Evaluation of binding and thermodynamic characteristicsof interactions between a citrus flavonoid hesperitin with proteinand effects of metal ions on binding

Ashwini H. Hegde • B. Sandhya • J. Seetharamappa

Received: 19 August 2010 / Accepted: 3 December 2010 / Published online: 16 December 2010

� Springer Science+Business Media B.V. 2010

Abstract The mechanism of interaction of a non-glyco-

sidic citrus flavonoid, hesperitin (HES) with bovine serum

albumin (BSA) was studied by UV–vis absorption, fluores-

cence, FT-IR, circular dichroism, fluorescence anisotropy

and synchronous fluorescence spectroscopy in phosphate

buffer of pH 7.4. Fluorescence data revealed that the fluo-

rescence quenching of BSA by HES was the result of the

formed complex of HES–BSA. The binding constants and

thermodynamic parameters at four different temperatures,

the location of binding, and the nature of binding force were

determined. The hydrogen bonds interactions were found to

be the predominant intermolecular forces to stabilize the

complex. The conformation of BSA was discussed by syn-

chronous fluorescence and CD methods. The alterations of

protein secondary structure upon complexation with HES

were evident from the gradual decrease in a-helicity. The

distance between the donor (BSA) and acceptor (flavonoid)

was calculated from the fluorescence resonance energy

transfer and found to be 1.978 nm. Common ions viz., Zn2?,

K?, Cu2?, Ni2?, Mn2? and Co2? were found to influence the

binding of flavonoid to protein.

Keywords Non-glycosidic citrus flavonoid �Bovine serum albumin � Secondary structure �Fluorescence anisotropy

Introduction

Flavonoids exist widely in vegetable kingdom as poly-

phenolic compounds. They are the constituents of fruits,

vegetables, nuts, grains, bark, roots, stems, flowers, plant-

derived beverages such as tea, wine, cocoa and fruit juices.

Several beneficial properties have been attributed to these

dietary compounds, including antioxidant, anti-inflamma-

tory, anti-allergic, anti-viral, anti-bacterial, anti-protozoal,

anti-fungal and anti-carcinogenic effects. The best descri-

bed property of almost every group of flavonoids is their

capacity to act as antioxidants able to scavenge free radi-

cals and reactive oxygen species which are associated with

several forms of tissue damage and disease, including

cancer, atherosclerosis, neuronal degeneration, rheumatoid

arthritis as well as with aging [1]. Knowledge of these

small molecule-protein interactions is important to under-

stand how proteins function in biological systems. The

pharmacological actions of drugs are produced in many

instances due to binding to protein molecules. Binding of

drug to plasma proteins or tissue proteins also has phar-

macological implications as it affects rates of absorption,

distribution and excretion, can lead to allergic and toxic

reactions and displace endogenous substances. Therefore,

studies on this aspect can provide information on the

structural features that determine the therapeutic effec-

tiveness of drugs and will be much helpful in clinical

research. In the present study, we have employed the

bovine protein as model target protein because of its lone-

standing interest in the protein community and 76%

structural homology with human serum albumin [2]. It is

composed of a single polypeptide chain with 585 amino

acids and two tryptophan residues located in subdomains

IA and IIA, respectively. BSA has a wide range of physi-

ological functions involving the binding, transport and

A. H. Hegde � B. Sandhya � J. Seetharamappa (&)

Department of Chemistry, Karnatak University,

Dharwad 580003, Karnataka, India

e-mail: jseetharam@yahoo.com

123

Mol Biol Rep (2011) 38:4921–4929

DOI 10.1007/s11033-010-0634-9

delivery of fatty acids, porphyrins, bilirubin, tryptophan,

thyroxin and steroids.

Hesperitin (HES) (30,5,7-trihydroxy-40methoxyflava-

none) (Fig. 1) is the aglycone form of hesperidin, occurring

in citrus fruits. It belongs to group of flavonones having

different biological and pharmacological functions com-

pared with quercetin and other types of flavonoids [3]. It

has antioxidant, anti-inflammatory, anti-allergic, hypolipi-

demic, vasoprotective and anti-carcinogenic actions.

Therefore, the study of interaction between the protein and

HES is imperative. We have employed spectroscopic

methods to understand the mechanism of interaction

between HES and BSA as these methods are sensitive and

relatively easy to use. They have advantages over con-

ventional methods such as affinity and size exclusion

chromatography, equilibrium dialysis, ultrafiltration and

ultracentrifugation, which suffer from lack of sensitivity,

long analysis time or both and use of protein concentrations

far in excess of the dissociation constant for the drug-

protein complex and for drug-protein interaction studies.

Fourier transform infrared (FT-IR), circular dichroism

(CD) and synchronous fluorescence spectroscopy gave

valuable insights on the overall secondary structure of

protein in various conditions [4–6].

Some researchers have reported the interactions of

proteins with flavonoids by spectral techniques [7–10].

However, these studies have not dealt with conformational

changes in secondary structure of protein by CD mea-

surements, fluorescence anisotropy and FTIR, the effects of

metal ions on binding or displacement experiments with

different site probes. Further, no attempt has been made to

investigate the interaction of HES with BSA. Hence, it was

thought worthwhile to investigate the mechanism of

interaction of HES with BSA by different spectroscopic

techniques viz., steady state fluorescence, synchronous

fluorescence, fluorescence anisotropy, UV–visible, circular

dichroism and FT-IR.

Experimental procedures

Reagents

BSA (Fraction V, fatty acid free) and HES were obtained

from Sigma Chemical Company, St. Louis, USA. A stock

solution of 250 lM BSA was prepared in 0.1 M phosphate

buffer of pH 7.4 containing 0.15 M NaCl. Due to limited

solubility of HES in phosphate buffer, the stock solution of

HES (1000 lM) was prepared using 10% DMSO as the

co-solvent. All other materials employed were of analytical

reagent grade and Millipore water was used throughout the

study.

Instrumental

Fluorescence measurements were performed on a spectro-

fluorimeter Model F-7000 (Hitachi, Japan) equipped with a

150 W Xenon lamp and a slit width of 5 nm. The CD

measurements were made on a JASCO-715 spectropolar-

imeter (Tokyo, Japan) using a 0.1 cm cell at 0.2 nm

intervals, with 3 scans averaged for each CD spectrum in

the range of 200–250 nm. The absorption spectra were

recorded on a double beam CARY 50-BIO UV–vis spec-

trophotometer (Varian, Australia) equipped with a 150 W

Xenon lamp and a slit width of 5 nm. FT-IR measurements

were performed at room temperature on a Thermo Nicolet-

5700 FTIR spectrometer (Waltham, MA, USA) equipped

with a germanium attenuated total reflection (ATR)

accessory.

Procedures

Based on preliminary experiments, the concentration of

BSA was kept constant at 2.5 lM while that of HES was

varied from 0 to 22.5 lM in both fluorescence and UV

absorption studies. Fluorescence spectra were recorded at

289, 294, 299 and 304 K in the range of 300–500 nm upon

excitation at 296 nm. Absorption spectra were noted down

in the range of 250–370 nm at room temperature. The CD

spectra of protein–flavonoid mixtures (1:0, 1:1 and 1:2)

were recorded in the range of 200–250 nm. The FT-IR

spectra of BSA in the presence and absence of the drug

were recorded at room temperature in the range of

1530–1670 cm-1. The displacement experiments were

performed using different site probes viz., warfarin, ibu-

profen and digitoxin for site I, II and III, respectively by

keeping the concentration of protein and probe constant

(2.5 lM each). The fluorescence spectra of flavonoid–BSA

system were recorded in presence of cations viz Ca2?,

Ni2?, Cu2?, Co2? and Zn2? upon excitation at 296 nm.

The overall concentration of BSA and common ions were

fixed at 2.5 lM.

O

OH

HO

O

OH

OCH3

Fig. 1 Strucure of hesperitin

4922 Mol Biol Rep (2011) 38:4921–4929

123

Results and discussion

Analysis of fluorescence quenching of serum

albumin by HES

Fluorescence measurements give information about the

molecular environment in a vicinity of the chromophore

molecules. By measuring the intrinsic fluorescence of BSA,

the accessibility of protein to the quencher can be esti-

mated. This information helps to predict the binding

mechanism of HES to BSA. Figure 2 shows the quenching

of fluorescence intensity of BSA upon the addition of a

series of concentrations of HES in physiological condition

(pH 7.4) at kex = 296 nm. The quenching mechanism is

usually classified as either dynamic quenching or static

quenching. The collisional quenching or dynamic

quenching results from collision between the fluorophore

and a quencher while static quenching results from non-

fluorescent complex formation in ground state of the

fluorophore.

HES caused the concentration dependent quenching of

the intrinsic fluorescence of BSA without changing the

emission maximum and shape of the peaks. The fluores-

cence quenching data were analyzed using the Stern–

Volmer equation [11] shown below:

F0=F ¼ 1þ Kqs0 Q½ � ¼ 1þ KSV Q½ � ð1Þ

where F and F0 are the steady-state fluorescence intensities

in the presence and absence of quencher, respectively. Kq is

the quenching rate constant, s0 is the average lifetime of the

protein in the absence of the drug and its value was reported

to be 10-8 s [12], KSV is the Stern–Volmer quenching

constant and [Q] is the concentration of quencher

(flavonoid). The plot of F0/F versus [Q] (Fig. 3a) yielded

the straight line. However, it did not define the quenching

type and additional information was required for this

determination. In order to determine the quenching type, the

effect of temperature on the interaction of HES with BSA

was studied. The KSV values increase with increase in

temperature for dynamic quenching while it is reverse for

static quenching. The values of KSV were observed to be

decreased with increase in temperature in the present

study. Further, the maximum scatter collisional quenching

constant of various quenchers with the biopolymer is

reported to be 2.0 9 1010 l mol-1 s-1 [12]. The higher

values of rate constants of the protein quenching procedure

initiated by HES indicated that the quenching was not

initiated by dynamic quenching, but probably by static

quenching resulting from the formation of HES–BSA

complex. For static quenching mechanism, the data were

analyzed using the modified Stern–Volmer equation shown

below:

F0= F0 � Fð Þ ¼ 1=faKa Q½ � þ 1=fa ð2Þ

where F and F0 are the fluorescence intensities of protein

with and without HES, respectively; Ka is the modified

Stern–Volmer association constant, [Q] is the concentra-

tion of quencher and fa is the fraction of the initial

0

50

100

150

200

250

300 350 400 450 500

Wavelength (nm)

Inte

nsit

y

1

10

Fig. 2 Quenching of fluorescence intensity of BSA (2.5 lM) upon

the addition of increasing amounts of HES: (1) 0, (2) 2.5, (3) 5, (4)

7.5, (5) 10, (6) 12.5, (7) 15, (8) 17.5, (9) 20 and (10) 22.5 lM

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

0.00E+00 2.00E-06 4.00E-06 6.00E-06 8.00E-06 1.00E-05 1.20E-05

[Q]

F 0/F

0

2

4

6

8

10

12

14

0.00E+00 2.00E+05 4.00E+05 6.00E+05 8.00E+05 1.00E+06

1/[Q]

F0/

F0-

F

a

b

Fig. 3 a The Stern–Volmer plot for binding of HES with BSA at

289 K (circle), 293 K (triangle), 299 (square) and 305 K (diamond)

and b Modified Stern–Volmer plot at 289 K (diamond), 293 K

(square), 299 (triangle) and 305 K (circle). Decrease in KSV values

with increase in temperature indicate the presence of static quenching

in the binding of HES to BSA

Mol Biol Rep (2011) 38:4921–4929 4923

123

fluorescence that is accessible to quencher. The plot of

F0/(F0 - F) versus 1/[Q] (Fig. 3b) yielded fa-1 as the

intercept on y axis and (fa K)-1 as the slope. The corre-

sponding results are shown in Table 1.

Analysis of binding equilibria

For static quenching interaction, the binding constant and

number of binding sites can be obtained from the equation

shown below [13]:

logðF0 � FÞ=F ¼ log Kþ n log Q½ � ð3Þ

where K and n are the binding constant and the number of

binding sites, respectively. Thus, the plot of log (F0 - F)/

F versus log [Q] (Figure not shown) yielded the values of

K and n (Table 1). From the values of n, it may be inferred

that there is one independent class of binding sites on BSA

for HES.

Binding mode

The thermodynamic parameters for protein interactions can

be accounted for the main forces contributing to protein

stability. The action force between a small molecule and a

biomolecule may include hydrogen bonding, van der Waals

force, electrostatic force and hydrophobic interactions [14].

Therefore, the thermodynamic parameters which depend

on temperatures were evaluated to further characterize the

acting forces between BSA and HES. The thermodynamic

parameters, enthalpy change (DH�), entropy change (DS�)

and free energy change (DG�) were evaluated using the

following equations:

logK ¼ �DH�=2:303RT þ DS�=2:303R ð4ÞDG� ¼ DH� � TDS� ð5Þ

where K and R are the binding constant and gas constant,

respectively. The plot of log K versus 1/T enabled the

determination of values of DH� and DS� (Figures not

shown). The corresponding results are shown in Table 1.

The positive entropy change occurs when the water

molecules that are arranged in an orderly fashion around

the ligand and the protein acquires a more random con-

figuration as a result of hydrophobic interactions. The

negative DG� meant that the binding process was sponta-

neous. In the present study, the negative DH� value may

not be attributed to electrostatic interactions as higher

negative DH� value would be observed whenever there is

hydrogen bonding [15, 16]. So, we propose that the

hydrophobic forces and hydrogen bonding play a signifi-

cant role in the binding of HES to BSA.

Binding site

Sudlow et al. [17] have suggested two distinct binding sites

on protein, sites I and II. Site I of BSA shows affinity for

warfarin, phenylbutazone etc., while site II for ibuprofen,

flufenamic acid etc. Digitoxin binding is independent of

sites I and II [18, 19]. In order to determine the specificity

of the HES binding, displacement experiments were per-

formed with warfarin, ibuprofen and digitoxin as per

Sudlow’s classification of the binding sites. The binding

constant values of HES–BSA were calculated to be

0.98 9 105, 1.24 9 105 and 1.32 9 105 M-1 in presence

of warfarin, ibuprofen and digitoxin, respectively. It was

evident that the warfarin (site I) showed a significant dis-

placement of flavonoid suggesting that both HES and

warfarin compete for the same binding site on BSA. Fur-

ther, due to the similarity of the structures of HES and

warfarin [20], we consider that the HES most likely binds

at the site I located in subdomain II A near Trp 214.

Energy transfer between flavonoid and protein

Fluorescence resonance energy transfer (FRET) is a dis-

tance dependent interaction between the different elec-

tronic excited states of fluorescent molecules in which

excitation energy is transferred from one molecular system

(donor) to another molecular system (acceptor) without

emission of a photon from the former molecular system.

The overlap of UV absorption spectrum of HES with

fluorescence emission spectrum of BSA is shown in Fig. 4.

The spectral studies suggested that HES interacted with

BSA. The distance, r between the tryptophan in BSA and

the bound flavonoid could be determined using FRET [21].

However, the r value calculated for flavonoid–BSA system

is actually the average distance between the bound

Table 1 Binding parameters of HES–BSA interaction

T(K) Modified Stern–Volmer

constant, (L mol-1)

R2 K (L mol-1) n DG� (KJ mol-1) DH� (KJ mol-1) DS� (J mol-1 K-1)

289 1.39 9 105 0.9993 5.96 9 105 1.13 -31.96

294 1.06 9 105 0.9988 2.5 9 105 1.07 -30.39 -164.568 0.4568

299 0.97 9 105 0.9979 1.29 9 105 1.03 -29.26

305 0.58 9 105 0.9822 0.15 9 105 0.87 -24.31

4924 Mol Biol Rep (2011) 38:4921–4929

123

flavonoid and the tryptophan residue. The efficiency of

energy transfer, E, is calculated using the equation:

E ¼ 1� F0=F ¼ R60=R6

0 þ r6 ð6Þ

where F and F0 are the fluorescence intensities of BSA in

presence and absence of HES, and R0 is the critical

distance when the transfer efficiency is 50%. It is

calculated as follows:

R60 ¼ 8:8 � 10�25 k2 N�4 U J ð7Þ

where k2 is the spatial orientation factor of dipole, N is the

refractive index of the medium, U is the fluorescence

quantum yield of donor and J is the overlap integral of

fluorescence emission spectrum of donor and absorption

spectrum of the acceptor. J is determined using the

following equation:

J ¼ RFðkÞeðkÞk4Dk=RFðkÞDk ð8Þ

where F(k) is the fluorescence intensity of the donor of

wavelength k, and e(k) is the molar absorption coefficient of

the acceptor at wavelength k. In the present case,k2 = 2/3,

N = 1.36 and U = 0.15 [10]. From Eqs. 6–8, we obtained

that J = 12.753 9 10-16 cm3 L mol-1, R0 = 1.86 nm, E =

0.4146 and r = 1.978 nm. The donor to acceptor distance,

r \ 8 nm [22] indicated that the energy transfer from BSA

to HES occurred with high probability.

The effect of metal ions on the binding

of flavonoid to protein

The binding reaction between a molecule and the serum

albumin would be influenced by some metal ions both in

vitro and in vivo. BSA has high affinity metal-binding site

at N-terminus [23]. So, the binding aspect of HES–BSA

was studied in presence of common ions viz., K?, Co2?,

Cu2?, Ni2? and Mn2? at 298 K. As evident from Table 2,

the binding constant values of HES–BSA decreased in

presence of these ions indicating the shortening of storage

time of drug in blood plasma. All these results indicated

that the quenching efficiency of drug was influenced by the

metal ions.

Effect of Zn2? on flavonoid–protein interaction

In blood plasma, zinc is bound and transported by albumin.

The concentration of zinc in blood plasma stays relatively

constant regardless of zinc intake. Zinc is found in nearly

100 specific enzymes. It is typically the second most

abundant transition metal in organisms. A study estimated

that about 10% of human proteins (2800) potentially bind

zinc, in addition to hundreds which transport and traffic

zinc. The metal ion also has a flexible coordination

geometry, which allows proteins using it to rapidly shift

conformations to perform biological reactions. In the

present study, the K value of HES–BSA decreased in

presence of different concentrations of Zn2? as shown in

Table 3. Therefore, the complexation of Zn2? with protein

reduced the binding affinity of HES to BSA and increased

the concentration of free HES. In other words, it shortened

the retention time HES in blood and facilitated the easy

release of the drug.

Alterations of protein secondary structure induced

by drug binding

UV–visible absorption studies

Generally, bathochromic and hypochromic shifts are

observed in the absorption spectra of proteins if they bind

to small molecules. The absorption maximum of free BSA

at 279 nm was found to be shifted to 285 nm (Fig. 5). This

slight red shift could be attributed to formation of complex

between protein and flavonoid [24]. Further, the UV

absorption intensity of BSA increased with increase in the

concentration of flavonoid.

0

50

100

150

200

250

300 350 400 450

Wavelength (nm)

Flu

ores

cenc

e In

tens

ity

0

0.05

0.1

0.15

0.2

Abs

orba

nce

x

y

Fig. 4 The overlap of the fluorescence spectrum of BSA [x] with

absorption spectrum of HES [y]. The distance, r between the

tryptophan in BSA (donor) and the bound flavonoid (acceptor) could

be determined with the aid of overlap of emission spectrum with

absorption spectrum

Table 2 Effects of common ions on binding of BSA with HES

System Association constant (M-1)

Without metal ion 1.29 ± 0.012 9 105

K? 1.17 ± 0.023 9 105

Co2? 1.20 ± 0.024 9 105

Cu2? 1.14 ± 0.015 9 105

Ni2? 0.98 ± 0.024 9 105

Mn2? 1.25 ± 0.026 9 105

Mol Biol Rep (2011) 38:4921–4929 4925

123

Synchronous fluorescence spectra

The synchronous fluorescence spectra give information

about the molecular environment in the vicinity of chro-

mosphere molecules [25]. These spectra were obtained by

simultaneously scanning excitation and emission mono-

chromators. When the Dk between excitation wavelength

and emission wavelength was 15 nm, the synchronous

fluorescence indicated characteristics of tyrosine residues

and when Dk was 60 nm, it provided the characteristic

information of tryptophan residues. Synchronous fluores-

cence spectra of BSA in presence of HES obtained when

Dk = 15 and 60 nm are shown in Fig. 6a, b, respectively.

It is apparent from figures that the emission maximum of

tyrosine residues did not exhibit a significant shift while a

marginal red shift (from 288 to 290 nm) was noticed when

Dk = 60. This indicated that the polarity around the tryp-

tophan residues was increased while the hydrophobicity

decreased [26]. However, the microenvironment around the

tyrosine residue was not affected during the binding pro-

cess [27].

Fluorescence anisotropy studies

Fluorescence polarization is a technique specially applied

to study molecular interactions. The anisotropy (r) is

defined as the difference between the fluorescence intensity

emitted parallel and perpendicular (IVV and IVH) divided by

the total intensity. Protein interactions can be detected

when one of the interacting partner is fused to a fluoro-

phore- upon binding of the partner molecule, a larger more

stable complex is formed which will tumble more slowly in

the solution, thus increasing the polarization of the emitted

light. Small fluorescent molecules have low polarization

values while large molecules caused by binding of other

molecules have large values.

In order to ascertain the binding of flavonoid to BSA, we

measured the fluorescence anisotropy of HES in the pres-

ence of different concentrations of BSA and it was calcu-

lated from fluorescence intensity measurement employing

a vertical excitation polarizer and a vertical horizontal

emission polarizer according to the equation as shown

below [28].

r ¼ IVV � GIVHð Þ = IVV þ 2GIVHð Þ ð9Þ

where IVV is the intensity of emitted light measured in the

direction parallel to excitation and IVH is the intensity of

emitted light measured in the direction perpendicular to

excitation. The G factor is the instrument grating correction

Table 3 Effect of Zn2? on binding of BSA with HES

Concentration of zinc Binding constant (K), M-1

0 lM 1.29 9 105

250 lM 1.002 9 105

500 lM 0.93 9 105

1 mM 0.87 9 105

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

250 270 290 310 330 350 370

Wavelength(nm)

Abs

orba

nce

10

1

Fig. 5 Absorption spectra of BSA and HES–BSA system, BSA

concentration was maintained at (1) 2.5 lM and HES concentration

was varied (2) 2.5, (3) 5, (4) 7.5, (5) 10, (6) 12.5, (7) 15, (8) 17.5, (9)

20 and (10) 22.5 lM. These spectra indicate the formation of a

complex between BSA and HES

0

20

40

60

80

100

120

140

280 290 300 310 320 330

Wavelength (nm)

Flu

ores

cenc

e In

tens

ity 1

10

0

400

800

1200

300 320 340 360 380Wavelength (nm)

Flu

ores

cenc

e In

tens

ity 1

10

a

b

Fig. 6 The effect of HES on the synchronous fluorescence spectra of

BSA, a Dk = 15 nm; b Dk = 60 nm. (1) [BSA] = 2.5 lM; (2)

[HES] = 2.5, (3) 5, (4) 7.5, (5) 10, (6) 12.5, (7) 15, (8) 17.5, (9) 20

and (10) 22.5 lM. A marginal red shift in the emission maximum at

Dk = 60 reveal the changes in the microenvironment of tryptophan

residue

4926 Mol Biol Rep (2011) 38:4921–4929

123

factor and is equal to IHV/IHH. Figure 7 presented the

variation of the fluorescence anisotropy value for HES with

the addition of BSA. The plot showed that the anisotropy of

HES increased from -0.0975 (in absence of BSA) to

0.0718 (in presence of 12.5 lmol L-1 BSA) and gradually

levelled after this concentration of protein. This revealed

that the flavonoid molecule was moved from aqueous

buffer phase to protein environment where enhanced

rigidity was observed. So, the rotational diffusion was

enhanced leading to lesser depolarization of its emission

and greater anisotropy.

Circular dichroism studies

CD, a sensitive technique to monitor the conformational

changes in the protein, is employed in the present study.

The CD spectra of proteins in the region of 200–250 nm

are sensitive to changes in the contributions of the sec-

ondary structures such as a-helices, b-sheets, unordered

structure, as well as the random coils to the overall

conformation.

The CD spectra of BSA in the absence and presence of

HES (lines 1–3) are shown in Fig. 8. The molar ratios of

BSA to HES maintained were 1:0, 1:1 and 1:2. The CD

spectra of BSA exhibited two negative minima at 208 and

220 nm, which are the typical characterization of the

a-helix structure of proteins. The addition of flavonoid to

protein decreased the band intensity at these wavelengths

without any significant shift in the peaks. This indicated

that the flavonoid changed the secondary structure of BSA

upon interaction with the drug.

The CD results are expressed in terms of mean residue

ellipticity (MRE) in deg cm2 dmol-1 according to the

following equation:

MRE ¼ observed CD mdegð Þ = Cpnl� 10� �

ð10Þ

where Cp is the molar concentration of the protein, n is the

number of amino acid residues and l is the path length. The

a-helical contents of free and combined BSA are calculated

from MRE values at 208 nm using the equation [29]:

a-helix %ð Þ ¼ �MRE208 � 4000ð Þ = 33000� 4000ð Þ½ �� 100 ð11Þ

where MRE208 is the observed MRE value at 208 nm, 4000

is the MRE of the a-form and random coil conformation

cross at 208 nm and 33,000 is the MRE value of a pure

a-helix at 208 nm. Using the above equation, the a-helicity

in the secondary structure of BSA was determined. It

decreased from 60.98% in free BSA to 51.16% in HES–

BSA. This revealed that there was a change in a-helicity of

protein upon interaction [30]. Further, the CD spectra of

BSA in the presence and absence of HES are observed to

be similar in shape indicating that the structure of BSA is

also predominantly a-helical even after binding to drug.

FT-IR analysis

Additional evidence regarding interaction between HES

and BSA was obtained from FT-IR spectroscopic results

[31]. IR spectrum of protein exhibits a number of amide

bands, which represent different vibrations of the peptide

moiety. Of all the amide bands of the peptide group, the

single most widely used one in studies of protein secondary

structures is the amide I. The amide I and amide II peaks

occur in the region of 1600–1700 and 1500–1600 cm-1,

respectively. Since amide I band is more sensitive to the

changes in protein secondary structure compared to amide

II, amide I band is more useful for the studies of secondary

structure. In the amide I region, the observed stretching

frequency of C=O hydrogen bonded to NH moieties is

dependent upon the secondary structure adopted by the

peptide chain. The FT-IR spectra of free BSA and HES–

BSA (Fig. 9a, b) in phosphate buffer solution were

-0.15

-0.1

-0.05

0

0.05

0.1

0.00E+00 8.00E-06 1.60E-05 2.40E-05 3.20E-05 4.00E-05

Concentration of BSA

Fuo

resc

ence

ani

sotr

opy

Fig. 7 Variation of the fluorescence anisotropy value of HES with

the addition of different concentrations of BSA (0–12.5 lM);

[HES] = 10 lM [kex = 320 nm]

-100

-80

-60

-40

-20

0

20

40

200 210 220 230 240 250

Wavelength (nm)

MR

E

3

1

Fig. 8 CD spectra of (a) BSA (5 lM) in absence (1) and presence of

5 (2) and 10 lM (3) of HES. The addition of HES to BSA decreases

the a-helicity of protein

Mol Biol Rep (2011) 38:4921–4929 4927

123

recorded at 298 K. The amide I and amide II peaks of BSA

were noticed to be shifted from 1564 to 1546.6 cm-1 and

from 1641.1 to 1648.8 cm-1 upon interaction with HES.

These results indicated that the flavonoid interacted with

the CO and CN groups in the protein polypeptides. The

flavonoid–protein system caused the rearrangement of the

polypeptide carbonyl hydrogen-bonding network and

finally the reduction of the protein a-helical structure.

Conclusions

In summary, the fluorescence quenching of BSA by HES is

a result of the static quenching mechanism. Thermody-

namic parameters reveal that the BSA molecules have a

relatively high affinity for the flavonoid through hydrogen

bonds. The interaction of BSA with the flavonoid results in

conformational changes affecting the secondary structure.

Acknowledgments We are grateful to the Council of Scientific and

Industrial Research, New Delhi, for financial assistance (No.

01(2279)/08/EMR-II dated 20-11-2008). One of the authors (AHH)

thanks the UGC, New Delhi for awarding the Fellowship for meri-

torious students in Science. Thanks are also due to the authorities of

the Karnatak University, Dharwad, for providing the necessary

facilities.

References

1. Finkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the

biology of ageing. Nature 408:239–247

2. Peters T (1996) All about albumin, biochemistry, genetics and

medical application. Academic Press, San Diego

3. Heim KE, Tagliaferro AR, Bobilya DJ (2002) Flavonoid anti-

oxidants: chemistry, metabolism and structure-activity relation-

ships. J Nutr Biochem 13:572–584

4. Lefevre T, Subirade M (2001) Conformation rearrangement of

b-lactoglobulin upon interaction with an anionic membrane.

Biochim Biophys Acta 1594:37–50

5. Xie MX, Jiang M, Li S, Liu Y (2004) Studies on the interaction of

b-1,2,3,4,6-penta-O-galloyl-D-glucopyranose with human serum

albumin. Acta Chim Sin 62:1460–1466

6. Wen M-G, Zhang X-B, Tian J-N, Ni S-H, Bian H-D, Huang Y-L,

Liang H (2009) Binding interaction of xanthoxylin with bovine

serum albumin. J Solut Chem 38:391–401

7. Sun Y, Zhang H, Sun Y, Zhang Y, Liu H, Cheng J, Bi S, Zhang H

(2010) Study of interaction between protein and main active

components in Citrus aurantium L. by optical spectroscopy.

J Lumin 130:270–279

8. Wang Y-Q, Zhang H-M, Zhang G-C, Tao W-H, Tang S-H

(2007) Interaction of the flavonoid hesperidin with bovine

serum albumin: a fluorescence quenching study. J Lumin

126:211–218

9. Xie M-X, Xu X-Y, Wang Y-D (2005) Interaction between hes-

peretin and human serum albumin revealed by spectroscopic

methods. Biochim Biophys Acta 1724:215–224

10. Jinyao Z, Fenglian R (2009) Influence of hydroxylation and

glycosylation in ring A of soybean isoflavones on interaction with

BSA. Spectrochim Acta A 72:682–685

11. Lakowicz JR (1999) Fluorescence quenching: theory and appli-

cations. Principles of fluorescence spectroscopy. Kluwer Aca-

demic, New York

12. Liu YM, Li GZ, Sun XF (2004) Study on the interaction between

colchicine and bovine serum albumins by fluorescence method.

Chin J Anal Chem 32:615–618

13. Feng XZ, Lin Z, Yang LJ, Wang C, Bai CL (1998) Investigation

of the interaction between acridine orange and bovine serum

albumin. Talanta 47:1223–1229

14. Khan SN, Islam B, Rajeswari MR, Usmani H, Khan AU (2008)

Interaction of anesthetic supplement thiopental with human

serum albumin. Acta Biochim Polon 55:399–409

15. Ross PD, Subramanian S (1981) Thermodynamics of protein

association reactions: forces contributing to stability. Biochem-

istry 20:3096–3102

16. Yue Y, Zhang Y, Zhou L, Qin J, Chen X (2008) In vitro study on

the binding of herbicide glyphosate to human serum albumin by

optical spectroscopy and molecular modelling. J Photochem

Photobiol B 90:26–32

17. Sudlow G, Birkett DJ, Wade DN (1975) The characterization of

two specific drug binding sites on human serum albumin. Mol

Pharmacol 11:824–832

18. Gerbanowski A, Malabat C, Rabiller C, Gueguen J (1999)

Grafting of aliphatic and aromatic probes on rapeseed 2S and 12S

proteins: influence on their structural and physicochemical

properties. J Agric Food Chem 47:5218–5226

19. Sengupta A, Hage DS (1999) Characterization of minor site

probes for human serum albumin by high-performance affinity

chromatography. Anal Chem 71:3821–3827

20. Yamasaki K, Maruyama T, Kragh-Hansen U, Otagiri M (1996)

Characterization of site I on human serum albumin: concept

about the structure of a drug binding site. Biochim Biophys Acta

1295:147–157

0.026

0.031

0.036

0.041

1530 1550 1570 1590 1610 1630 1650 1670

Wavelength cm-1

Abs

orba

nce

1564

1641

.1

0.012

0.017

0.022

0.027

0.032

0.037

1530 1550 1570 1590 1610 1630 1650 1670

Wavelength cm-1

Abs

orba

nce 15

46.6

1648

.8

a

b

Fig. 9 FT-IR spectra of free BSA (a) and difference spectra [(BSA

solution ? HES solution)-(HES solution)] in buffer of pH 7.4 in the

region of 1700–1500 cm-1. The shift in amide I and amide II peaks of

BSA indicate the interaction between BSA and HES

4928 Mol Biol Rep (2011) 38:4921–4929

123

21. Forster T, Sinanoglu O (1996) Modern quantum chemistry, vol 3.

Academic Press, New York

22. Jurasekova Z, Marconi G, Sanchez-Cortes S, Torreggiani A

(2009) Spectroscopic and molecular modeling studies on the

binding of the flavonoid luteolin and human serum albumin.

Biopolymers 91:917–927

23. Predki PF, Harford C, Brar P, Sarkar B (1992) Further charac-

terization of the N-terminal copper(I1)-and nickel(I1)-binding

motif of protein. Biochem J 287:211–215

24. Cui F, Zhang Q, Yan Y, Yao X, Qu G, Lu Y (2008) Study of

characterization and application on the binding between 5-iodo-

uridine with HSA by spectroscopic and modeling. Carbohydr

Polym 73:464–472

25. Zhou J, Wu X, Gu X, Zhou L, Song K, Wei S, Feng Y, Shen J

(2009) Spectroscopic studies on the interaction of hypocrellin A

and haemoglobin. Spectrochim Acta Part A 72:151–155

26. Klajnert B, Bryszewska M (2002) Fluorescence studies on

PAMAM dendrimers interactions with bovine serum albumin.

Bioelectrochemistry 55:33–35

27. Stan D, Matei I, Mihailescu C, Savin M, Matache M, Hillebrand

M, Baciu I (2009) Spectroscopic investigations of the binding

interaction of a new indanedione derivative with human and

bovine serum albumins. Molecules 14:1614–1626

28. Bentley KL, Thompson LK, Klebe RJ, Horowitz PM (1985)

Fluorescence polarization: a general method for measuring ligand

binding and membrane microviscosity. BioTechniques 3:356–365

29. Lu ZX, Cui T, Shi QL (1987) Applications of circular dichroism

and optical rotatory dispersion in molecular biology, 1st edn.

Science Press, Beijing

30. Mandal P, Ganguly T (2009) Fluorescence spectroscopic char-

acterization of the interaction of human adult hemoglobin and

two isatins, 1-methylisatin and 1-phenylisatin: a comparative

study. J Phys Chem B 113:14904–14913

31. Li Y, He WY, Dong Y, Sheng F, Hu Z (2006) Human serum

albumin interaction with formononetin studied using fluorescence

anisotropy, FT-IR spectroscopy and molecular modeling meth-

ods. Bioorg Med Chem 14:1431

Mol Biol Rep (2011) 38:4921–4929 4929

123