Inhibition of Ribonuclease A by polyphenols present in green tea

proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS

Inhibition of Ribonuclease A by polyphenolspresent in green teaKalyan S. Ghosh, Tushar K. Maiti, Joy Debnath, and Swagata Dasgupta*

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

INTRODUCTION

Mammalian ribonucleases are noncytosolic endonucleases, which cleave ribonucleic

acid (RNA) in a mechanism that involves a transphosphorylation step followed by

hydrolysis.1,2 Ribonucleases may be cytotoxic when they adsorb specifically to certain

cells, enter the cytosol, degrade RNA, and consequently inhibit protein syntheses that

results in cell death. Ribonuclease inhibitors, either synthetic or natural have been

intensively sought after for therapeutic purposes. This is largely because a growing

number of ribonuclease type enzymes such as angiogenin,3 eosinophil-derived neuro-

toxin (EDN),4 and bovine seminal RNase A5 exhibit biological activities that have been

shown to play important roles in human disease processes.6

The use of natural compounds as possible functional inhibitors of proteins is cur-

rently gaining importance and the focus on ribonucleases with unusual biological prop-

erties has generated renewed interest. The usual toxicity associated with synthesized

compounds is eliminated to a great extent by the consideration of natural inhibitors.

Epidemiological studies show a lower incidence of cancer among regular drinkers of

green tea.7,8 Green tea extracts, which have a marked antioxidant effect,9 contain the

polyphenols (�)-epicatechin (EC), (�)-epicatechin gallate (ECG), (�)-epigallocatechin

(EGC), and (�)-epigallocatechin gallate (EGCG) (Fig. 1).10

The ribonucleolytic center of RNase A is constituted of multiple subsites (P1, B1, and

B2) (Fig. 2) that bind to the phosphate, nucleobase, and sugar components of the RNA

molecule.11 The most important binding site is the P1 binding site (comprised of His

12, Lys 41, His 119) where cleavage of the phosphodiester bond occurs. Thus inhibition

of the ribonucleolytic activity of RNase A may be possible by directly blocking the

active site12 that would lead to a disruption of enzymatic activity. Apart from

the amino acid residues directly involved in the catalytic process, residues present in

the various subsites are known to play an indirect role in the catalytic mechanism.11

In a previous report we have shown that EGCG acts as a noncompetitive inhibitor of

RNase A, indicating that it does not interact directly with the ribonucleolytic site.13

Apart from an agarose gel based assay and enzyme kinetics conducted for EGCG earlier,

this study has been conducted with the other major components present in green tea

with the inclusion of further spectroscopic and theoretical studies for EGCG. The inter-

actions of the polyphenols with RNase A have been studied by UV–Vis spectroscopy,

Fourier transformed infrared (FTIR), and circular dichroism (CD). Our studies using

similar techniques to investigate the interaction of EGCG with human serum albumin

(HSA) and of catechin and EC with human and bovine serum albumins have been

reported.14,15 Binding parameters obtained from these experiments have been calcu-

Grant sponsor: Council of Scientific and Industrial Research (CSIR), India; Grant number: 37(1188)/04/EMR-II

Tushar K. Maiti’s current address is Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel 76100.

*Correspondence to: Dr. Swagata Dasgupta, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India.

E-mail: [email protected]

Received 7 December 2006; Revised 8 February 2007; Accepted 16 February 2007

Published online 10 July 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21484

ABSTRACT

We report the effect of the

natural polyphenolic com-

pounds from green tea on the

catalytic activity of Ribonucle-

ase A (RNase A). The com-

pounds behave as non-

competitive inhibitors of the

protein with inhibition con-

stants ranging from 80–1300

lM. The dissociation con-

stants range from 50–150 lM

for the RNase A–polyphenol

complexes as determined by

ultraviolet (UV) and circular

dichroism (CD) studies. We

have also investigated the

changes in the secondary

structure of RNase A on com-

plex formation by CD and

Fourier transformed infrared

(FTIR) spectroscopy. The pres-

ence of the gallate moiety has

been shown to be important

for the inhibition of enzymatic

activity. Docking studies for

these compounds indicate that

the preferred site of binding is

the region encompassing resi-

dues 34–39 with possible

hydrogen bonding with Lys 7

and Arg 10. Finally we have

also looked at changes in the

accessible surface area of the

interacting residues on com-

plex formation for an insight

into the residues involved in

the interaction.

Proteins 2007; 69:566–580.VVC 2007 Wiley-Liss, Inc.

Key words: green tea poly-

phenols; Ribonuclease A; non-

competitive inhibition; FTIR;

circular dichroism; FlexX sin-

gle molecule docking.

566 PROTEINS VVC 2007 WILEY-LISS, INC.

lated to determine the types of interactions involved. To

substantiate our experimental studies we have also per-

formed docking studies to obtain a clearer insight into

the residues involved in the interaction. This study is

pertinent because RNase A is a convenient model system

of the ribonuclease superfamily. The focus on the ribonu-

cleolytic site in the development of small molecule inhib-

itors is because of its key role in the biological function

of these proteins.16 This study shows that apart from the

ribonucleolytic site, it is reasonable to target other sites

in the rational development of inhibitors of ribonuclease

type proteins.

EXPERIMENTAL

Materials

Bovine pancreatic RNase A, yeast tRNA, 20,30-cCMP,

EC, ECG, EGC, and EGCG were from Sigma Aldrich.

D2O was from Acros Organics and all other reagents

were from SRL India. UV measurements were made

using a Perkin Elmer UV–Vis spectrophotometer (Model

Lambda 25). Concentrations of the solutions were deter-

mined spectrophotometrically using the following data:

for RNase A e278.5 ¼ 9800 M�1 cm�1,17 for 20,30-cCMP

e268 ¼ 8500 M�1 cm�1,18 and e270 values for EC, ECG,

EGC, and EGCG are 4290, 12443, 2088, 11,920 M�1

cm�1 19 respectively.

Methods

Agarose gel-based assay

Inhibition of RNase A by the four polyphenols was

checked qualitatively by the degradation of tRNA in an

agarose gel. In this method, 20 lL of RNase A (1.5 lM)

was mixed with 20 lL each of 0.30 mM EGCG, EGC,

ECG, EC to a final volume of 50 lL and the resulting

solutions were incubated for 6 h at 378C. Twenty micro-

liter aliquots of the incubated mixtures were then mixed

with 20 lL of tRNA solution (5.0 mg/mL tRNA, freshly

dissolved in RNase free water) and incubated for another

30 min. Ten microliter of sample buffer which contains

10% glycerol and 0.025% bromophenol blue was added

to the mixture. Fifteen microliter from each solution was

extracted and loaded onto a 1.1% agarose gel. The gel

was run using 0.04M Tris-Acetic acid-EDTA (TAE) buffer

(pH 8.0). The undegraded tRNA was visualized by ethi-

dium bromide staining under UV light.

Precipitation assay

Inhibition of the ribonucleolytic activity of RNase A

was assayed by the precipitation assay as described by

Bond.20 In this method 10 lL of RNase A (5.6 lM) was

mixed with varying concentrations (0–0.12 mM) of EC,

ECG, EGC, and EGCG to a final volume of 100 lL and

incubated for 2 h at 378C. Twenty microliter of the

Figure 1Structures of the four major polyphenols present in green tea.

Figure 2Schematic representation of the active center cleft in the RNase A substrate

complex. B1, R1, and P1 correspond to the base, ribose and phosphate

recognition sites that comprise the main binding site where the catalytic reaction

takes place.

Inhibition of RNase A by Green Tea Polyphenols

DOI 10.1002/prot PROTEINS 567

resulting solutions from the incubated mixtures were

then mixed with 40 lL of tRNA (5 mg/mL tRNA freshly

dissolved in RNase A free water), 40 lL of phosphate

buffer of pH 6.5 containing 5 mM EDTA and 0.5 mg/

mL HSA. After incubation of the reaction mixture at

258C for 30 min, 200 lL of ice-cold 1.14N perchloric

acid containing 6 mM uranyl acetate was added to

quench the reaction. The solution was then kept in ice

for another 30 min and centrifuged at 48C at 12,000

rpm for 5 min. Hundred microliter of the supernatant

was taken and diluted to 1 mL. The change in absorb-

ance at 260 nm was measured and compared with a

control set.

Inhibition kinetics

The inhibition of RNase A by the polyphenols was

assessed individually by a spectrophotometric method as

described by Anderson et al.18 The assay was performed

in oligo vinylsulphonic acid free (using the method as

described by Smith et al.21) 0.1M Mes-NaOH buffer,

pH 6.0 containing 0.1M NaCl using 20,30-cCMP as the

substrate. The inhibition kinetics for EGCG has been

previously reported from this laboratory.13 For EC,

ECG, and EGC the substrate concentrations ranged

from 0.10 to 0.30 mM and the inhibitor concentration

ranges for EC, ECG, and EGC were from 0 to �0.25

mM. The RNase A concentration used was 12 lM. The

inhibition constants were determined from initial veloc-

ity data. The reciprocal of initial velocity was plotted

against the reciprocal of substrate concentration (Line-

weaver–Burk plot) at a constant inhibitor concentration

according to the equation:

1

V¼ Km

Vmax

1 þ I½ �Ki

� �1

S½ � þ1

Vmax

1 þ I½ �Ki

� �

where V is the initial velocity, [S] the substrate concen-

tration, [I] the inhibitor concentration, Km the Michaelis

constant, Ki the inhibition constant, and Vmax the maxi-

mum velocity. A rearranged form of this equation leading

to the Eadie–Hofstee plot has been used to confirm the

Km, Ki, and Vmax values. In this plot, usually used for

low substrate concentrations in enzyme kinetics, V vs. V/

[S] is plotted to obtain the kinetic constants.

Determination of association constants ofRNase A–Polyphenol complexes

The RNase A–polyphenol binding constants were

determined by spectrophotometric methods according to

published methods.22–24 By assuming that there is only

one type of interaction between the polyphenols and

RNase A in aqueous solution, the overall binding con-

stant can be written as follows considering the equilib-

rium.

RNase A þ Polyphenol , RNase A � Polyphenol

K ¼ ½RNase A � Polyphenol�½RNase A�½Polyphenol�

The double reciprocal plot of 1/(A � A0) versus 1/[L]

is linear, where A0 is the initial absorbance of the free

protein and A is the absorbance at different ligand con-

centrations at 280 nm. The binding constant K can be

estimated from the ratio of the intercept to the slope.

For EC, ECG, EGC, and EGCG the polyphenol concen-

trations were varied from 3.5 to 50 lM. The RNase A

concentration was 4.4 lM for EC, ECG, and EGC. For

EGCG, the protein concentration was kept at 3.5 lM.

CD measurements

CD measurements were made on a Jasco-810 auto-

matic recording spectrophotometer, using a path length

of 10 mm at 258C. The spectra were recorded in the

range of 190–240 nm with a scan rate of 50 nm/min and

a response time of 4 s. For baseline correction, CD spec-

tra of buffer (20 mM phosphate buffer of pH 7.0)

containing variable concentrations of polyphenols were

collected and were subtracted from each sample spectra.

The polyphenol concentrations were varied from 0.05 to

0.55 mM with the RNase A concentration at 11.2 lM.

For EGCG, the RNase A concentration was 14.8 lM. For

comparison with the results from FTIR studies, the CD

spectra of RNase A ligand complexes were collected at

protein to ligand molar ratios of 1:0.5 and 1:1.5. Second-

ary structures were determined using DICHROWEB,25

an online server for protein secondary structure analyses

from CD spectroscopic data.

Calculation of binding constant from CD data

We used the ellipticity ratio (Y222/Y208)26 as a con-

venient parameter (response) to follow changes in the

secondary structure of RNase A upon addition of the

four ligands using the following relationship as described

by Zhai et al.27

R ¼ R0 þ DRsat

½L�Kd þ ½L� ; DRsat ¼ Rsat � R0

where R is the observed response at a given polyphenol

concentration, R0 is the response at zero polyphenol con-

centration, and Rsat is the response at saturation. Kd is

the effective site dissociation constant, and [L] is the free

ligand concentration, which, under the conditions of the

experiment, is approximately equal to the total polyphe-

nol concentration, added to the cuvette. We have also

used this expression to determine the dissociation con-

K.S. Ghosh et al.

568 PROTEINS DOI 10.1002/prot

stants of the complexes formed between EC and catechin

with human and bovine serum albumins.15 It should be

noted that Kd is not the actual site dissociation constant,

but an effective site dissociation constant that reflects the

shape of the binding curve and not the number of sites

present on the macromolecule. If n equivalent and inde-

pendent sites exist on a macromolecule, the number of

bound ligands can be equated to n[L]/(Kd þ[L]). Data

were fit to the above equation using Microcal Origin 6.0.

Fourier transformed infrared studies

For the FTIR studies 15–18 mg/mL RNase A was dis-

solved in 20 mM phosphate buffer of pD 7.2 in 99.9%

D2O. RNase A–polyphenol complex solutions were pre-

pared by mixing the ligands and RNase A maintaining

final ligand-protein molar ratios of 0.5:1 and 1.5:1. FTIR

measurements were carried out at 258C on a Nexus-870

FTIR spectrometer (Thermo Nicolet Corporation)

equipped with a germanium-attenuated total reflection

accessory, a DTGS KBr detector and a KBr beam splitter.

All spectra were taken via the attenuated total reflection

(ATR) method with resolution of 4 cm�1, with 256 scans

each. A control buffer spectra (20 mM phosphate buffer

of pD 7.2) was also recorded under identical conditions.

The background was corrected before every sample.

IR spectra processing procedure

The relative amounts of secondary structural elements

of RNase A and the RNase A complexes with four cate-

chins were determined following the method of Byler and

Susi.28 The spectra obtained after buffer subtraction were

smoothed by an 11 point Savitsky–Golay smooth function

to reduce noise. The protein secondary structure content

was determined from the amide I band located between

1700 and 1600 cm�1. Fourier self-deconvolution and sec-

ondary derivative calculations were applied to estimate the

number and position of the component bands. Based on

these parameters a multiple Gaussian curve-fitting process

was carried out in the region 1700–1600 cm�1 of the am-

ide I band to quantify the area of each component. The

secondary structural elements were obtained from the area

under the Gaussian curve. The resultant fit is analyzed as

follows: each Gaussian band is assigned to a secondary

structure according to the frequency of its maximum: a-

helix (1650–1662 cm�1), b-sheet (1610–1632 cm�1), ran-

dom coil (1636–1644 cm�1), turn (1665–1680 cm�1), and

b-antiparallel (1680–1692 cm�1).29,30 The areas of all the

component bands assigned to a given conformation are

then summed and divided by the total area. The number

obtained is taken as the proportion of the polypeptide

chain in that particular conformation.28

Docking studies

The crystal structure of RNase A (PDB entry 1FS331)

was downloaded from the Protein Data Bank.32 We have

chosen 1FS3 for the docking studies since this is the

structure of the wild type bovine pancreatic RNase A. This

should be able to appropriately complement our experi-

mental results that have been conducted with the same pro-

tein. The 3D structures of green tea polyphenols were gen-

erated by Sybyl6.92 (Tripos, St. Louis) and their energy-

minimized conformations were obtained with the help of

the TRIPOS force field using Gasteiger–Huckel charges

with a gradient of 0.005 kcal/mole. The FlexX software as

part of the Sybyl suite was used for docking of the polyphe-

nols to RNase A. The ranking of the generated solutions is

performed using a scoring function that estimates the free

binding energy DG of the protein-ligand complex.33

PyMol34 was used for visualization of the docked confor-

mations. The theoretical value of the dissociation constants

(Kd) of the docked structures of the protein ligand com-

plexes was computed using PEARLS, which computes inter-

action energies for receptor ligand systems.35

Accessible surface area calculations

The accessible surface area (ASA) of RNase A (uncom-

plexed) and their docked complexes with EC, ECG, EGC,

EGCG were calculated using the program NACCESS.36

The structures corresponding to the minimum score as

obtained from the FlexX analysis of the protein-ligand

docked structures were chosen in each case. Composite

coordinates of the polyphenols and RNase A were gener-

ated to form the docked complex. The change in ASA

for residue, i was calculated using: DASAi ¼ ASAiRNase A�

ASAiRNase A�polyphenol. If a residue lost more than 10 A2

ASA when going from the uncomplexed to the com-

plexed state it was considered as being involved in the

interaction.

RESULTS AND DISCUSSION

The use of natural product based compounds for the

inhibition of RNase A that may subsequently be used for

the rational design of more potent inhibitors is the basis

of this study. This approach to understand the inhibition

of ribonucleases has been investigated by an initial look

at the effect of the green tea polyphenols on RNase A.

The notion being that the inhibition of the ribonucleo-

lytic activity of RNase A, information on which is largely

available, would allow us to be able to rationally design

inhibitors for proteins belonging to the ribonuclease

superfamily e.g. angiogenin, that are known to possess

unusual biological properties.3

The inhibition of the ribonucleolytic activity of RNase

A was initially checked by an agarose gel based assay,

where the degradation of tRNA by RNase A was moni-

tored [Fig. 3(A)]. The most intense band observed in

lane 1 is due to the presence of the control, tRNA.

The faint intensity of the band in lane 2 is due to the

degradation of tRNA by RNase A. The differential inten-



sity of bands in lanes 3–6 indicates the degree of RNase

A inhibition by EGCG, EGC, ECG and EC respectively.

These results qualitatively show that the compounds are

capable of effectively inhibiting the ribonucleolytic activ-

ity of RNase A. The concentrations of the polyphenols

being the same in each lane, the EGCG lane appeared

more intense than the other three components. This

indicates that the major polyphenolic constituent of

green tea, EGCG acts as the most potent inhibitor

of RNase A. The inhibition of the ribonucleolytic activity

of RNase A by the polyphenols was further confirmed

quantitatively by a precipitation assay. The plots of rela-

tive ribonucleolytic activity versus inhibitor concentration

[Fig 3(B)] give us a comparative idea about the inhibi-

tory powers of the individual polyphenols. Considering a

0.04 mM concentration of EC, ECG, EGC and EGCG the

percent inhibition is found to be 4.4, 12.7, 6.9, and 18.4,

respectively. It is interesting to note that polyphenols

containing the gallate moiety are more effective inhibi-

tors. This feature is also apparent in the other studies

discussed later.

To determine the nature of inhibition and the inhibi-

tion constants, kinetic experiments were conducted. The

reciprocal of reaction velocity was plotted against the

reciprocal of substrate concentration for three different

inhibitor concentrations for each polyphenol. The inhibi-

tion constant values obtained for EC, ECG and EGC are

1336 � 2.5, 392 � 23, and 671 � 18 lM respectively

have been calculated from both the Lineweaver–Burk and

Eadie–Hofstee plots. The unchanged slopes of the lines

in Eadie–Hofstee plots (Fig. 4) are indicative of a non-

competitive type of inhibition. The inhibition constant in

case of EGCG for RNase A is 81 lM as obtained from a

previous study in this laboratory.13 We find that all the

inhibitors behave in a noncompetitive fashion. The order

of values obtained for the inhibition constants correlate

well with those obtained from the precipitation assay.

Our results indicate that the ribonucleolytic inhibitory

power of the catechins follows the order EGCG > ECG

> EGC > EC. Following this study the overall binding

constants for the RNase A–polyphenol complexes were

determined spectrophotometrically. From Figure 5 the

association constants obtained for the EC, ECG, EGC,

EGCG complexes with RNase A are 6.66 � 0.01 3 103,

1.26 � 0.03 3 104, 7.89 � 0.01 3 103, and 2.04 � 0.02

3 104 M�1 respectively. The association constant value

for EGCG is highest, which implies that it forms the

most stable complex with RNase A. This is in good

agreement with the results of the inhibition studies (aga-

rose gel, precipitation assay, and inhibition kinetics),

where EGCG was also found to be the most potent in-

hibitor. It may also be noted that the order of values for

the association constants is EGCG > ECG > EGC > EC.

In both studies, we observe that polyphenols with a gal-

late moiety exhibit a greater potency for inhibition.

The results from the kinetic experiments that indicate

a noncompetitive mode of inhibition prompted us to

further probe the effects of these compounds on the sec-

ondary structure of RNase A by CD and FTIR experi-

ments. CD spectra of RNase A exhibit two negative

bands in the ultraviolet region at 208 and 222 nm that

are characteristic of a-helices in proteins (Fig. 6). The

CD spectroscopic studies indicate that polyphenol bind-

ing to RNase A results in perturbations of the protein

secondary structure upon complexation (Table I). The

CD spectra of free RNase and its complexes with EC,

ECG, EGC, and EGCG are shown in Figure 6. The CD

spectra of RNase A at various concentrations of EC,

ECG, EGC and EGCG were analyzed in terms of the

ratio of ellipticity at 222 to 208 nm. The variations of

Y222/Y208 with the addition of ligands are shown in Fig-

ure 7 for the four compounds. The ellipticity ratio

increased hyperbolically with increasing concentration of

the ligand, yielding dissociation constants (Kd) of 141 �9, 80 � 3, 112 � 12, and 40 � 1 lM for EC, ECG, EGC,

and EGCG, respectively. The binding stoichiometries for

EC, ECG, EGC, and EGCG were found 1.31, 1.16, 1.13,

Figure 3A. Agarose gel based assay for the inhibition of RNase A by the green tea

polyphenols. Lane1: tRNA; Lane 2: RNase A and tRNA; Lane 3: EGCG, RNase

A and tRNA; Lane 4: EGC, RNase A and tRNA; Lane 5: ECG, RNase A and

tRNA; Lane 6: EC, RNase A and tRNA. B. Relative ribonucleolytic activity plots

of inhibition of RNase A by EC (-~-), ECG (-^-), EGC (-n-) and EGCG

(-3-).

K.S. Ghosh et al.


and 1.09 as obtained from fitting results. The results

show that the dissociation constants for protein ligand

complexes are consistent with the inhibition constants of

the polyphenols. The reciprocal of the association con-

stants obtained from UV–Vis experiments are found to

be 150 � 0.32, 79 � 2.7, 127 � 0.28, 49 � 0.68 lM for

RNase A-EC, RNase A-ECG, RNase A-EGC, and RNase

A-EGCG, respectively. These correlate well with the

values obtained from CD experiments—the order of

potency remaining the same.

The difference between the Kd and Ki values for these

polyphenols can be explained when we consider the fol-

lowing scheme of simple noncompetitive inhibitors:

E þ S $ ES ! P

þ þI I

l lEI þ S $ ESI

where E, S, and I represent enzyme, substrate and inhibi-

tor respectively. In considering Ki, the inhibitor, I inter-

acts not only with E but is also in equilibrium with two

other species ES and ESI. However, for the Kd calcula-

Figure 4Eadie-Hofstee plots for inhibition of RNase A by (A) EC: concentrations are

0.25 mM (~), 0.13 mM (n), 0 mM (l); (B) ECG: concentrations are 0.19

mM (~), 0.09 mM (n), 0 mM (l); (C) EGC: concentrations are 0.19 mM

(~), 0.09 mM (n), 0 mM (l). RNase A concentration: 12.0 lM.

Figure 5The plot of 1/(A � A0) versus (1/L) for RNase A–Polyphenol complexes at 280

nm. EC (-~-), ECG (-^-), EGC (-n-), and EGCG (-3-). RNase A

concentration: 4.4 lM for EC, ECG and EGC and 3.5 lM for EGCG.



Figure 6CD spectra of RNase A and its complexes with (A) EC, (B) ECG, (C) EGC and (D) EGCG. RNase A (free) (__) and RNase A with 0.0556 mM (---); 0.074 mM (-~-)

ligand. RNase A concentration: 11.2 lM for EC, ECG and EGC and 14.8 lM for EGCG.


K.S. Ghosh et al.

tions by UV and CD methods, in the absence of any sub-

strate, we consider the equilibrium: E þ I $EI where I is

the ligand in these cases.

Secondary structure analyses for CD data was per-

formed using the SELCON method in DICHROWEB for

the RNase A–polyphenol complexes to directly compare

with the FTIR results (Table I). The trends are similar

though the content of secondary structural elements dif-

fers as discussed below. Apart from differences in sample

preparation for the two methods, where a hydrated film

was used for FTIR and an aqueous solution was used for

CD measurements, a major difference lies in the spectro-

scopic signals themselves. FTIR signals arise from the

vibrational modes whereas CD spectra are obtained from

electronic transitions that may be the cause for the differ-

ence in content of their structural information. Similar

differences between FTIR and CD spectroscopic results

for free RNase A have been reported elsewhere.24,37

The percent change in secondary structure content is,

however, well correlated for both studies. The results

indicate that there is a certain degree of order brought

into the structure of the protein on ligand binding,

which is most apparent in ECG where the largest per-

centage increase in a-helicity is observed. This is fol-

lowed by EC and EGCG. For EGC on the other hand,

there is a substantial increase in the b-sheet content on

binding. The difference in the binding pattern of EGC is

also highlighted in the FTIR difference spectra and dock-

ing studies (discussed later).

Hydrogen bonding and the coupling between transi-

tion dipoles are key factors that play a crucial role in

governing the conformational sensitivity of the amide

bands of proteins. The protein amide I and amide II

bands at 1645–1650 cm�1 (mainly C¼¼O stretching) and

1548–1560 cm�1 (C��N stretching coupled with N��H

bending) respectively, are correlated with structural

changes in proteins.28 The difference spectra [(enzyme

solution þ polyphenol solution)—(enzyme solution)]

provide information about the conformational changes

that arise upon interaction between the protein and

ligand. The results are shown in Figure 8. The protein

C��H stretching vibrations exhibited no spectral change

upon ligand interaction and therefore cancel out on spec-

tral subtraction. We observe that the peak positions of

the amide I and II bands in the two difference spectra of

each polyphenol shift, with a simultaneous change in the

relative intensity. This is suggestive of a change in the

secondary structure of the protein. In general, the spec-

tral ranges from 1610–1632 cm�1, 1636–1644 cm�1,

1650–1662 cm�1, and 1665–1680 cm�1 in the amide I

region are attributed to b-sheet, random coil, a-helix,

and turn structures, respectively.29,30

In the difference spectra of EC at lower concentrations,

the observed peak at 1632 cm�1 with two shoulders at

1646 and 1658 cm�1 indicate that the b-sheet, random

structure, and a-helix content of the protein are affected.

At a higher concentration of EC, the peak maximum

shifts to 1649 cm�1 with two shoulders at 1627 and 1678

cm�1, which implies that the a-helical region is further

stabilized. This observation is similar to what is found in

the CD studies. In case of ECG, at low concentrations,

the peak at 1640 cm�1 with a shoulder at 1657 cm�1 is

associated with a change in random coil and a-helix

components respectively. At higher concentrations, the

changes in the peak and the shoulder positions become

more prominent reflecting the changes in content of b-

sheet (1625 cm�1), a-helix (1653 cm�1), and turn (1676

cm�1). The maximum change observed at 1653 cm�1

can be attributed to the stabilization of a-helix of RNase

A. Changes observed in the secondary structural compo-

nents of RNase A on interaction with EGC are distinctly

different from the other polyphenols as is evident from

the difference spectra. The maximum of the amide I

band in the EGC complex at a lower concentration is at

1645 cm�1, which is indicative of a change in the unor-

dered region of the protein. With an increase in concen-

tration the peak maximum shifted to 1632 cm�1. The

difference spectra for EGC are also characterized by an

overall lower absorbance change compared with the other

polyphenols. The maximum change for EGC is observed

in the b-sheet region indicating the stabilization of this

structural element. For EGCG, at higher concentration,

the major changes in absorbance are at 1638 and

1627 cm�1 with two shoulders at 1651 and 1671 cm�1

Table ISecondary Structure Analyses of Free RNase A (in italics) and RNase A Complexes With Four Polyphenols With Protein:Ligand Ratios at 1:1.5 Determined by FTIR and

CD Techniques

a-helix (%) b-sheet (%) Random coil (%) Turn (%)

FTIR CD FTIR CD FTIR CD FTIR CD

RNase A 23.2 15.0 41.8 42.0 23.7 26.0 10.7 18.0RNase A: EC 28.3 20.0 43.9 44.0 18.6 19.0 9.2 16.0RNase A: ECG 33.2 22.5 42.7 44.0 11.3 16.0 6.9 17.0RNase A: EGC 21.4 14.5 51.4 52.0 16.9 20.0 9.5 13.0RNase A: EGCG 26.8 18.0 43.0 42.0 20.3 24.0 9.8 16.0

The numbers in bold are the values showing the most change in each column. Underlined values correspond to values lower that that of free RNase A.



corresponding to the increase in the a-helix and random

structures at the expense of b-sheet region. It was found

that the polyphenols EC, ECG, and EGCG preferentially

stabilize the a-helix and EGC stabilizes b-sheet con-

formations of RNase A compensating from random

structure. The distinctive changes in secondary structural

elements are more prominent in the deconvoluted

spectra of RNase A–polyphenol complexes (Fig. 9). The

increase in a-helix content of RNase A has also been

observed when it interacts with 30-azido-30-deoxythymi-

dine (AZT).24

Quantitative analyses of the protein secondary struc-

ture for free RNase A and its polyphenol complexes in

D2O are given in Table I. For free RNase A, the maxi-

mum of the amide I band is at 1629 cm�1, which indi-

cates that the major contribution to the amide I band is

due to the b-sheet of RNase A, which is similar to a pre-

vious observation.38 The amide II band arises mainly

from an out-of-phase combination of N��H in plane

bending and C��N stretching vibrations of peptide link-

ages.39 Differences in the intensity of the amide II region

were observed at 1550 cm�1 for EC, 1546 cm�1 for ECG,

Figure 7Variation of Y222/Y208 with ligand concentration for (A) EC (B) ECG, (C) EGC and (D) EGCG. RNase A concentration: 11.2 lM for EC, ECG and EGC and 14.8 lM

for EGCG.

K.S. Ghosh et al.


1572 cm�1 for EGC, and 1544 cm�1 for EGCG at the

higher protein: ligand ratio. The increase in intensity

observed in the difference spectra between 1544 and

1550 cm�1 for all polyphenols can be attributed to the

interaction of the polyphenols with the backbone of the

protein. Interestingly the amide II regions are substan-

tially broadened for ECG and EGCG in comparison to

EC or EGC. The presence of the gallate moiety has previ-

ously been shown to have an inhibitory effect on several

proteins.40–42 A recent study reported that the ester-

bonded gallate catechins, EGCG and ECG, isolated from

green tea are potent inhibitors of dihydrofolate reductase

activity in vitro.43 We also note that the presence of the

gallate moiety of ECG and EGCG (ring D) plays a crucial

Figure 8FTIR difference spectra of RNase complexes with (A) EC, (B) ECG, (C) EGC and (D) EGCG at two different concentrations, RNase A : polyphenol 1:0.5 (��) and

1.1.5 (–*–) (left hand axis) with FTIR spectra of free RNase A (—) (right hand axis). RNase A concentration: 16 mg/mL.



Figure 9Deconvoluted spectra of (A) RNase A; (B) RNase A-EC complex; (C) RNase A-ECG complex; (D) RNase A-EGC complex; (E) RNase A-EGCG complex. The percentage

of a-helix, b-sheet, and random coil are indicated in the legend for each panel.

K.S. Ghosh et al.


role in the interaction, which is reflected in the broaden-

ing of the amide II band in the difference spectra. This

implies that there is a substantial effect on the peptide

backbone N��H in plane bending and C��N stretching

vibrations on interaction with ECG and EGCG. This ob-

servation has also been correlated with docking studies

(discussed later). The overall observed spectral changes

are indicative of a nonspecific interaction signifying that

the binding involves different parts of the protein as the

concentration of the polyphenols is increased.

Attempts to crystallize the protein with the polyphe-

nols have been unsuccessful so far. Soaking experiments

of RNase A crystals with 100 mM solutions of the poly-

phenols as well as RNase A crystals grown in the presence

of either 5 or 10 mM of polyphenols did not show any

binding of the compounds to RNase A (Leonidas DD.

Personal communication.) Hence protein-ligand docking

studies were performed to obtain some insight into the

amino acid residues involved in the interactions of the

polyphenols. Model development for the rational design

of proteasome inhibitors based on docking studies of tea

polyphenols has been reported earlier in this journal.44

Our earlier studies with synthetic inhibitors of RNase A

revealed docking poses similar to those obtained from

structural studies (RMSD 0.47 A). Interestingly, these

inhibitors were competitive in nature and were found to

dock to the active site, which was further confirmed

from structural studies (PDB entry 2G8R).12,45 The

mode of binding observed in the docking experiments

for the polyphenols with RNase A is also correlated with

the experimental studies. The docking poses shown in

Figure 10 reveal that the polyphenols do not dock to the

ribonucleolytic site of RNase A (1FS3), which is in agree-

ment with our experimental observations. This could

also be the probable reason why crystallization of the

protein ligand complexes has not been possible. From

the docking results, we find that a nonregular region of

the protein comprising a turn and a loop encompassing

residues 34–39 is primarily involved in the interactions.

An inspection of the structure of the protein revealed

that Asn 34 and Asp 38 possess helical /,w angles that

can act as turn elements in the polypeptide chain.46 Asn

34 lies on the right hand side of the Ramachandran plot

and Asp 38 in the a-helical region. It may also be men-

tioned that the docking poses indicate interactions with

the backbone atoms of the nonregular region comprising

residues 34–39. This substantiates the observation made

in the difference spectra that the increase in intensity

between 1544 and 1550 cm�1 is due to the interaction of

the polyphenols with the backbone of the protein. Possi-

ble hydrogen bonding distances for the polyphenols with

the protein are given in Table II. For EC and EGC the

oxygen atoms of the A ring are involved in the interac-

tion whereas in ECG and EGCG the A ring is oriented

away from the protein and interactions are preferred

with the gallate moiety (ring D). In case of ECG and

EGCG docked to RNase A, the orientation of the D rings

are reversed. In ECG Arg 10 is close to the ester moiety

whereas the B ring oxygen atoms interact with Glu 2.

The D ring ��OH groups of ECG interact with Arg 39.

For EGCG, the D ring is close to Glu 2 and the B ring is

oriented such that it interacts with the side chains of Arg

10 and Arg 39. Lys 7 and Arg 10 are part of the cation

cluster of the binding subsite adjacent to the active site

and are known play an indirect role in C>p hydrolysis.11

The docking suggestions indicate interactions with Arg

10, which corroborates the noncompetitive nature of in-

hibition observed experimentally.

To further probe the nature of the ligand binding

pockets we calculated the change in ASA of the interact-

ing amino acid residues in the complexed and uncom-

plexed forms. The results of these calculations are given

in Table III. As expected there is no change in the ASA

of the catalytic residues, His 12 and His 119 that form

part of the P1 binding site. There is only a slight change

in ASA of Lys 41 for all the ligands. This indicates that

Figure 10Stereoview of the docked conformations of the polyphenols with Ribonuclease A

(1FS3). The protein conformations have been kept identical for comparison.

His12 and His 119 have been marked to indicate the location of the catalytic

site. Possible hydrogen bonds are shown as dashed lines. Panels correspond to A:

EC; B: ECG; C: EGC; and D: EGCG.



the interactions observed with Arg 39 cause Lys 41 to be

less accessible to the solvent, which in turn may inhibit

the catalytic activity of RNase A indirectly. For EC, Asp

38 and Arg 39 lose maximum ASA on complex forma-

tion. The residues losing the most ASA on complex for-

mation with ECG are Asn 34 and Lys 37. For EGC, Arg

10, Asn 34, and Arg 39 seem to the most affected. For

EGCG which was found to inhibit the enzymatic activity

of RNase A with an inhibition constant of 81 lM, in

addition to changes observed at Lys 37 and Asp 38, addi-

tional changes in ASA are found at Lys 1. Lys 1 from the

B3 subsite of RNase A, which has purine specificity, (Fig.

2) plays a role in the hydrolysis of RNA.47,48 For RNA

the recognition preferentially occurs through B2R2 P1

(the main binding site). The phosphate of the second

nucleoside binds to P2 while the base and ribose interact

Table IIDistances (in A) Between Polar Neighbors of RNase A (PDB code 1FS3) and the Polyphenols

RNase A EC ECG EGC EGCG

Lys 1 Nf 2.96 [D(30-O)]Glu 2 Oe1 2.94 [B(30-O)] 2.79 [A(5-O)] 2.70 [D(30-O)]Glu 2 N 2.81 [D(40-O)]Lys 7 Nf 3.00 [A(7-O)]Arg 10 Nh1 2.70 [D(10-C¼O)] 2.59 [C(3-O)] 3.04 [B(50-O)]Arg 10 Nh2 2.65 [A(5-O)] 2.27 [D(10-C¼O)] 3.13 [C(1-O)]Gln 11 Oe1 2.40 [A(7-O)] 3.13 [B(40-O)]

3.25 [B(50-O)]Asn 34 Nd2 3.08 [A(7-O)]Asn 34 O 2.36[A(5-O)] 3.06 [D(10-C¼O)] 2.74 [C(1-O)]

3.39 [C(3-O)]Leu 35 O 3.06 [D(40-O)] 2.96 [B(40-O)] 3.05 [B(30-O)]Lys 37 N 2.73 [D(50-O)] 2.95 [B(50-O)]Asp 38 Od1 2.53 [B(30-O)]Asp 38 N 2.84 [D(50-O)] 2.48 [B(50-O)]Arg 39 Nh1 2.65 [C(1-O)] 2.88 [D(30-O)] 2.55 [B(30-O)] 2.72 [B(30-O)]

2.65 [D(40-O)] 2.90 [B(40-O)]Arg 39 Ne 3.16 [D(40-O)] 3.09 [B(40-O)]Arg 39 N 3.27 [D(50-O)] 2.87 [B(50-O)]Arg 39 O 2.89 [D(50-O)] 2.38 [B(40-O)]

2.82 [D(40-O)] 2.91 [B(50-O)]Lys 41 N 3.39 [B(40-O)]

Table IIIChange in Accessible Surface Area (DASA) in A2 of Interacting Residues of RNase A (Uncomplexed), and its Complexes With EC, ECG, EGC, and EGCG

Amino acid residues

DASA

Location of the residueEC ECG EGC EGCG

Lys 1 0.00 21.27 0.00 33.61 UnorderedGlu 2 0.00 17.85 22.97 16.99 UnorderedLys 7 16.59 21.33 12.24 20.54 H1Arg 10 7.89 25.63 37.17 16.71 H1His 12 0.00 0.00 0.00 0.00 H1Asn 34 9.69 35.93 32.35 21.56 T2Leu 35 12.53 12.53 12.53 12.53 Loop between T2 and S1Lys 37 17.10 39.98 13.27 34.19 Loop between T2 and S1Asp 38 36.03 19.43 8.72 32.35 Loop between T2 and S1Arg 39 39.49 24.08 25.43 24.66 Loop between T2 and S1Lys 41 7.50 3.74 3.63 3.90 UnorderedHis 119 0.00 0.00 0.00 0.00 S7SDASA 153.35 227.13 173.22 222.48SDASA/Mol.Wt 0.53 0.51 0.57 0.49Kd (lM) (PEARLS) 47.10 0.48 15.10 0.32Kd (klM) (CD) 141 � 9 80 � 3 112 � 12 40 � 1Kd (lM) (UV) 150 � 0.32 79 � 2.7 127 � 0.28 49 � 0.68

Total change in ASA (SDASA) of RNase A complexes and their calculated dissociation constants as obtained from PEARLS.34

Dissociation constants from experimental studies have also been included.

H: a-Helix; T: Turn; S: b-Sheet.

K.S. Ghosh et al.


with two additional binding sites, B3 and R3 respectively.

Interaction of the D ring 30 oxygen atom of EGCG with

Lys 1 may be a probable cause for the observed noncom-

petitive inhibition. The loss in ASA has been summed for

each polyphenol and normalized with respect to molecu-

lar weight. Calculations for the expected values of the

dissociation constants from docking studies have been

obtained using PEARLS.35 The order of Kd values calcu-

lated using PEARLS follows the same order as obtained

from our experimental studies. We find a direct correla-

tion between the experimental and theoretical data with

correlation constants ranging from 0.83 to 0.99.

The results presented in this paper have been com-

bined and summarized in Figure 11. The specific interac-

tions identified from docking studies that support our

experimental findings imply that the gallate moiety is

indeed a feature worth further investigation in terms of

design potential. The importance of the gallate moiety

has been further investigated by considering metal com-

plexes of ECG and EGCG with Cu(II) where we found

that the complexes also inhibit the ribonucleolytic activ-

ity of the protein.49 Our speculations about the interac-

tions and their effect on the activity of the protein may

be further substantiated by structural studies of the

RNase A–polyphenol complexes which is the subject of a

further study. With knowledge of the specific ligand

binding sites one may thus rationally design derivatives

of the compounds based on green tea that may act as

inhibitors of the proteins. Further studies exploiting the

multiple hydroxyl groups present in the gallate moiety

and their metal chelating properties is currently in pro-

gress. This study is expected to provide guidelines for the

design of more potent inhibitors of RNase A that could

in turn be extrapolated to members of the superfamily

with unusual biological properties.

ACKNOWLEDGMENTS

TKM and JD thank CSIR, New Delhi for fellowships.

REFERENCES

1. Richards FM, Wyckoff HW. The enzymes, 3rd ed. New York: Aca-

demic press; 1971. pp 647–806.

2. Raines RT. Ribonuclease A. Chem Rev 1998;98:1045–1066.

3. Fett JW, Strydom DJ, Lobb RR, Alderman EM, Bethune JL, Riordan

JF, Vallee BL. Isolation and characterization of angiogenin, an

angiogenic protein from human carcinoma cells. Biochemistry

1985;20:5480–5486.

4. Sorrentino S, Glitz DG, Hamann KJ, Loegering DA, Checkel JL,

Gleich GJ. Eosinophil-derived neurotoxin and human liver ribonu-

clease: identity of structure and linkage of neurotoxicity to nuclease

activity. J Biol Chem 1992;267:14859–14865.

5. Matousek J. Ribonucleases and their antitumor activity. Comp Bio-

chem Phys C 2001;129:175–191.

6. Loverix S, Steyaert J. Ribonucleases: from prototypes to therapeutic

targets? Curr Med Chem 2003;10:779–785.

7. Balentine D. Tea and health. Crit Rev Food Sci 1997;37:691–692.

8. Dreostic IE, Wargovich MJ, Yang CS. Inhibition of carcinogenesis

by tea: the evidence from experimental studies. Crit Rev Food Sci

1997;37:761–770.

9. Cai Y, Ma L, Hou L, Zhou B, Yang L, Liu Z. Antioxidant effects of

green tea polyphenols on free radical initiated peroxidation of rat

liver microsomes. Chem Phys Lipids 2002;120:109–117.

10. Khokhar S, Venema DP, Hollman PCH, Dekker M, Jongen WMF. A

RP-HPLC method for the determination of tea catechins. Cancer

Lett 1997;114:171–172.

11. Nogue’s MV, Vilanova M, Cuchillo CM. Bovine pancreatic Ribonu-

clease A as a model of an enzyme with multiple substrate binding

sites. Biochim Biophys Acta 1995;1253:16–24.

12. Maiti TK, De S, Dasgupta S, Pathak T. 30-N-Alkylamino-30-deoxy-

ara-uridines: a new class of potential inhibitors of Ribonuclease A

and angiogenin. Bioorg Med Chem 2006;14:1236–1246.

13. Ghosh KS, Maiti TK, Dasgupta S. Green tea polyphenols as inhibi-

tors of Ribonuclease A. Biochem Biophys Res Commun 2004;325:

807–811.

14. Maiti TK, Ghosh KS, Dasgupta S. Interaction of (�)-epigallocate-

chin-3-gallate with human serum albumin: fluorescence, fourier

transform infrared, circular dichroism and docking studies. Proteins

2006;64:355–362.

15. Roy D, Dutta S, Maity SS, Ghosh S, Ghosh KS, Dasgupta S. Inter-

action of two stereoisomeric flavonoids with serum albumins: per-

turbation of tryptophan residues. Communicated.

16. Russo A, Acharya RK, Shapiro R. Small molecule inhibitors of

RNase A and related enzymes. Methods Enzymol 2001;341:629–648.

17. Sela M, Anfinsen CB. Some spectrophotometric and polarimetric

experiments with ribonuclease. Biochim Biophys Acta 1957;24:229–

235.

18. Anderson DG, Hammes GG, Walz FG. Binding of phosphate

ligands to Ribonuclease A. Biochemistry 1968;7:1637–1645.

19. Pelillo M, Cuvelier ME, Biguzzi B, Toschi TG, Berset C, Lercker G.

Calculation of the molar absorptivity of polyphenols by using liquid

chromatography with diode array detection: the case of carnosic

acid. J Chromatogr A 2004;1023:225–229.

Figure 11Comparison of experimental and theoretical data; the Kd and Kin values for the

experimental studies are in lM. The panels correspond to studies reported in

this paper. Kd values obtained from UV, CD, and docking studies have being

combined.



20. Bond MD. An in vitro binding assay for angiogenin using placental

ribonuclease inhibitor. Anal Biochem 1988;173:166–173.

21. Smith BD, Soellner MB, Raines RT. Potent Inhibition of Ribonuclease

A by Oligo(vinylsulfonic Acid). J Biol Chem 2003;278:20934–20938.

22. Klotz MI, Hunston LD. Properties of graphical representations of

multiple classes of binding sites. Biochemistry 1971;10:3065–3069.

23. Klotz MI. Numbers of receptor sites from Scatchard graphs: facts

and fantasies. Science 1982;217:1247–1249.

24. Gaudreau S, Novetta-dellen A, Neault JF, Diamantoglou S, Tajmir-

riahi HA. 30-Azido-30-deoxythymidine binding to Ribonuclease A:

model for drug-protein interaction. Biopolymers 2003;72:435–441.

25. Whitmore L, Wallace BA. DICHROWEB, an online server for pro-

tein secondary structure analyses from circular dichroism spectro-

scopic data. Nucleic Acids Res 2004;32:W668–W673.

26. Greenfield NJ. Methods to estimate the conformation of proteins

and polypeptides from circular dichroism data. Anal Biochem 1996;

235:1–10.

27. Zhai X, Srivastava A, Drummond DC, Daleke D, Lentz BR. Phos-

phatidylserine binding alters the conformation and specifically

enhances the cofactor activity of bovine factor Va. Biochemistry

2002;41:5675–5684.

28. Byler DM, Susi H. Examination of the secondary structure of pro-

teins by deconvolved FTIR spectra. Biopolymers 1986;25:469–487.

29. Dong AC, Huang P, Caughey WS. Protein secondary structures in

water from second-derivative amide I infrared spectra. Biochemistry

1990;29:3303–3308.

30. Susi H, Byler DM. Resolution-enhanced Fourier transform infrared

spectroscopy of enzymes. Methods Enzymol 1986;130:290–311.

31. Chatani E, Hayashi R, Moriyama H, Ueki T. Conformational strict-

ness required for maximum activity and stability of bovine pancre-

atic Ribonuclease A as revealed by crystallographic study of three

Phe120 mutants at 1.4 A resolution. Protein Sci 2002;11:72–81.

32. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig

H, Shindyalov IN, Bourne PE. The Protein Data Bank. Nucleic

Acids Res 2000;28:235–242.

33. Rarey M, Kramer B, Lengauer T, Klebe G. A fast flexible docking

method using an incremental constraction algorithm. J Mol Biol

1996;261:470–489.

34. DeLano WL. The PyMOL molecular graphics system. San Carlos,

CA: DeLano Scientific, 2004. Available at http://pymol.sourceforge.

net/

35. Han LY, Lin HH, Li ZR, Zheng CJ, Cao ZW, Xie B, Chen YZ.

PEARLS: program for energetic analysis of receptor-ligand system. J

Chem Inf Model 2006;46:445–450.

36. Hubbard SJ, Thornton JM. ‘‘NACCESS’’ Computer Program. Depart-

ment of Biochemistry and Molecular Biology, University College,

London, 1993.

37. Seshardi S, Oberg KA, Fink AL. Thermally denatured Ribonuclease

A retains secondary structure As shown by FTIR. Biochemistry

1994;33:1351–1355.

38. Torrent J, Rubens P, Ribo M, Heremans K, Vilanova M. Pressure

versus temperature unfolding of Ribonuclease A: An FTIR spectro-

scopic characterization of 10 variants at the carboxy-terminal site.

Protein Sci 2001;10:725–734.

39. Krimm S, Bandekar J. Vibrational spectroscopy and conformation

of peptides, polypeptides and proteins. Adv Protein Chem 1986;38:

181–364.

40. Zhang Y, Rock CO. Evaluation of epigallocatechin gallate and

related plant polyphenols as inhibitors of the FabG and FabI reduc-

tases of bacterial type II fatty-acid synthase. J Biol Chem 2004;279:

30994–31001.

41. Chung JY, Huang C, Meng X, Dong Z, Yang CS. Inhibition of acti-

vator protein 1 activity and cell growth by purified green tea and

black tea polyphenols in H-ras-transformed cells: structure-activity

relationship and mechanisms involved. Cancer Res 1999;59:4610–

4617.

42. Lee WJ, Shim JY, Zhu BT. Mechanisms for the inhibition of DNA

methyltransferases by tea catechins and bioflavonoids. Mol Pharma-

col 2005;68:1018–1030.

43. Navarro-Peran E, Cabezas-Herrera J, Hiner ANP, Sadunishvili T,

Garca-Canovas F, Rodrıguez-Lopez JN. Kinetics of the inhibition of

bovine liver dihydrofolate reductase by tea catechins: origin of

slow-binding inhibition and pH studies. Biochemistry 2005;44:

7512–7525.

44. Smith DM, Daniel KG, Wang Z, Guida WC, Chan T, Dou QP.

Docking studies and model development of tea polyphenol protea-

some inhibitors: applications to rational drug design. Proteins 2004;

54:58–70.

45. Leonidas DD, Maiti TK, Samanta A, Dasgupta S, Pathak T, Zogra-

phos SE, Oikonomakos NG. The binding of 30-N-piperidine-4-car-

boxyl-30-deoxyara-uridine to Ribonuclease A in the crystal. Bioorg

Med Chem 2006;14:6055–6064.

46. Pal M, Dasgupta S. The nature of the turn in omega loops of pro-

teins. Proteins 2003;51:591–606.

47. Pare’s X, Llorens R, Arus C, Cuchillo CM. The reaction of bovine

pancreatic Ribonuclease A with 6-chloropurineriboside 50-mono-

phosphate. Evidence on the existence of a phosphate-binding sub-

site. Eur J Biochem 1980;105:571–579.

48. Alonso J, Nogues MV, Cuchillo CM. Modification of bovine pan-

creatic Ribonuclease A with 6-chloropurine riboside. Arch Biochem

Biophys 1986;246:681–689.

49. Ghosh KS, Maiti TK, Mandal A, Dasgupta S. Copper complexes of

(�)-epicatechin gallate and (�)-epigallocatechin gallate act as

inhibitors of Ribonuclease A. FEBS Lett 2006;580:4703–4708.

K.S. Ghosh et al.


Inhibition of Ribonuclease A by polyphenols present in green tea

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