Binding of naturally occurring hydroxycinnamic acids to bovine serum albumin

doi:10.4236/ns.2010.26071

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Vol.2, No.6, 563-570 (2010) Natural Science

http://dx.doi.org/10.4236/ns.2010.26071

Binding of naturally occurring hydroxycinnamic acids to

bovine serum albumin

Lucie Trnková1,2, Iva Boušová1*, Vladimír Kubíček1, Jaroslav Dršata1,2

1Charles University in Prague, Faculty of Pharmacy, Hradec Králové, Czech Republic; *Corresponding Author: [email protected]

2University of Hradec Králové, Faculty of Education, Hradec Králové, Czech Republic

Received 22 February 2010; revised 12 April 2010; accepted 13 May 2010.

ABSTRACT

Hydroxycinnamic acids (HCAs) possess numer-

ous biological effects including antioxidant, an-

tiallergic, antimicrobial, and immunomodulatory

activities and due to these properties are widely

used in folk medicine. Nevertheless, they can

interact with protein molecules and cause some

structural and functional changes. The possib-

ility of HCAs binding to bovine serum albumin

(BSA) under physiological conditions was inve-

stigated by the UV-VIS absorption spectroscopy

and fluorescence quenching method. Apart

from rosmarinic acid, all tested HCAs quenched

tryptophan fluorescence of BSA in the studied

range of concentrations (0-20 µM) mainly by

static quenching mechanism (formation of non-

fluorescent HCA-BSA complexes). The binding

constants, number of binding sites and free en-

ergy changes were determined. The binding

affinities of HCAs were ranked in the order:

chlorogenic acid > sinapic acid ≥ caffeic acid >

ferulic acid > o-coumaric acid > p-coumaric acid

≥ m-coumaric acid, which was confirmed by

spectral overlaps of BSA emission spectrum

with absorption spectrum of HCA. All free en-

ergy changes possessed negative sign indicat-

ing the spontaneity of HCA-BSA interaction.

Keywords: Bovine Serum Albumin;

Hydroxycinnamic Acid; Fluorescence Quenching;

Protein-Ligand Binding

1. INTRODUCTION

Recently, considerable attention has been focused on the

study of the interaction between small molecules (drugs)

and biological macromolecules (e.g. proteins), especially

discussing the thermodynamic quality, binding force qu-

ality, and mechanism of interactions [1-3]. These studies

play crucial role in promoting research on proteins be-

cause they can provide useful information for study of

pharmacological and biological effects of drugs as well

as conformational changes of proteins caused by drugs.

Serum albumin is one of the most abundant proteins

in circulatory system of a wide variety of organisms and

one of the most extensively studied proteins at all [4,5].

Bovine serum albumin (BSA) consists of 583 amino

acids in a single polypeptide chain cross-linked with 17

disulfide bonds. It is composed of three homologous

domains (I-III), each of which comprises of two subdo-

mains (A and B). BSA has two tryptophan residues,

which significantly contribute to the intrinsic fluores-

cence of this protein: Trp-134 is located near the surface

in domain IB and Trp-212 is buried in a hydrophobic

(non-polar) pocket in the internal part of domain IIA [6].

HSA differs from BSA by 24% of primary structure and

the most important difference from spectroscopic point

of view seems to be presence of only one tryptophan

residue (Trp-214) in its molecule [4,5]. Serum albumin

possesses a wide range of physiological functions in-

volving the binding, transport and deposition of many

endogenous and exogenous ligands present in blood cir-

culation [4,7]. Perhaps, its most outstanding property is

the ability to bind a variety of ligands. It is well known

that many drugs bind to serum albumin and their effec-

tiveness depends on the binding ability [5,8]. On the

other hand, drugs can cause various changes in protein

conformation influencing its physiological function and

such impaired proteins may be consequently pathologi-

cally accumulated in body tissues.

Plant polyphenols represent a heterogeneous group of

natural compounds with one or more hydroxyl groups

attached to the benzene ring. These substances possess

several important physiological roles in plants, such as

defense against herbivores and pathogens, pigmentation,

and attraction of pollinating insects [9]. The most widely

distributed polyphenolic compounds in plant tissues are

hydroxycinnamic acids. Some of the most common

L. Trnková et al. / Natural Science 2 (2010) 563-570

564

naturally occurring HCAs are p-coumaric acid, ferulic

acid, sinapic acid, and caffeic acid. These can be found

in a free form but more often in various conjugated

forms resulting from enzymatic hydroxylation, O-glyco-

sylation, O-methylation or esterification [10,11]. Hy-

droxycinnamic acids have been reported to possess an-

timicrobial, antiallergic and anti-inflammatory activities,

as well as antimutagenic and immunomodulatory effects

[12,13] and due to these properties are widely used in

folk medicine. They exert also antioxidant and anti-radi-

cal activities [14-16]. Their biological effects are strong-

ly dependent on the number and position of hydroxyl

groups [15]. Just the presence of hydroxyl groups sug-

gests the possibility of HCAs binding with molecules of

proteins.

Spectroscopic techniques, such as ultraviolet-visible

(UV-VIS) absorption spectroscopy [17], fluorescence

spectroscopy [1,6], circular dichroism [18], and attenu-

ated total reflectance-Fourier transform infrared spec-

troscopy [19] are commonly used tools to observe con-

formational changes in structure of proteins because of

non-destructive measurements of substances in low

concentration under physiological conditions, high sen-

sitivity, rapidity and ease of implementation. Fluores-

cence spectroscopy is widely used to study mechanism

of the binding between drugs and plasma proteins [6].

Nowadays, the investigation of the binding of natu-

rally occurring polyphenolic compounds with various

proteins attracts a great attention. Several spectroscopic

studies on the interaction between bovine serum albumin

and cinnamic acid [1], ferulic acid [20,21], chlorogenic

acid [19,22] or various flavonoids [18,19,24] have been

published. Also several studies dealing with the interac-

tion of human serum albumin (HSA) with derivatives of

cinnamic acid or flavonoids have been carried out

[2,3,17,25-29]. The data obtained in several studies con-

cerning the HCAs-BSA binding parameters (especially

caffeic, chlorogenic, and ferulic acid) are hardly compa-

rable because these studies were performed under vari-

ous conditions (e.g. pH, temperature).

The aim of the presented work was to study interac-

tions of eight naturally occurring hydroxycinnamic acids

with bovine serum albumin under physiological condi-

tions (pH 7.4; 37℃) using UV-VIS absorption spectros-

copy and fluorescence quenching method, reveal their

character, evaluate structure-activity relationships, and

compare obtained results with already published spec-

troscopic data on interaction of HCAs with BSA or HSA.

The presented study contributes to the current knowl-

edge in the area of protein-ligand binding, particularly

bovine serum albumin-hydroxycinnamic acids interac-

tions.

2. EXPERIMENTAL

2.1. Chemicals

Bovine serum albumin and all hydroxycinnamic acids

were obtained from Sigma-Aldrich GmbH, Germany.

The chemical structures of tested HCAs are presented in

Figure 1. All other chemicals were of analytical grade.

2.2. Preparation of Stock Solutions

Bovine serum albumin was dissolved in sodium phosp-

hate buffer (pH 7.4; 0.1 M; 0.05% sodium azide) in ord-

er to yield solutions with concentration 16 µM and 2 µM

for UV-VIS absorption and fluorescence spectroscopic

experiments, respectively. Individual HCAs were dis-

solved in anhydrous methanol in order to yield 10 mM

stock solutions. BSA and HCA solutions were prepared

fresh before each measurement.

2.3. UV-VIS Absorption Spectroscopy

The UV-VIS spectra were recorded by a spectropho-

tometer Helios β (Spectronic Unicam, United Kingdom)

in a 10 mm quartz cuvette. Quantitative analysis of the

potential interaction between HCAs and BSA was per-

formed by the spectroscopic titration. Briefly, solution of

BSA (16 µM) was titrated in cuvette by successive addi-

tions of HCA solution (10 mM) to a final concentration

of 50 µM (the drug to protein molar ratios were 0; 0.25;

0.5; 0.75; 1.0; 1.25; 1.5; 1.75; 2.0; 2.5; and 3.125) and

the absorption spectra were recorded from 190 to 550

nm at 37℃.

2.4. Fluorescence Spectroscopy

Fluorescence spectra were recorded using a luminescen-

ce spectrometer LS-50B (Perkin Elmer, United Kingdom)

in a 10 mm quartz Suprasil fluorescence cuvette (Hellma,

Figure 1. Chemical structures of tested hydroxycinnamic ac-

ids.

L. Trnková et al. / Natural Science 2 (2010) 563-570

565

Germany). Fluorescence emission spectra of individual

HCA solutions (25 µM) in sodium phosphate buffer (pH

7.4; 0.1 M; 0.05% sodium azide) were recorded. Quan-

titative analysis of the potential interaction between

HCA and BSA was performed by the fluorimetric titra-

tion. Briefly, solution of BSA (2 µM) was titrated in

cuvette by successive additions of HCA solution (10

mM) to a final concentration of 20 µM (the drug to pro-

tein molar ratios were 0; 1.25; 2.5; 3.75; 5.0; 6.25; 7.5;

8.75; and 10.0). Fluorescence emission spectra were

recorded from 300 to 530 nm with excitation at 295 nm

while stirring. The excitation and emission slits were

both set to 5 nm and scanning speed to 200 nm/min. All

experiments were carried out at 37℃. Fluorescence in-

tensity was read at emission wavelength of 350 nm.

2.5. Principles of Fluorescence Quenching

The intensity of fluorescence can be decreased as a res-

ult of a wide variety of processes. Such declines in in-

tensity are called quenching and can be caused by diff-

erent molecular interactions. Dynamic quenching occurs

when the excited-state fluorophore is deactivated upon

contact with some other molecule (quencher) and no

molecule is chemically altered during this process. In the

case of static quenching, a non-fluorescent complex is

formed between molecules of fluorophore and quencher.

The static and dynamic quenching can be distinguished

by the Stern-Volmer analysis. In the case the quenching

is either purely static or dynamic, the plot shows linear

dependence. When the plot of Stern-Volmer diagram

shows exponential dependence, both static and dynamic

quenching are exerted [6].

Dynamic quenching of fluorescence is described by

the well-known Stern-Volmer equation as follows.

][1][1/ 00 QKQkFF Dq 







(1)

In this equation, F0 and F are the fluorescence intensi-

ties of BSA in the absence and presence of quencher,

respectively, [Q] is the quencher concentration, kq is the

bimolecular quenching constant, and τ0 is the lifetime of

the fluorophore in the absence of quencher (τ0 is about

5.10-9 s, as to Reference [6]). The Stern-Volmer quench-

ing constant is given by kqτ0. In case the quenching is

known to be dynamic, the Stern-Volmer constant will be

presented by KD otherwise this constant will be de-

scribed as KS [2,6]. The dynamic quenching depends on

diffusion, while static quenching does not. One criterion

for distinguishing the type of quenching is the fact that

the bimolecular quenching constant kq is larger than dif-

fusion-limited rate constant of the biomolecule (1 × 1010

M-1 · s-1) [6], so the static mechanism is the main reason

that causes the fluorescence quenching (formation of a

complex).

When small molecules bind independently to a set of

equivalent sites on a macromolecule, the equilibrium

between free and bound molecules is given by the equa-

tion:

]log[log/)log( 0QnKFFF b





(2)

where Kb represents binding constant for quencher-pro-

tein interaction, n the number of binding sites per BSA,

and F0, F, have the same meaning as in (1) [2]. The val-

ues of Kb and n could be determined from the intercept

of y-axe and slope by plotting log (F0 – F)/F against log

[Q]. Utilizing Kb, the free energy change (ΔG0) value

can be estimated from the following equation [30]:

KRTGln

0 (3)

The negative sing ΔG0 value confirms the spontaneity

of binding.

3. RESULTS

3.1. Spectroscopic Study of Interactions

between BSA and HCAs

The UV-VIS absorption spectra of BSA titrated by indi-

vidual HCAs solution were monitored in order to ex-

plore the structural changes of BSA caused by addition

of these compounds. Spectral shifts were observed in all

HCA-BSA systems with rising concentration of tested

compound. Six HCAs (p-coumaric, caffeic, ferulic,

sinapic, chlorogenic, and rosmarinic acid) induced move

of Trp absorption maximum (280 nm) to longer wave-

lengths which is called bathochromic (red) shift. Maxi-

mal spectral shift was about 6 nm. The opposite phe-

nomenon (blue shift) occurred in the absorption spec-

trum of BSA after interaction with o- or m-coumaric

acid. Absorbance maximum moved about 5 nm towards

shorter wavelengths in both cases (data not shown).

3.2. Fluorescence Quenching of BSA in the

Presence of HCAs

Quenching of protein intrinsic (tryptophan) fluorescence

was employed for more detailed study of HCA-BSA

binding. Fluorescence emission spectra were recorded

upon excitation at 295 nm, which is attributed to trypto-

phan residues only. Four individual HCAs (o-coumaric,

caffeic, sinapic and ferulic acid) possessed remarkable

emission maximum at 498, 432, 428 and 414 nm, resp-

ectively. The most significant fluorescence intensity

showed o-coumaric acid. Fluorescence intensities of

HCA-BSA systems were read at emission wavelength of

350 nm, where the emission maximum of BSA was lo-

cated. Protein solution was titrated by successive addi-

tions of individual HCA solutions and its fluorescence

L. Trnková et al. / Natural Science 2 (2010) 563-570

566

intensity gradually decreased with rising concentration

of HCA. This may indicate that the microenvironment

around tryptophan residues in BSA molecule was altered

due to the interaction with tested compound. Fluores-

cence emission spectrum of p-coumaric acid-BSA sys-

tem is shown in Figure 2.

Red shifts of tryptophan emission maximum (350 nm)

in dependence on increasing concentration of tested co-

mpounds were found in the case of o-coumaric, sinapic,

chlorogenic, and rosmarinic acid. Emission maximum

was slightly shifted towards longer wavelength by 2 nm

for both o-coumaric and sinapic acid-BSA systems, and

by 4 and 5 nm for chlorogenic and rosmarinic acid-BSA

system, respectively. Other four tested HCAs did not

cause any spectral shift. Emission spectra of o-coumaric,

caffeic, ferulic, and sinapic acid involved isosbestic

point, which might indicate that studied compounds exist

both in bound and free form that are in equilibrium. The

bound form exerts fluorescence whereas the unbound

form does not (Figure 3).

It was noticed that emission spectra of these HCA-

BSA systems above 430 nm corresponding with emis-

sion spectra of individual HCAs.

The type of fluorescence quenching of HCA-BSA

systems was distinguished using the Stern-Volmer dia-

grams in the range of HCA concentrations of 0-20 µM.

It was confirmed that the static quenching mechanism is

the main reason of protein fluorescence quenching and

consecutively the KS and kq (1) were determined from

the slope of the linear regression curve of F0/F versus [Q]

(Table 1). The representative Stern-Volmer diagram of

o-coumaric-BSA system is displayed in the inset of Fig-

ure 3. Rosmarinic acid exhibited exponential depend-

ence (Figure 4) indicating that both types of quenching

Figure 2. Fluorescence emission spectra of BSA (2 μM) in the

absence and in the presence of increasing amounts of p-coum-

aric acid (0-20 μM) in sodium phosphate buffer (pH 7.4; 0.1 M;

0.05% sodium azide) at λex = 295 nm and 37℃. The inset

shows the corresponding Stern-Volmer diagram of the p-coum-

aric acid-BSA system (λem = 350 nm), R2 = 0.9908.

Figure 3. Fluorescence emission spectra of BSA (2 μM) in the

absence and in the presence of increasing amounts of o-cou-

maric acid (0-20 μM) in sodium phosphate buffer (pH 7.4; 0.1

M; 0.05% sodium azide) at λex = 295 nm and 37℃. The inset

shows the corresponding Stern-Volmer diagram of the o-cou-

maric acid-BSA system (λem = 350 nm), R2 = 0.9921.

Table 1. The Stern-Volmer quenching constants (KS) and the

bimolecular quenching constants (kq) of the system of HCA-

BSA at 37℃.

Tested compound KS ± S.D.a

[× 104 l.mol-1]

kqb ± S.D.a

[× 1013 M-1s-1]

o-coumaric acid 5.95 ± 0.155 1.19 ± 0.031

m-coumaric acid 5.96 ± 0.170 1.19 ± 0.034

p-coumaric acid 7.13 ± 0.190 1.43 ± 0.038

caffeic acid 4.30 ± 0.263 0.86 ± 0.053

ferulic acid 4.86 ± 0.090 0.97 ± 0.018

sinapic acid 4.25 ± 0.209 0.85 ± 0.042

chlorogenic acid 5.36 ± 0.195 1.07 ± 0.039

astandard deviation (mean value of three independent experiments); bkq

= KS/τ0; τ0 = 5.10-9 s. [6]

Figure 4. The Stern-Volmer diagram of the rosmarinic acid-

BSA system obtained by the titration with rosmarinic acid at

37℃. [BSA] = 2 µM, [rosmarinic acid] = 0-20 µM, pH 7.4, λex =

295 nm, λem = 350 nm. The inset shows the corresponding

fluorescence quenching spectra.

L. Trnková et al. / Natural Science 2 (2010) 563-570

567

were asserted and for this reason the KS (kq) of the ros-

marinic acid-BSA system was not determined.

It was noticed that emission spectra of these HCA-

BSA systems above 430 nm corresponding with emis-

sion spectra of individual HCAs.

The type of fluorescence quenching of HCA-BSA

systems was distinguished using the Stern-Volmer dia-

grams in the range of HCA concentrations of 0-20 µM.

It was confirmed that the static quenching mechanism is

the main reason of protein fluorescence quenching and

consecutively the KS and kq (1) were determined from

the slope of the linear regression curve of F0/F versus [Q]

(Table 1). The representative Stern-Volmer diagram of

o-coumaric-BSA system is displayed in the inset of

Figure 3. Rosmarinic acid exhibited exponential depen-

dence (Figure 4) indicating that both types of quenching

were asserted and for this reason the KS (kq) of the ros-

marinic acid-BSA system was not determined.

3.3. Binding Parameters and Binding Mode

of BSA-HCA Complexes

Except for the rosmarinic acid-BSA system, the binding

constants (Kb), binding sites (n), and free energy changes

(∆G0) of all other HCA-BSA systems have been deter-

mined according to the Eqs. (2) and (3), respectively.

Obtained values are presented in Table 2 and represen-

tative example of binding parameters determination for

sinapic acid is displayed in Figure 5. The binding affin-

ity was strongest for chlorogenic acid and ranked in the

order chlorogenic acid > sinapic acid ≥ caffeic acid >

ferulic acid > o-coumaric acid > p-coumaric acid ≥

m-coumaric acid. This order of binding affinities of

HCA to BSA was confirmed also by spectral overlaps of

BSA emission spectrum with absorption spectrum of

individual HCAs. Example of spectral overlap for

chlorogenic acid is shown in Figure 6. The negative

Table 2. The binding constants (Kb), the number of binding sites

(n) and the free energy change (∆G0) of the HCA-BSA system at

37℃ which showed the static quenching mechanism.

Tested

compound

Kb ± S.D.a

[× 105 l.mol-1] n ± S.D.a ΔG0 ± S.D.a

[kJ.mol-1]

o-coumaric acid 3.34 ± 0.720 1.17 ± 0.012 –32.73 ± 0.563

m-coumaric acid 1.31 ± 0.045 1.08 ± 0.002 –30.36 ± 0.088

p-coumaric acid 1.81 ± 0.728 1.10 ± 0.027 –30.98 ± 1.096

caffeic acid 4.16 ± 1.659 1.18 ± 0.011 –33.12 ± 1.088

ferulic acid 3.39 ± 0.802 1.18 ± 0.019 –32.75 ± 0.621

sinapic acid 4.19 ± 0.117 1.21 ± 0.004 –33.36 ± 0.072

chlorogenic acid 6.67 ± 0.837 1.23 ± 0.016 –34.55 ± 0.325

astandard deviation (mean value of three independent experiments)

Figure 5. Logarithmic plots of fluorescence quenching of BSA

treated with different concentrations of sinapic acid at physio-

logical conditions (37℃; pH 7.4). [BSA] = 2 µM, [sinapic acid]

= 0-20 µM, λex = 295 nm and λem = 350 nm. R2 = 0.9988.

Figure 6. Overlap between the fluorescence emission spectrum

of BSA and the absorption spectrum of chlorogenic acid at

physiological conditions (37℃; pH 7.4). [BSA] = 2 µM, [chlo-

rogenic acid] = 2 µM, λex = 295 nm and λem = 350 nm.

value of ΔG0 indicating spontaneous process of HCA-

BSA binding was determined for all studied interactions

(Table 2).

4. DISCUSSION

Red shift in absorption maximum of tryptophan residues

indicates changes in its microenvironment, where the

polypeptide strand of BSA molecule is less extended and

the hydrophobicity around Trp is increased. The con-

formational stability, rigidity, mechanical strength, and

contributions of electrostatic interactions are enhanced

by absence of water in the molecular interior [31]. On

the other hand, blue shift implies that the BSA polypep-

tide strands are more extended and the hydrophocibity of

Trp vicinity is decreased.

The changes in tryptophan microenvironment polarity

are probably related to chemical structure of each HCA,

namely to the number and position of hydroxyl groups.

Hydrophobicity around tryptophan residues rose with

increasing number of hydroxyl groups in the molecule of

L. Trnková et al. / Natural Science 2 (2010) 563-570

568

HCA. However, the position of hydroxyl groups on the

benzene ring seemed to be also important. Only o- and

m-monosubstituted derivatives of cinnamic acid (o-cou-

maric and m-coumaric acid) caused increase in the po-

larity of Trp environment, while p-monosubstituted (p-

coumaric acid), di- and tri-substituted derivatives show-

ed opposite effect.

Emission spectra of BSA were measured using excita-

tion wavelength at 295 nm to ensure that the light caused

excitation only of tryptophan residues. These are highly

susceptible to any change in their local environment re-

sulting in appearance of a substantial spectral shift [6].

In contrast to absorption spectroscopy, the red shift of

Trp emission band is caused by decrease in hydrophobic

property of its environment in protein molecule suggest-

ing that tryptophan residue has been brought to more

hydrophilic environment [3] and protein secondary

structure has been changed [26].

Red shift in emission spectra of four studied hydroxy-

cinnamic acids (o-coumaric, sinapic, chlorogenic, and

rosmarinic acid), which indicated that binding of these

compounds to BSA was associated with changes in the

dielectric environment of at least one of its two indole

rings, was observed. Only slight blue shift and no shift

were noticed during interaction of cinnamic acid with

BSA and HSA, respectively [1,2]. It can be expected that

changes found in emission spectra of HCA-BSA systems

were connected with alterations in microenvironment of

Trp-134 because Trp-212 in BSA molecule is in the

similar position as Trp-214, where cinnamic acid caused

no spectral shift of its emission band. Increasing number

of hydroxyl groups in the molecule of HCA was accom-

panied by decline of hydrophobicity around Trp. The

significant changes in BSA emission spectrum were ob-

served after its interaction with chlorogenic or rosma-

rinic acid. Other authors described red shifts by 3 to 18

nm in HSA spectrum upon reaction with p-coumaric,

ferulic, sinapic, and chlorogenic acid [2,3,25] but the

concentrations applied in these experiments were higher

(up to 150 µM) than those used in presented work.

The natural lifetime for the biological macromolecules

(τ0) is generally given as 10-8 s [1,3]. However, the value

for BSA is more precisely estimated as 5 × 10-9 s [6].

The latter mentioned value was used in this study for

calculation of bimolecular quenching constants (kq)

which reflect efficiency of quenching or the accessibility

of the fluorophores to the quencher. The fact that the

value of kq is higher than the value of the diffu-

sion-limited rate constant of the biomolecule (1 × 1010

M-1 · s-1) is one of the criterions for determination of

static mechanism of quenching [1,6].

Only small but significant differences among kq val-

ues for the tested HCA-BSA systems were observed. All

tested HCAs exerted better quenching effect than cin-

namic acid (2.26 × 1012 M-1 · s-1 for τ0 = 10-8 s) [1]. Ros-

marinic acid caused the most outstanding decrease in

fluorescence intensity of BSA in the range of studied

concentrations but it exhibited exponential dependence

in the Stern-Volmer diagram and thus was not consid-

ered in overall comparison. Coumaric acids showed

stronger quenching activity than the other more substi-

tuted HCAs. This effect was probably dependent upon

the position of hydroxyl group on aromatic ring and

p-position was determined as the most suitable location

of hydroxyl group. The kq value of chlorogenic acid was

similar to kq of coumaric acids and slightly higher than

kq of more substituted derivatives (Table 1). It was

probably caused by the presence of five hydroxyl groups

in its molecule and their spatial arrangement [3]. The

lowest quenching effect was observed for sinapic and

caffeic acid followed by ferulic acid. Presence of meth-

oxy group seemed to be important for quenching activity

too. Ferulic acid showed higher effect than caffeic acid,

while the kq of sinapic acid was slightly lower compared

to caffeic acid. This was perhaps caused by steric hin-

drance in molecule of sinapic acid. The obtained bimol-

ecular quenching constant for chlorogenic acid-BSA

system is in good agreement with data found in literature

[19]. However, published data are inconsistent and even

in one case no quenching by chlorogenic acid was ob-

served [20]. More studies were published for some

HCA-HSA systems. The highest kq was obtained for

chlorogenic acid followed by caffeic and sinapic, while

ferulic and p-coumaric acid possessed lower kq values.

Data obtained for HCA-BSA systems in this study cor-

responded with these findings by other authors [2,3].

In general, the binding constant Kb reflects the power

of ligand-protein association and thus can be used for

comparison of binding affinities of structurally-related

ligands to protein molecule connected with alterations of

its secondary structure. Number of binding sites is an-

other important parameter that contributes to better un-

derstanding of ligand-protein interaction [1-3].

The binding constant Kb for cinnamic acid-BSA sys-

tem mentioned in the literature [1,2] is lower than values

obtained in our experiments for HCA-BSA systems,

which may confirm significance of hydroxyl groups in

the process of binding. Moreover, binding affinity of

cinnamic acid is higher for BSA than for HSA, which

indicates that also binding of HCAs to BSA may be

more pronounced [2]. It was demonstrated that interac-

tion of HCAs with protein molecule depends mainly on

the size and structure of ligand, especially on the number

and position of hydroxyl groups on the aromatic ring

[20,32]. Hydroxyl groups of studied compounds form

hydrogen bonds with amino acid residues in the protein

L. Trnková et al. / Natural Science 2 (2010) 563-570

569

molecule. Another important factor influencing ligand-

protein binding is aromaticity of the ligand molecule

because hydrophobic interactions are formed between

aromatic rings of ligand and amino acid residues [25].

Chlorogenic acid with two aromatic hydroxyls and three

hydroxyls on cyclohexane ring exerted the strongest

binding affinity because this compound can form hy-

drogen bonds with protein more easily than other

less-substituted HCAs, e.g. ferulic acid. Similar results

were published also for ferulic acid-HSA and chloro-

genic acid-HSA system [3]. It is evident that the hy-

droxyls substituted on aromatic ring of the HCAs play

an important role in the changes of BSA secondary

structure. Monosubstituted HCAs possessed the lowest

binding affinities of all HCAs studied. The differences

among binding affinities of sinapic, ferulic and caffeic

acid were not statistically significant.

The binding parameters of chlorogenic and ferulic

acid with bovine serum albumin have been intensively

studied by other authors, while no information about

other HCAs was found in the literature. The value of Kb

reported by Tang et al. [19] for chlorogenic acid-BSA

system using fluorescence quenching method is lower

than value obtained in performed experiments, but the

authors used higher concentrations of chlorogenic acid

and different experimental conditions. Moreover, Rawel

et al. [20,21] reported that chlorogenic acid does not

quench Trp fluorescence in BSA and determined its

binding constant by Hummel-Dreyer/size exclusion

chromatography which showed significantly lower value

of Kb in comparison with results presented by Tang et al.

[19]. Non-covalent interactions of chlorogenic acid with

BSA have been studied by Prigent et al. [33], who re-

ported that these interactions decrease with the increas-

ing temperature while pH and ionic strength had no

pronounced effect. Zhang et al. [34] reported the Kb for

ferulic acid-BSA system determined by affinity capillary

electrophoresis which is in good agreement with our

result. By contrast, Rawel et al. [20] determined binding

constant of ferulic acid-BSA system by fluorescence

quenching method and Hummel-Dreyer/size exclusion

chromatography, where first method gave similar results

to our data and Kb obtained by second method was sig-

nificantly lower. However, several studies dealing with

binding of p-coumaric, caffeic, ferulic, sinapic acid, and

chlorogenic acid with HSA have been published

[2,3,25].

The results showed that the numbers of binding sites

ranged between 1.08 and 1.23 suggesting that one

molecule of BSA was associated with one molecule of

HCA in the drug to protein ratio up to 10 for the tested

HCAs apart. The number of binding sites rose with incre-

asing number of hydroxyl groups in the ligand molecule.

The free enthalpy had negative sign for all studied in-

teractions which indicates the spontaneity of the interac-

tion between BSA and hydroxycinnamic acids. The rela-

tively strong binding enthalpy underlines the stability of

BSA-HCA complexes from the energetic point of view.

These findings are supported by data found in literature

[1,20,21].

5. CONCLUSIONS

Apart from rosmarinic acid, all tested HCAs quenched

tryptophan fluorescence of BSA in the studied range of

concentrations (0-20 µM) mainly by static quenching

mechanism and thus showed the formation of non-fluo-

rescent HCA-BSA complexes. For this reason the ros-

marinic acid-BSA system was not concluded in the

overall assessment of binding affinities. The obtained

results suggest that the binding affinity and number of

binding sites depend on the number and position of hy-

droxyl groups in the molecule of HCA. Disubstituted

and trisubstituted derivatives exhibited stronger binding

affinity than monosubstituted derivatives. The number of

binding sites for all HCAs ranged from 1.08 to 1.23

suggesting that one molecule of BSA associates with one

molecule of HCA. All HCA-BSA interactions were

spontaneous processes based on ΔG0. The results imply

that HCAs could be stored and transported in blood by

serum albumin which may influence their biological and

pharmacological activities. On the other hand, physio-

logical functions of this protein could be altered by

ligand binding.

6. ACKNOWLEDGEMENTS

The presented study was supported by the Specific research 2009 of

the University of Hradec Králové.

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