lysozyme

HEWL) as well as ribonuclease A (RNase A) [5–9]. Earlier reportsrom this laboratory have investigated the interaction of the GTPsith ribonuclease A (RNase A) and of EGCG with HSA and HEWL

6,8,9]. The gallate moiety is implicated as the preferred site ofnteraction for ECG and EGCG for RNase A [8]. HSA has also beenhown to prevent oxidation of EGCG due to the formation of achiff-base between the carbonyl group of EGCG and the �-NH2 ofysine in the HSA backbone which results in stabilization of EGCG5]. For the binding of EGCG to the active site of HEWL, the interac-ion involves the hydroxyl groups of ring A (5-OH), B (3′ and 4′-OH)

nd D (3′ and 4′-OH) of EGCG which are within hydrogen bondingistance of Arg 61, Trp 62, Trp 63 and Arg 112 of HEWL [9].

∗ Corresponding author. Tel.: +91 3222 283306; fax: +91 3222 255303.E-mail address: swagata@chem.iitkgp.ernet.in (S. Dasgupta).

141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2012.11.031

Protein misfolding generates toxic fibrillar deposits which arethe main causative agents of several debilitating disorders likeAlzheimer’s, Parkinson’s, Huntington’s diseases [10]. Independentof their structural dissimilarity and sequence, most proteins areable to self assemble under certain biophysical conditions whichresults in the formation of fibrillar plaques having identical corecrossed �-sheet structure [11]. Inhibition of amyloid fibrils thusopens up a scope for the therapeutic treatment of neurological dis-orders which involves either preventing the proteins to undergoself association, or disintegration of mature fibrils. The neuropro-tective property associated with GTPs was found to be apparentlyindependent of their classical anti-oxidative and metal chelatingproperty, which includes variation in cell signaling molecules andcell endurance pathways [12]. Studies have shown that the pres-ence of vicinal dihydroxyphenyl moieties, irrespective of theirposition in the aromatic rings play a key role in their inhibitionproperty [13]. It has been suggested that inhibitors are able toaffect the assembled conformation of the fibrillar species insteadof the native state of the protein thus ensuring that their bind-ing is conformation dependent rather than sequence dependent[14]. Polyphenols acquire a three-dimensional pharmacophoreconformation which might be responsible for their binding with�-amyloid that is capable of resisting amyloid formation [15].

EGCG has been reported to inhibit amyloid formation of mutanttransthyretin (TTR) which is responsible for familial amyloidotic

polyneuropathy in the cellular system and also disrupts preformedfibrils in vitro [16]. EGCG was also able to inhibit amyloid like fibrilformation of Merozoite surface protein 2 (MSP2) and disaggrega-tion of pre formed fibrils by remodeling their structure into smaller

S. Ghosh et al. / International Journal of Biological Macromolecules 54 (2013) 90– 98 91

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ragments [17,18]. EGCG is also capable of binding with nativelynfolded amyloid � peptide and �-synuclein and prevents forma-ion of toxic fibrillar species [19]. EGCG inhibits and disintegrates

ature fibrils of Islet amyloid polypeptide which is related to type diabetes [20]. However, only preformed HEWL fibrils (fibrillationarried out at pH 2) were converted into amorphous aggregates inhe presence of tea catechins where EGCG plays the most effectiveole [21].

HEWL a globular protein of 129 amino acid residues has two dif-erent domains which are cross linked by means of four disulfideonds [22] and is known to achieve molten globule conformationt high pH (12.75) [23]. HEWL was found to form fibrils at pH 12.7n the presence of tertiary butanol [24]. Moreover, the presencef salts exerts stabilization in several ways such as electrostatichielding of charges and selective binding of ions to proteins [25].ecent studies have reported that HEWL undergoes amyloid for-ation at pH 12.2 [26,27]. In addition to this, in the presence of 2 M

Cl at pH 12.7, enhanced hydrophobic exposure was observed forEWL [28]. Self assembly of HEWL was also found to occur underild UV illumination [29].We have shown earlier that the HEWL fibrillation process results

n the complete reduction of Cu(II) to Cu(I) in an article publishedn this journal [30]. Recently inhibition and disintegration of HEWLbrillation by salen-manganese complexes at acidic pH has alsoeen reported in this journal [31]. In the present article to elucidatehe role of GTPs during fibrillation, we have monitored the fibril-ation propensity of HEWL at pH 12.75 (200 mM KCl/NaOH) in thebsence and presence of three GTPs, EGC, EGCG and ECG (Fig. 1).e have characterized the fibrillar species using fluorescence

pectroscopy, circular dichroism spectroscopy, turbidity assay, flu-rescence and high resolution transmission electron microscopy.e have shown for the first time the potent inhibitory property of

CG at pH 12.75 in comparison to EGCG. We speculate this variation

tea polyphenols.

might be due to autoxidation of GTPs at alkaline pH which resultsin the formation of different autoxidized products which in turnresults in their differential contribution toward inhibition of fibril-lation. It has been found earlier that aromatic interactions betweenphenolic compounds in inhibitor molecules and aromatic residuesin the amyloidogenic core are the major reasons behind polyphenolmediated inhibition of fibrillation [32]. Our study points towarddiverse structural aspects of polyphenols that could be furtherexplored with respect to their role in interactions with proteins ingeneral and in the fibrillation process of proteins in particular.

2. Materials and methods

2.1. Materials

Hen egg white lysozyme (HEWL), Thioflavin T (ThT), 8-anilino-1-naphthalenesulfonic acid (ANS), (−)-epigallocatechin gallate(EGCG), (−)-epicatechin gallate (ECG), and (−)-epigallocatechin(EGC) were purchased from Sigma Chemical Co. (St. Louis, USA)and used as received. All other chemicals were obtained from SRL(India).

2.2. Preparation of fibrillar solutions

Stock HEWL solutions were prepared by dissolving HEWL indouble distilled water and the concentration determined using amolar extinction coefficient of 37,646 M−1 cm−1 at 280 nm [33]. Theprotein solution at pH 12.75 (maintained using 200 mM KCl/NaOH)was incubated at 37 ◦C for 6 h in absence and presence of GTPs to

form fibrils with pH measurements carried out on a EUTECH (pH510) pH meter. The pH of each set was measured before and afterincubation and found to be stable. The fibrillar solutions were keptat ambient temperature for further analyses. In each set, the final

9 f Biological Macromolecules 54 (2013) 90– 98

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rotein and GTP concentrations were 150 �M (1:1 molar ratio).liquots were diluted with 20 mM of pH 12.75 solution in eachtudy to maintain the desired concentration in the final workingolution.

.3. ThT and Trp fluorescence study

The ThT (an amyloid specific dye) [34,35] intensity was mea-ured after incubation at 37 ◦C for 6 h in all cases. Aliquots (27 �l)ithdrawn from the respective sets and 10 �l of ThT (1 mM) were

ncubated in 20 mM of pH 12.75 solutions for 2 min and scannedn a Horiba Jobin Yvon Fluoromax 4 spectrofluorimeter. For thehT kinetic study, aliquots (27 �l) were withdrawn at differentime intervals for 0–12 h from HEWL, HEWL-EGCG and HEWL-CG sets respectively and incubated with ThT (10 �l, 1 mM) for

min and scanned. Protein and dye concentrations were kept at �M and 5 �M, respectively in each case. Excitation and emissionaxima were kept at 450 nm and 485 nm, respectively. Slit width

nd integration time were fixed at 5 nm and 0.3 s. Each spectrumas corrected with respect to the corresponding blank. Tryptophan

Trp) fluorescence was monitored using a protein concentration of �M keeping excitation at 295 nm, to ensure no excitation of Tyresidues.

.4. ANS binding assay

After incubation at 37 ◦C for 6 h, a 27 �l aliquot from each set wasixed with 4 �l ANS (stock concentration 5 mM) in the presence

f 20 mM pH 12.75 solution and incubated in the dark for 1 h atmbient temperature. Protein and ANS concentrations were 2 �Mnd 10 �M, respectively Solutions were scanned in a Horiba Jobinvon Fluoromax 4 spectrofluorimeter from 400 to 600 nm keepinghe excitation wavelength at 370 nm. Slit width and integrationime were fixed at 5 nm and 0.2 s. Each spectrum was correctedith respect to the corresponding blank.

.5. Turbidity assay

Aliquots drawn from each set after 6 h incubation at 37 ◦C (keep-ng protein concentration at 5 �M) were scanned in a Shimadzu450 UV-vis spectrophotometer at 350 nm using a quartz cuvettef 1 cm path length. The increase in the absorbance value at 350 nms a measure of the scattering of light by aggregated particles [36].

.6. Circular dichroism spectroscopy

Far-UV circular dichroism spectra were acquired using a Jasco-10 spectrophotometer. A quartz cuvette of 0.1 cm path length wassed. Far-UV CD spectra were accumulated keeping the proteinoncentration at 20 �M and scanned between 190 and 240 nm at5 ◦C at a scan rate of 50 nm/s. Protein secondary structure contentas estimated using an online server, DICHROWEB [37].

.7. Fluorescence microscopy

Samples for fluorescence microscopy were prepared by mix-

ng 10 �l of ThT (1 mM) with 5 �l of each solution set, placed on

glass slide and covered with a cover slip. Images were observednder a Leica DM 2500M microscope equipped with a fluorescencettachment. For ThT excitation an emission filter cube no 2 (Leica I31513878, BZ: 01) was used. The images were captured with a LeicaFC 310 FX camera attached with the microscope. All observationsere performed at 10×/0.25 (N PLAN EPI).

Fig. 2. Histogram of ThT fluorescence of HEWL in absence and presence of GTPsafter incubation at 37 ◦C for 6 h at pH 12.75.

2.8. High resolution transmission electron microscopy

HEWL fibrillar solutions were diluted to a concentration of30 �M and placed on TEM grids. Samples were negatively stainedwith an aqueous solution of uranyl acetate [1% (w/v)], air driedand scanned in a JEM 2100 high resolution transmission electronmicroscope operating at an accelerating voltage of 200 kV.

3. Results and discussion

HEWL is known to adopt a molten globule state at alkalinepH [23]. Apart from forming fibrils at alkaline pH [24,26,27], thepresence of salts facilitates hydrophobic exposure under theseconditions [28]. Tea catechins are naturally occurring compoundswhich have been reported to be proficient amyloid inhibitors.They have also been found to possess the potency to disaggre-gate preformed fibrils [21]. We have investigated the effect of thesepolyphenols on HEWL fibrillation at alkaline pH (pH 12.75) wherethe protein attains a molten globule state. HEWL fibrillation wascarried out at pH 12.75 by incubating the protein at 37 ◦C for 6 hfollowed by incubation at ambient temperature. Results show thatthe GTPs are capable of inhibiting HEWL fibrillation. The inhibitoryefficacy of the GTPs was monitored using spectroscopic and micro-scopic techniques.

3.1. ThT fluorescence and kinetic studies

Incubation of HEWL at pH 12.75 at 37 ◦C produces fibrillarspecies that showed high fluorescence intensity. To monitor theextent of inhibition caused by GTPs on HEWL fibrillation we haveperformed ThT fluorescence after incubation at 37 ◦C for 6 h. HEWLfibrils without GTPs show intense fluorescence around 485 nmwhereas the presence of GTPs decreases ThT fluorescence signif-icantly. The ability to inhibit HEWL fibrillation follows the orderECG > EGCG > EGC. The relative decrease in ThT intensity in case ofECG was 51% whereas that in case of EGCG and EGC was 43% and29%, respectively (Fig. 2).

To further assess the inhibitory effect of the two major polyphe-nols, the ThT fluorescence intensity of HEWL in the absence andpresence of EGCG and ECG was monitored over a time period of0–12 h. The results shown in Fig. 3(a) clearly indicate the effect

of EGCG and ECG on fibril formation. The lower intensity of ThTfluorescence in presence of EGCG and ECG indicates that fibrilformation is inhibited, with ECG being more effective in the inhi-bition. The ThT fluorescence kinetics data obtained during HEWL

S. Ghosh et al. / International Journal of Biological Macromolecules 54 (2013) 90– 98 93

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ig. 3. (a) Time evolution of ThT fluorescence over a period of 0–12 h during HEWL fif ksp (h−1) [rate constant of spontaneous fibril formation] for HEWL fibril, HEWL-E

brillation in the absence and presence of EGCG and ECG wereubjected to a nonlinear least squares curve-fitting to a stretchedxponential function F = F∝ + �F exp[−(ksp × t)n] where F, F∝ andF are the observed fluorescence intensity at time t, final fluo-

escence intensity and fluorescence amplitude respectively and ksp

epresents the rate of spontaneous fibril formation [38–40]. A valuef n between 0 and 1 denotes kinetics with multiple exponentialunctions that signifies numerous events linked with fibrillation.

value of n > 1 is representative of a sigmoidal transition with annitial lag phase which involves different intermediate species. Theurve fitting employed here have been used to extract importantnformation regarding complex kinetic reactions [38,39].

The n values obtained for HEWL Fibril, HEWL-EGCG and HEWL-CG are 1.49 ± 0.27, 0.75 ± 0.26 and 0.92 ± 0.25 respectively andhe corresponding ksp values are 0.28 ± 0.023 h−1, 0.22 ± 0.051 h−1,nd 0.18 ± 0.027 h−1. In the absence of EGCG and ECG, the value of

> 1 indicates HEWL fibrillation proceeds via a classical nucleationathway with an initial lag phase. However, in the presence of EGCGnd ECG, n is less than unity (n < 1), which points toward a fibrilla-ion pathway that involves several events. These observations areimilar to studies on HSA fibrillation where at two different pH val-es two different aggregation pathway have been proposed on theasis of different n values [41]. Fig. 3(b) shows the ksp values (h−1) ofEWL Fibril, HEWL-EGCG and HEWL-ECG solutions from which it isvidently clear that the rate of spontaneous fibril formation is least

n the presence of ECG making ECG the more effective inhibitor.revious studies have revealed the unique inhibitory property ofGCG for various cases [16–21]. Furthermore, it has been noticedhat the antioxidant property of polyphenols is correlated with

ig. 4. (a) Trp fluorescence spectra of native HEWL, and HEWL in the absence and presencmission spectra of HEWL fibrils in absence and presence of GTPs. The control correspond

tion in the absence and presence of EGCG and ECG at pH 12.75 at 37 ◦C. (b) Variationnd HEWL-ECG solutions.

the anti-amyloidogenic activity but there is also evidence wherethis correlation is found to the contrary [42]. The plausible reasonbehind this trend of GTPs found in this study has been discussedlater in detail.

3.2. Trp fluorescence study

The intrinsic (Trp) fluorescence is quenched in the presence ofthe polyphenols (Fig. 4(a)). We observed that the Trp fluorescenceof native HEWL decreased on fibrillation along with a red shift in�max ∼ 346 nm to 351 nm. This is indicative of an increased polarenvironment around tryptophanyl residues upon fibrillation [43].Trp fluorescence is reduced further in the presence of EGCG andto a greater extent in the presence of ECG. It has been observedearlier that aromatic stacking interactions are one of the gover-ning factors of amyloid formation and amyloidogenic sequences arefound to be enriched with aromatic amino acids, particularly Trp[32,44,45]. The ability of the phenolic rings of polyphenols to adopta specific stacking interaction mode with amyloidogenic aromaticresidues renders them as effective inhibitors against fibrillation[32,45]. Previous studies published in this journal, have demon-strated that interactions between aromatic rings of polyphenolsand aromatic residues of proteins are responsible for the quench-ing of Trp fluorescence [46,47]. Thus the significant reduction in Trpfluorescence observed in presence of EGCG and ECG is most likely

due to aromatic interactions between Trp residues of HEWL andaromatic phenolic rings of polyphenols indicating the importantrole of aromatic interactions in the inhibition process. The plausi-ble reason behind the greater extent of quenching observed in case

e of EGCG and ECG after incubation at 37 ◦C for 6 h at pH 12.75. (b) ANS fluorescences to ANS binding with native HEWL.

9 f Biological Macromolecules 54 (2013) 90– 98

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Table 1Secondary structure parameter (% �-sheet content) of HEWL in the absence andpresence of GTPs (estimated by online server DICHROWEB using CONTIN analysisprogram).

System % �-sheet

HEWL Fibril 37HEWL-EGC 33

a maximum indicating greater aggregation, which decreases inthe presence of GTPs the most effective inhibitor being ECG. Thisis in accordance with the ThT binding and CD studies that also

Fig. 5. Far UV-CD spectra of HEWL fibrils in the absence and presence of GTPs at pH12.75. Inset shows comparative far UV CD spectra of native HEWL, HEWL fibril andHEWL-ECG solutions.

4 S. Ghosh et al. / International Journal o

f ECG has been discussed later in detail. At pH 12.75, deprotonatedydroxyl groups of the GTPs [48] interact with fibrils via negativelyharged oxygen atoms that may also contribute to its inhibitoryotency.

.3. ANS binding study

ANS is a fluorescent dye that binds with high affinity toydrophobic surfaces of proteins with the emission maximumhowing a blue shift with increase in fluorescence intensity on bind-ng to low polarity regions of a protein surface. ANS binding is thusn important tool used for detection of the molten globular states ofroteins and an indicator of hydrophobic patches in proteins [49].o investigate the relative exposure of hydrophobic regions dur-ng alkali-salt induced fibrillation of HEWL, ANS binding studies

ere performed. There is no significant binding of ANS to nativeEWL in absence of KCl whereas at pH 12.6 and 2 M KCl, a promi-ent increase in intensity occurs with a significant blue shift from10 to 480 nm [28]. We observed a notable blue shift (∼50 nm)rom 520 to 473 nm (Fig. 4(b)) in ANS fluorescence when incubatedith HEWL fibrils. This is indicative of an exposure of hydrophobic

egions due to the formation of molten globule like intermediates.he presence of GTPs, however, results in a significant depreciationf ANS intensity indicating that there is a decrease in hydropho-ic exposure. In addition, the extent of blue shift observed for theTPs is to a lesser extent compared to the fibrils alone. For EGCnd EGCG the blue shift is ∼40 nm showing a similar exposure ofhe hydrophobic region. In case of ECG the blue shift of ∼30 nm isccompanied by the maximum reduction in fluorescence intensityf the GTPs (Fig. 4(b)). Since polyphenols are known to be involvedn hydrophobic interactions with proteins [50], the importance ofhis interaction in relation to the inhibition of fibril formation haseen discussed where it has been found as one of the key factorsehind polyphenol driven inhibition [32,51]. This means that theTPs bind with the hydrophobic regions of the fibrillar species thusaking them less available for binding with ANS resulting in a

ecrease in fluorescence intensity. This reduction in the availableydrophobic exposed regions in the presence of GTPs is manifested

n an inhibition of fibrillation.

.4. Circular dichroism study

The alteration in secondary structure associated with fibrilla-ion primarily involves �-helix to �-sheet conversion which can be

onitored effectively using circular dichroism spectroscopy [52].TPs are not likely to cause any significant changes themselves

o the conformation of the native HEWL. Our earlier studies withGCG and HEWL did not show any significant change in the CDpectrum of the native protein [9]. The representative CD spectraFig. 5) of HEWL fibrils in the presence of GTPs show a significantall in mdeg value (more negative) at 208 nm. This is indicative ofncreased helicity which is most prominent in the presence of ECG.t appears that there is a decrease of the fibrillar species whichould imply the likely existence of a greater extent of monomericorm of HEWL in the presence of ECG (inset of Fig. 5). This is asso-iated with a concomitant decrease in �-sheet content (listed inable 1) that was found to be highest in case of ECG as expected.e have found that % of relative lowering in �-sheet content is

20% in the presence of ECG in comparison to HEWL fibrillar solu-ion. This conformational change indicates that GTPs prevent HEWLrom acquiring a predominant �-sheet structure. Perturbation of-sheet content is possibly due to a contribution of both autoxi-

ation of GTPs and interaction with deprotonated hydroxyl groupsf GTPs with the fibrillar species. Polyphenols have been reportedo undergo complexation with proteins which increases on heatingue to the exposure of favorable binding sites for polyphenols [50].

HEWL-EGCG 32HEWL-ECG 30

Apart from hydrophobic interactions, HEWL binds with polyphe-nols via specific amino acid residues such as proline and Trp [9,50].It is possible that all these possible types of interactions of polyphe-nols with HEWL are responsible for the alteration in secondarystructure content i.e. decrease in �-sheet content. This is reflectedin the reduced extent of fibrillation in the presence of GTPs.

3.5. Turbidity assay

The self association process of HEWL in presence of GTPs wasinvestigated using a turbidity assay at 350 nm. An increase in theabsorbance value at 350 nm indicates greater aggregation due toan increment in the scattering of light by aggregated particles [36].Fig. 6 shows that in the absence of GTPs, the absorbance value is

Fig. 6. Absorbance at 350 nm of HEWL fibrils in absence and presence of GTPs after6 h incubation at 37 ◦C to assess turbidity.

S. Ghosh et al. / International Journal of Biological Macromolecules 54 (2013) 90– 98 95

Fig. 7. Fluorescence microscopic and high resolution transmission electron microscopic images of HEWL fibrils formed in absence and presence of GTPs after incubation at37 ◦C at pH 12.75 for 6 h followed by incubation at ambient temperature for 10 days. Scale bars represent 100 �m and 0.2 �m, respectively; (a and e) HEWL fibril, (b and f)in the presence of EGC, (c and g) in the presence of EGCG, and (d and h) in the presence of ECG.

96 S. Ghosh et al. / International Journal of Biological Macromolecules 54 (2013) 90– 98

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emonstrate that GTPs prevent HEWL to undergo self assemblyith ECG being the most effective.

.6. Fluorescence microscopy and high resolution transmissionlectron microscopy

Fluorescence microscopic images clearly indicate that maxi-um fibrillation occurs in the absence of GTPs whereas in the

resence of GTPs the extent of fiber formation is less with a dis-inct minimum for ECG (Fig. 7(a)–(d)). Similarly HRTEM imagesFig. 7(e)–(h)) demonstrate that thin wormlike fibers are formed inhe absence of GTPs. In the presence of EGC, the extent of fiber for-

ation is less. Fibrillation is reduced notably in case of EGCG, wheremorphous aggregates are visible. ECG was capable of inhibiting theormation of fibrillar species significantly, confirming that it acts ashe most potent inhibitor amongst the GTPs at pH 12.75.

.7. Inhibition potency of GTPs

All experiments performed here reveal that ECG is the mostotent inhibitor among the GTPs studied. The reason behind its

EGCG, and ECG at alkaline pH.

potency can be explained considering several factors mentionedbelow. �-Amyloid neurotoxicity has been related with the produc-tion of free radicals that can be suppressed in presence of effectiveradical scavengers [53]. Reports are available where better rad-ical scavengers have been shown to be good amyloid inhibitors[54,55]. The radical scavenging property of GTPs lies in the orderof ECG > EGCG > EGC [56,57], which accounts for better inhibitoryactivity of ECG than the other two polyphenols used. A similar kindof observation has been found earlier in case of rifampicin whereits amyloid inhibitory property (in case of A�1–40 and HEWL) hasbeen attributed to its potent radical scavenging activity [54,55].Moreover, the radical scavenging activity of GTPs is known to be pHdependent and increases with increasing pH [58]. This is because ofthe deprotonation of the different phenolic hydroxyl groups whichnot only results in an increase in the anionic character but alsoenhances the electron donation capacity of GTPs [58].

The studies reported here have been performed at alkaline

pH, where polyphenols are known to be autoxidized resulting inthe formation of various radical species (Fig. 8) [59]. It has beenreported earlier that alkaline autoxidation of GTPs results in theoxidation of the B ring mainly for both EGC and EGCG whereas the

S. Ghosh et al. / International Journal of Biological Macromolecules 54 (2013) 90– 98 97

kali-sa

Dhiappihccta[irrgrbiHtAmtqrUtifmtsifleTpmt

Fig. 9. A schematic diagram showing inhibition of al

ring (i.e. the gallate ring) is oxidized in case of ECG [59]. Studiesave also shown that the presence of hydroxyl groups in the B ring

s most likely responsible for their better inhibitory property [13]. Itppears therefore that oxidation of the B ring lowers the inhibitoryroperty of EGCG and enables ECG to be a better inhibitor at alkalineH which is in line with its radical scavenging property. In addition,

t has been found that the aromatic interactions (�–� stacking) andydrophobic interaction among the side chains of proteins play arucial role in amyloid formation and provide stability [44]. Thusompounds offering sufficient aromatic and hydrophobic interac-ion possibilities are able to perturb the intermolecular interactionmong the protein molecules and in turn act as efficient inhibitors60,61]. During the fibrillation process at alkaline pH, attenuationn Trp fluorescence was observed. Further reduction in Trp fluo-escence takes place in the presence of EGCG with an even greatereduction in the presence of ECG. Earlier investigations have sug-ested that the aromatic ring of polyphenol interact with aromaticesidues of proteins such as tryptophan which might be the reasonehind attenuation in Trp fluorescence intensity [46,47]. The potent

nhibitory property of two manganese-salen derivatives againstEWL fibrillation involves both aromatic and hydrophobic interac-

ion [31]. For the EGCG inhibition studies of the aggregation of the�1–40 peptide, the aromatic interaction between EGCG and aro-atic residues of peptide were also shown to play pivotal role in

he inhibition [62]. Therefore it appears that the Trp fluorescenceuenching observed is due to aromatic interactions between Trpesidues of HEWL and phenolic rings of EGCG and ECG in our case.nder alkaline conditions employed, variation in the ring oxida-

ion of EGCG and ECG is likely to be the cause of the better aromaticnteraction of ECG (with the unoxidized B ring) which accountsor its greater inhibitory property as mentioned earlier (Fig. 8). The

olten globule formation of HEWL exposes hydrophobic regions inhe protein as demonstrated by the enhanced fluorescence inten-ity on ANS binding. We have found that the GTPs that are involvedn hydrophobic interactions with HEWL, are able to reduce the ANSuorescence intensity significantly. This reduces the hydrophobicxposure of HEWL which in turn resists self aggregation of HEWL.

here are evidences in favor of hydrophobic interaction betweenolyphenols and hydrophobic region of proteins being one of theajor driving forces responsible for the prevention of fibril forma-

ion [32,51], which is in agreement with these studies.

lt mediated fibrillation of HEWL in presence of ECG.

4. Conclusion

We show here for the first time the effective inhibitory activityof tea polyphenols in an alkali-salt induced aggregation environ-ment for hen egg white lysozyme (HEWL). A schematic diagramrepresenting the inhibition pathway (in presence of ECG) of HEWLhas been shown in Fig. 9. The most effective of the ones studiedbeing ECG in preventing the fibrillation process. The ThT bindingassay indicates maximum aggregation in the absence of polyphe-nols and minimum in presence of ECG. ThT kinetics of HEWL,HEWL-EGCG and HEWL-ECG solutions indicates different aggrega-tion pathways in the absence and presence of GTPs. Moreover, thelowest ksp value (0.18 ± 0.027 h−1) obtained in case of ECG confirmsit to be most potent inhibitor. Trp fluorescence suggests involve-ment of aromatic interaction which is more pronounced in thepresence of ECG. The reduction in hydrophobic exposure of HEWLon fibrillation in the presence of GTPs as observed in the ANS bind-ing assay suggests reduced fibrillation. The CD study indicates anincrease in helicity in the presence of GTPs, most significant inthe case of ECG. Turbidity assay shows aggregation propensity ofHEWL is least in the presence of ECG. Furthermore, the microscopicimages clearly show growth and distribution of fibers where ECGwas found to inhibit fibrillation remarkably. The reason behind thegreater potency of ECG as compared to the other two polyphenolsis attributed to combination of its greater radical scavenging abil-ity along with significant aromatic and hydrophobic interactions.Moreover, autoxidation of GTPs and their interactions via nega-tively charged oxygen atoms with HEWL fibrils plays moderate rolein the inhibition of HEWL fibrillation. Further elucidation of struc-ture activity relations of such inhibitors is required to enhance ourknowledge of inhibition of aggregated fibrillar species that play amajor role in neurodegenerative diseases.

Acknowledgements

SD is grateful to Department of Science and Technology, Govern-ment of India. The authors would like to acknowledge the CentralResearch Facility, IIT Kharagpur for providing experimental facil-ities. SG and NKP are supported by senior research fellowships

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8 S. Ghosh et al. / International Journal o

rom the Council of Scientific and Industrial Research, India andIT Kharagpur respectively.

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