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Journal of Molecular Structure 831 (2007) 144–150

Daidzein interaction with human serum albumin studied usingoptical spectroscopy and molecular modeling methods

Ying Li a,b, WenYing He b, Huanxiang Liu b, Xiaojun Yao b, Zhide Hu a,b,*

a Hainan Provincial Key Lab of Fine Chemicals, Hainan University, Haikou 570228, Chinab Department of Chemistry, LanZhou University, LanZhou, GanSu province 730000, China

Received 12 March 2006; received in revised form 20 July 2006; accepted 24 July 2006Available online 14 September 2006

Abstract

In this work, fluorescence anisotropy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy andmolecular modeling methods were used to characterize optical properties of the Daidzein–HSA complex and to gain information onthe binding mechanism at molecular level. Daidzein is weakly fluorescent in aqueous buffer medium, with an emission maximum at466 nm. Binding of Daidzein with HSA leads to dramatic enhancement in the fluorescence intensity and anisotropy (r), along with sig-nificant changes in the fluorescence excitation and emission profiles. The binding constant (K = (5.9 ± 0.6) · 104 M�1) and the standardfree energy change (DG � �27.5 kJ/mol) for Daidzein–HSA interaction have been calculated from the relevant fluorescence data. Thesecondary structure compositions of free HSA and its Daidzein complexes were estimated by the FT-IR spectra and the curve-fittedresults of amide I band, which are in good agreement with the analyses of CD spectra. Furthermore, the displacement experiments indi-cated that Daidzein can bind to the site I of HSA which is also in agreement with the result of molecule modeling study.� 2006 Elsevier B.V. All rights reserved.

Keywords: Daidzein; Human serum albumin; Fluorescence anisotropy; CD spectroscopy; FT-IR spectroscopy; Molecular modeling

1. Introduction

Daidzein (structure shown in Fig. 1), one of the majorbioactive components belong to isoflavones, is naturaloestrogenic compound present in large amounts (up to1–4 mg/g) in seeds, fruits and vegetables [1,2]. Phyto-oes-trogens have oestrogen-like acting and many interfere withthe reproductive cycle in animals [2]. During the last dec-ade, an interest in the study of this compound has increaseddue to it potential effects as health protecting dietary fac-tors in human populations. Recently, Daidzein was shownto decrease the incidence of hot flushes and to reduce oste-oporosis in menopausal women. Today, Daidzein is com-mercialized for the prevention of female menopausaltrouble.

Serum albumins have been one of the most studied pro-teins for many years. They are the most abundant of the

0022-2860/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.molstruc.2006.07.034

* Corresponding author. Tel.: +86 931 8912540; fax: +86 931 8912582.E-mail address: huzd@lzu.edu.cn (Z. Hu).

protein in blood plasma, accounting for about 60% ofthe total protein corresponding to a concentration of42 g/L [3,4] and provide about 80% of the osmotic pressureof blood [3]. Albumins have been used as a model proteinfor many and diverse biophysical and physicochemicalstudies. They play an important role in the transport anddeposition of a variety of endogenous and exogenous sub-stances in blood [5]. Recently, the three-dimensional struc-ture of human serum albumin has been determinedthrough X-ray crystallographic measurements [6]. Theglobular protein consists of a single polypeptide chain of585 amino acid residues and has many important physio-logical functions [7]. The globular protein is composed ofthree structurally similar domains (I, II and III), each con-taining two subdomains (A and B), and is stabilized by 17disulfide bridges. Aromatic and heterocyclic ligands werefound to bind within two hydrophobic pockets in subdo-mains II A and III A, which site I and site II [4,6]. Thereare typical sites of coordination for several substances suchas amino acids, fatty acids, hormones and drugs [4]. The

Fig. 1. The chemical structure of Daidzein.

Y. Li et al. / Journal of Molecular Structure 831 (2007) 144–150 145

multiple binding sites underlie the exceptional ability ofHSA to interact with many substances and make this pro-tein an important regulator of intercellular fluxes and phar-macokinetic behavior of many drugs. The studies on thebinding of drugs to HSA may provide information of struc-tural features that determine the therapeutic effectiveness ofdrugs, and become an important research field in life sci-ences, chemistry and clinical medicine. Therefore a studyon the binding of Daidzein to HSA is very significant. Sofar, none of the investigations determine in detail theDaidzein–HSA binding constant and the effects of Daidz-ein complexation on the protein structure.

Protein–ligand interactions play an important role in avariety of biological processes. To characterize theseinteractions at the molecular level, optical techniqueshave become valuable tool because these methods aresensitive and relatively easy to use. Among these, fluores-cence anisotropy was found to be intrinsically high [8]and sensitive [9,10] to relevant environmental changes.The present work was undertaken in an attempt toassess, for the first time, the usefulness of Daidzein asan extrinsic optical probe for protein exploiting its fluo-rescence anisotropy as useful parameter. FT-IR, a pow-erful technique for the study of hydrogen bonding, hasrecently become very popular for structural characteriza-tion of proteins. The most important advantage of FT-IR spectroscopy for biological studies is that spectra ofalmost any biological system can be obtained in a widevariety of environments.

In this paper, the fluorescence anisotropy (r) parame-ter dramatically reveals the binding characteristics ofDaidzein with the protein. And based on fluorescenceanisotropy measurements on Daidzein, the binding con-stant for the Daidzein–HSA complex has been calculatedas (5.9 ± 0.6) · 104 M�1. We have also estimated thestandard free energy change (DG� � �27.5 kJ/mol) forthe association reaction. Furthermore, FT-IR differencespectrum technology and CD spectrum were used forillustrating the changes of protein secondary structure.The changes of secondary structure were quantitativelyanalyzed by the curve fitting method. In addition, a com-parative study of HSA interaction with Daidzein andphenylbutazon (PB), fluofenamic acid (FA) or digitoxin(Dig) should help understand preferential binding atthe molecular level.

2. Experimental

2.1. Materials

Human serum albumin (fatty acid free), purchased fromSino-American Biotechnology Company (China), was usedwithout further purification and its molecular weight wasassumed to be 66500. Daidzein, phenylbutazon (PB), fluo-fenamic acid (FA) and digitoxin (Dig) were of analyticalgrade, and purchased from the National Institute for Con-trol of Pharmaceutical and Bioproducts (China). HSA wasdissolved in the pH 7.40 buffer solution (3.0 · 10�5 M) andHSA stock solution was kept in the dark at 277 K. Theprotein concentration was determined spectrophotometri-cally using the extinction coefficient of 36,500 M�1 cm�1

at 280 nm. Daidzein, phenylbutazon, fluofenamic acidand digitoxin were prepared into (1.0 · 10�3 M) stock solu-tions by dissolving appropriate amount of the compoundsin ethanol because ethanol has not fluorescence and doesnot affect the determinations. The concentration of Daidz-ein was determined by the molar absorption coefficients(daidzein = 26 · 103 M�1 cm�1) [11]. All the stock solu-tions were then diluted with buffer to obtain the actualassay solutions. NaCl (0.5 M) solution was used to keepthe ion strength of solutions at 0.1 M. Buffer consists ofTris (0.2 M) and HCl (0.1 M), and the pH wasadjusted to 7.4. The pH was checked with a suitably stan-dardized pH meter. The temperature of the experimentswas 298 K.

2.2. Apparatus and methods

The absorption and steady state fluorescence anisotropymeasurements were performed using RF-5301PC spectro-fluorophotometer (Shimadzu), the excitation and emissionband widths were both 5 nm. Steady state anisotropy (r) isdefined by:

r ¼ ðIVV � GIVHÞ=ðIVV � 2GIVHÞwhere IVV and IVH are the intensities obtained with theexcitation polarizer oriented vertically and the emissionpolarizer oriented in vertical and horizontal orientation,respectively. The G factor is defined as:

G ¼ IHV=IHH

I refers to the similar parameters as mentioned above forthe horizontal position of the excitation polarizer.

Spectra were recorded with an Olis DMS1000CD(USA). For measurements in the far-UV region (200–250 nm), a quartz cell with a path length of 0.2 cm wasused. Three scans were accumulated at a scan speed of30 nm/min, with data being collected at every nm from200 to 250 nm. Sample temperature was maintained at298 K. The protein concentration used in our experimentwas 3.0 · 10�6 M.

FT-IR measurements were carried out at roomtemperature on a Nicolet Nexus 670 FT-IR spectrometer

Fig. 2. The fluorescence emission spectra of Daidzein–HSA system (a)1.0 · 10�5 M Daidzein; (b)–(f) 1.0 · 10�5 M Daidzein in the presence of2.0 · 10�6 M, 6.0 · 10�6 M, 1.0 · 10�5 M, 2.0 · 10�5 M, 3.0 · 10�5 M,HSA. (kex = 345 nm, kem = 466 nm; pH = 7.4; T = 298 K).

Fig. 3. Variation of fluorescence anisotropy (r) of Daidzein with HSAconcentration. ([Daidzein] = 2.0 · 10�5 M; kex = 345 nm, kem = 466 nm;pH = 7.4; T = 298 K). Each data point indicates the average of threedeterminations where each bars indicate the standard deviation.

146 Y. Li et al. / Journal of Molecular Structure 831 (2007) 144–150

(America) equipped with a Germanium attenuated totalreflection (ATR) accessory, a DTGS KBr detector and aKBr beam splitter. All spectra were taken via the Attenuat-ed Total Reflection (ATR) method with resolution of4 cm�1 and 60 scans. Spectra processing procedures: spec-tra of buffer solution were collected at the same condition.Then, subtract the absorbance of buffer solution from thespectra of sample solution to get the FT-IR spectra of pro-teins. The subtraction criterion was that the original spec-trum of protein solution between 2200 and 1800 cm�1

was featureless [12]. Fourier self-deconvolution and sec-ondary derivative were applied to this range respectivelyto estimate the number, position and width of componentbands. Based on these parameters curve-fitting processwas carried out by Galactic Peak solve to get the bestGaussian-shaped curves that fit the original protein spec-trum. After the identifying individual bands with its repre-sentative secondary structure of HSA is calculated by areaof their respective component bands.

The crystal structure of HSA in complex with R-warfa-rin was taken from the Brookhaven Protein Data Bank(entry codes 1h9z) [13]. The potential of the 3-D structuresof HSA was assigned according to the Amber 4.0 force fieldwith Kollman-all-atom charges. The initial structures of allthe molecules were generated by molecular modeling soft-ware Sybyl 6.9 [14]. The geometries of these compoundswere subsequently optimized using the Tripos force fieldwith Gasteiger–Marsili charges. FlexX program was usedto calculate the interaction modes between the ligandsand HSA.

3. Results and discussion

3.1. Daidzein–HSA binding interaction

The fluorescence spectrum of Daidzein is shown inFig. 2. The fluorescence intensity of Daidzein was dramaticenhancement when the solution of HSA was added, whichindicates Daidzein can bind to the HSA. The maximumwavelength of Daidzein shifted from 466 to 459 nm afterthe addition of HSA, so a slight hypsochromic shift ofthe maximum emission wavelength was observed. Theobservations reflected that the microenvironment aroundthe fluorophore in the protein solution is quite differentfrom that of pure aqueous phase. Following the fluoromet-ric work of Daidzein in pure solvents, the blue shift in thefluorescence spectrum of the fluorophore with an additionof HSA is ascribed to a lowering in the polarity of the pro-tein environment compared to that of the bulk aqueousphase.

The changes in the fluorometric behavior of the fluoro-phore with the addition of HSA in buffered aqueous solu-tion can be rationalized in terms of binding of the drugwith the protein leading to a less polar microenvironmentaround the fluorophore. A small increase in the fluores-cence yield of Daidzein in HSA is also ascribed to a lower-ing in the polarity in the near vicinity of Daidzein. A blue

shift in the fluorescence maximum also suggests a reductionin the polarity of the microenvironment [15].

To ascertain the binding of Daidzein with HSA, thefluorescence anisotropy of Daidzein in the presence ofHSA was measured. Fig. 3 represents the variation of fluo-rescence anisotropy (r) value for Daidzein with increase inHSA concentration. The plot shows a marked increase inthe fluorescence anisotropy of the fluorophore while mov-ing from aqueous to HSA environment. The increase inanisotropy of daidzein bound HSA with increase in proteinconcentration, indicates the reduction of freedom of rota-tion of daidzein bound HSA. Increase in anisotropy couldbe due to decreased Brownian motion or energy transferbetween identical chromophores. The high anisotropy val-ue (r = 0.18) suggests that Daidzein is binding at a motion-al restricted site on HSA [11]. After the HSA concentration

Y. Li et al. / Journal of Molecular Structure 831 (2007) 144–150 147

reaches 15 lM no significant increase in binding of Daidz-ein with HSA takes place with further increase in HSA con-centration. In order to see the interaction between Daidzeinand the protein, the binding constant value was determinedfrom the fluorescence intensity considering the followingequation as developed by Bhattacharya et al. [16,17].

1=DF ¼ 1=DF max þ ð1=K½L�Þð1=DF maxÞwhere DF = FX � F0 and DFmax = F1 � F0 where F0,FX

and F1 are the fluorescence intensities of Daidzein in theabsence of HSA, at an intermediate concentration ofHSA, and at the saturation of interaction, respectively; K

being the binding constant and [L] the protein concentra-tion. Rearranging the above equation we have the follow-ing form:

ðF1 � F 0ÞðF X � F 0Þ ¼ 1þ ðK½L�Þ�1

The linearity in the plot of (F1 � F0)/(FX � F0) against[L]�1 confirms a one-to-one interaction between the twopartners (Fig. 4).

The slope of the plot led to the determination of thebinding constant between the two and the value obtainedwas (5.9 ± 0.6) · 104 M�1. The standard free energychange (DG� = �RT lnK) comes out to be approximately�27.5 kJ/mol at 298 K which indicates the spontaneity ofbinding of Daidzein of HSA.

3.2. Changes of the protein’s secondary structure induced

by drug binding

In order to gain a better understanding in physicochem-ical properties of Daidzein governing its spectral behaviorand to draw relevant conclusions on the Daidzein–HSAbinding mechanism, CD and FT-IR spectroscopic mea-surements were performed on HSA and the Daidzein–HSA complex. If the change of protein structure includedthe transforming of protein secondary structure in thedrug–HSA complex, it can be reflected in the infrared

Fig. 4. Plot of (F1 � F0)/(FX � F0) against [L]�1 ([Daidz-ein] = 2.0 · 10�5 M; kex = 345 nm, kem = 466 nm; pH = 7.4; T = 298 K).

absorption spectra or CD spectra. The CD spectra ofHSA and Daidzein–HSA complex were shown in Fig. 5.The CD spectra of HSA consist of two negative bands inthe ultraviolet region at 209 and 220 nm, which is typicalcharacterization of a-helix structure of class protein [18].The reasonable explanation is that the negative peaksbetween 208 and 209 nm and 222–223 nm are both contrib-uted to n fi p* transfer for the peptide bond of a-helical[19]. As shown in Fig. 5, the interaction between Daidzeinand HSA caused only a decrease in band intensity at allwavelength of the far–UV CD without any significant shiftof the peaks, indicating that this drug induces a sightdecrease in the helical structure content of protein.Decrease in the a-helical content in protein is dependenton the molar ratio Daidzein/HSA.

Evidence regarding drug–protein complexation comesfrom IR spectroscopic results. Because there was no majorspectral shifting for the protein Amide I peak positionoccur in the range 1600–1700 cm�1(mainly C@O stretch)and Amide II bands � 1548 cm�1 (C–N stretch withN–H bending mode) [20,21]. Upon drug interaction the dif-ference spectra [(protein solution + drug solution)� (drugsolution)] were obtained in order to monitor the intensityvariations of these vibrations. The results are shown inFig. 6. Similarly, the curve-fitting procedure was used todetermine the protein secondary structure in the presenceof Daidzein, and the results are presented in Fig. 7. Inthe present of Daidzein, the intensities of the Amide Idecreased in the spectra of free HSA (Fig. 6). The reduc-tion in the intensity of Amide I is related to continueddrug–protein interaction and the decreased a-helical struc-tures in favor of b-sheet and turn structure, which will dis-cussed in detail. The spectra indicate that Daidzeininteracts with the C@O and C–N groups in the proteinpolypeptides. The Daidzein–protein complexes caused therearrangement of the polypeptide carbonyl hydrogenbonding network and finally the reduction of the proteina-helical structure.

Fig. 5. CD spectra of the Daidzein–HSA System at different drug to HSA(3.0 · 10�6 M) ratios. pH = 7.4; T = 298 K.

Fig. 6. FT-IR spectra and difference spectra [(HSA solution + Daidzeinsolution) � (Daidzein solution)] of the Daidzein–HSA complexes atdifferent drug to HSA ratios in buffer solution in the region of 1800–1300 cm�1. Drug to HSA (3.0 · 10�5 M) ratios: A 0:1; B 1:1; C 2:1;pH = 7.4.

Fig. 7. The curve-fit amide I region with secondary structure determina-tion of the Daidzein–HSA complexes at different drug to HSA ratios inbuffer solution in the region of 1700–1600 cm�1. Drug to HSA(3.0 · 10�5 M) ratios: A 0:1; B 1:1; C 2:1; pH = 7.4.

148 Y. Li et al. / Journal of Molecular Structure 831 (2007) 144–150

Before estimation of percentage content of each second-ary structure, the component bands should be assigned.The component bands of Amide I were attributed accord-ing to the well-established assignment criterion [21]. Thesebands may be tentatively assigned to b-sheet (1610–1640 cm�1), to random coil (1640–1650 cm�1), to a-helix(1650–1659 cm�1), and to b-turn structure (1660–1700 cm�1) [21–24]. Based on the above assignments, thepercentages of each secondary structure of HSA were cal-culated from the integrated areas of the component bandsin Amide I. A quantitative analysis of the protein second-ary structure for the free HSA and its complex in H2O isgiven in Fig. 7. The free HSA contained major amountsof a-helical (54.4%); b-sheets (38.7%); turn structure(6.9%). Upon Daidzein complexation, the a-helical struc-ture was reduced from 54.4% to 39.8%, the b-turnincreased from 6.9% to 18.4%, and b-sheet was changedfrom 38.7% to 35.9% (Table 1). The reduction of a-helicesin favor b-sheet and b-turn structures is indicative of a par-tial unfolding of the protein in the presence of Daidzein.Similar conformational transitions from an a-helix to b-sheet structure were observed for the protein unfoldingupon protonation and heat denaturation [25].

3.3. Molecular modeling study of the interaction between

Daidzein and HSA

Descriptions of the 3-D structure of crystalline albu-min have revealed that HSA is comprised of three struc-

turally homologous domains (I–III): I (residues 1–195),II (196–383), III (384–585), each containing two subdo-mains (A and B). The principal regions of ligand binding

Table 1The effect of Daidzein on the components, assignments of the curve-fitted amide I of HSA before and after treatment with Daidzein differentconcentrations

Structure components Free HSA Daidzein (30 lM) Daidzein (60 lM)

a-helix 54.4%(1656 cm�1) 48.0%(1659 cm�1) 39.8%(1655 cm�1)b-sheet 38.7%(1616 cm�1) 40.4%(1633,1610 cm�1) 35.9%(1639,1623 cm�1)b-turn 5.4%(1672,1694 cm�1) 11.6%(1695 cm�1) 18.4%(1678,1699 cm�1)

Table 2The comparison of binding constant of Daidzein of HSA and after the addition of the site probe, 298 K

K(without the site probe) K(with PB) K(with FA) K(with Dig)

(5.9 ± 0.6) · 104 M�1 (5.1 ± 0.6) · 104 M�1 (6.5 ± 0.3) · 104 M�1 (6.3 ± 0.3) · 104 M�1

Y. Li et al. / Journal of Molecular Structure 831 (2007) 144–150 149

sites of albumin are located in hydrophobic cavities insubdomains IIA and IIIA. It is suggested that HSAhas binding sites of compounds within hydrophobic cav-ities in subdomains IIA and IIIA, which are consistentwith sites I and II, respectively, and one tryptophan res-idue (Trp-214) of HSA is in subdomain IIA [4]. Severalstudies have shown that HSA is able to bind manyligands in several binding sites [6,26]. These is a largehydrophobic cavity present in subdomain IIA that manydrugs can bind at. Sudlow et al. [27] have suggested twomain distinct binding sites on HSA, sites I and II. Site Iof HSA shown affinity for warfarin, PB, etc., while site

Fig. 8. The interaction mode between Daidzein and HSA. The residues of HSAand stick model. The hydrogen bond between Daidzein and HSA is represent

II for ibuprofen, FA, etc. Later studies indicated thatDig binding is independent of sites I and II [28], andbinds to what was nominated as site III. To establishwhich binding site of HSA that Daidzein in located,competition displacement experiments were used. Ternarymixture of PB, FA, or Dig and Daidzein with HSA werestudied. As shown in Table 2, the binding constant wasremarkably decreased after addition of PB, while theaddition of FA and Dig have little change the bindingconstants. These results indicate that PB can displacethe Daidzein but FA and Dig have little effect on thebinding of Daidzein to HSA. From these data, we dem-

are represented using line and the ligand structure is represented using balled using a dashed line.

150 Y. Li et al. / Journal of Molecular Structure 831 (2007) 144–150

onstrated PB displaces Daidzein from its site of HSA.This probably indicated that Daidzein has one reactivesite of HSA, that is, high affinity site (site I).

In order to further study which binding site of HSA thatDaidzein in located, SGI FUEL workstation was used to cal-culate the partial binding parameters of the HSA–Daidzeinsystem, and the best energy ranked result was shown inFig. 8. Daidzein has a flavone nucleus made up of two ben-zene rings (A and B) linked through a heterocyclic pyraneC ring. These aromatic rings involved in hydrophobic inter-actions with hydrophobic pockets of domain IIA of HSA. Asshown in Fig. 8, Daidzein is situated within subdomain IIAin Sudlow’s site I formed by six helices. The C ring of Daidz-ein is inserted in the hydrophobic cavity of site I, and it isimportant to note that the tryptophan residue of HSA(Try-214) is in close proximity to C ring suggesting the exis-tence of hydrophobic interaction between them. However,the interaction between Daidzein and HSA is not exclusivelyhydrophobic in nature since the several ionic and polar resi-dues in the proximity of the ligand playing important role instabilizing the negatively charged Daidzein molecule via H-bonds and electrostatic interaction. For instance, Lie290 isin suitable position to form intermolecular H-bond interac-tion with 9-OH. Additionally, His242, Arg222 are able toform intermolecular H-bond with the 12-O and 4-O.

4. Conclusions

In the present work, the binding of Daidzein to HSAunder physiological conditions has been studied by fluores-cence anisotropy in combination with Fourier transforminfrared, circular dichroism spectroscopy and molecularmodeling methods. According to fluorescence data, thebinding constant for the reaction at 298 K were obtained.The results of the calculations using of a Sillicon GraphicsOcatane2 workstations and displacement experiments indi-cate that Daidzein binds within the subdomain IIA of theprotein (site I). In addition, FT-IR and CD spectroscopyshown that the secondary structure of HSA changed afterDaidzein bound to HSA.

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