interaction of caffeine with bovine serum albumin: determination of binding constants and the...

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* E-mail: [email protected]; Tel.: 0086-027-68756667; Fax: 0086-027-68754067 Received June 17, 2010; revised July 29, 2010; accepted October 21, 2010. Project supported by the National Natural Science Foundation of China (Nos. 20873096, 20621502, 20803019).

Chin. J. Chem. 2011, 29, 433—440 © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 433

Interaction of Caffeine with Bovine Serum Albumin: Determination of Binding Constants and the

Binding Site by Spectroscopic Methods

Wu, Qionga,b(吴琼) Jiang, Fenglei*,b(蒋风雷) Li, Chaohongc(李超宏) Hu, Yanjunb(胡艳军) Liu, Yib(刘义)

a Key Laboratory for the Synthesis and Application of Organic Functional Molecules (Ministry of Education), College of Chemistry and Chemical Engineering, Hubei University, Wuhan, Hubei 430062, China

b State Key Laboratory of Virology, Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei 430072, China

c Key Laboratory for Oral Biomedical Engineering (Ministry of Education), School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430072, China

The interaction of caffeine with bovine serum albumin (BSA) under physiological condition was investigated by fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy. Fluorescence data revealed that the fluorescence quenching of BSA by caffeine was a result of the formation of BSA-caffeine complex. The binding constants Ka at different temperatures and corresponding thermodynamic parameters ∆H, ∆G and ∆S were calcu-lated. The spectroscopic measurements and the thermodynamic parameters suggested that van der Waals interaction and hydrogen bonds were the predominant intermolecular forces to stabilize the complex. The conformational change of BSA induced by caffeine has been analyzed by means of CD and synchronous fluorescence spectroscopy. Furthermore, it is observed from the probe of competitive experiments that the binding location of caffeine with BSA could be the same as warfarin binding site I of BSA, which was also revealed by fluorescence anisotropy.

Keywords caffeine, bovine serum albumin, fluorescence spectroscopy, UV-vis spectroscopy, site competitive binding

Introduction

Serum albumins are the most abundant proteins in the circulatory system of a wide variety of organisms. Being the major macromolecule contributing to the os-motic blood pressure,1 they can play a dominant role in drug disposition and efficacy.2 Many drugs and other bioactive small molecules bind reversibly to albumin and other serum components, which then function as carriers. Serum albumin often increases the apparent solubility of hydrophobic drugs in plasma and modu-lates their delivery to cells in vivo and in vitro. Bovine serum albumin (BSA) is considered to be a heart-shaped helical monomer composed of three homologous do-mains named I, II and III, and each domain includes two subdomains called A and B to form a cylinder. Crystal structure analyses have revealed that the drug binding sites are located in subdomains IIA and IIIA. The ge-ometry of the pocket in IIA is quite different from that found in IIIA. BSA has two tryptophan moieties (Trp 134 and Trp 212), located in subdomains IA and IIA, respectively.1 It is important to study the interaction of the drugs with BSA for the interaction of drugs with

BSA influences the drugs’ pharmacology and pharma-codynamics.

Caffeine (Figure 1) belongs to a class of compounds called methylxanthines. It is an abundant alkaloid of exogenous origin in human plasma.3 Caffeine is proba-bly the most frequently ingested pharmacologically ac-tive substance in the world. Stimulations of the central nervous system, cardiac muscle, respiratory system and diuresis are only some of the numerous physiological effects that it exerts.4-6 Extensive research on caffeine connected to various diseases has not identified any health hazard of normal caffeine consumption.7,8 It is widely accepted that the distribution, metabolism and efficacy of many drugs can be altered based on their affinity to serum albumin. Consequently, the determina-tion and understanding of caffeine interacting with se-rum albumin are important for the therapy and design of the drug. Investigating the influence of the drug on pro-tein not only provides the pharmacological action of caffeine, but also can illuminate its binding mecha-nisms.

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434 www.cjc.wiley-vch.de © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Chin. J. Chem. 2011, 29, 433—440

Figure 1 Structure of caffeine, warfarin and ibuprofen.

In the present work, BSA is selected as our protein model because of its medical importance, low cost, ready availability, unusual ligand-binding properties, and the results of all the studies are consistent with the fact that bovine and human serum albumins are ho-mologous proteins.9,10 We have studied the interaction of caffeine with BSA at three temperatures (298, 304, and 310 K) under physiological condition. Spectro-scopic data were used to quantify the binding constants of caffeine to BSA and the action distance which was based on the Förster theory of non-radiative energy transfer (FET). Synchronous fluorescence and circular dichroism (CD) spectroscopy revealed that the change of protein conformation resulted from the caffeine binding to BSA. What is more, the interaction of the mainly acting forces and the binding site of the location were characterized by optical spectroscopy.

Experimental

Materials

BSA was purchased from Sigma and used without further purification. Caffeine was obtained from the Second Reagent Co., Ltd (Shanghai, China). Warfarin was obtained from Medicine Co. Ltd., Jiangshu in China. Ibuprofen was presented by the company of Hubei Bio-cause Heilen Pharmaceutical Co. Ltd. of China. All starting materials were analytical reagent grade and Mil-lipore pure water (10 µS/cm at 25 ℃ ) was used throughout the experiments. All solutions were prepared in 0.05 mol•L－1, pH＝7.4, phosphate buffer solution (PBS) or ethanol.

Fluorescence measurements

Fluorescence spectra were measured with an F-2500 Spectrofluorimeter (Hitachi, Japan) equipped with a 1.0 cm quartz cell and a thermostatic bath. The excitation wavelength was 290 nm, and the emission spectra were recorded at 298, 304 and 310 K in the range of 300—450 nm. The widths of excitation and emission slits were both set to 2.5 nm. To quantify the binding con-stants of caffeine to BSA, a 2.0 mL solution containing 2.0×10－6 mol•L－1 BSA was titrated by successive ad-ditions of caffeine solution using trace syringes, and the fluorescence intensity was measured. All experiments

were measured at each temperature with recycle water keeping the temperature constant. The appropriate blanks corresponding to the buffer were subtracted to correct background of fluorescence. The results obtained were analyzed by using the Stern-Volmer equation or modified Stern-Volmer equation to calculate binding constants.

UV absorbance measurements

For each BSA sample in the titration experiment with caffeine, the UV-vis absorption spectrum was recorded using a TU-1901 spectrophotometer (Puxi Analytic In-strument Ltd. Beijing, China) equipped with 1.0 cm quartz cells. The range of wavelength was from 190 to 450 nm. To accomplish this, a 2.0 mL solution of 2.0×10－6 mol•L－1 BSA was titrated by successive additions of caffeine solution.

CD measurements

Circular dichroism (CD) measurements were per-formed on a J-810 Spectropolarimeter (Jasco, Tokyo, Japan) at 298 K. CD measurements of BSA in the ab-sence and presence of caffeine (1∶5, 1∶10) were re-corded in the range of 260—200 nm. The instrument was controlled by Jasco’s Spectra ManagerTM software. Quartz cells having path lengths of 0.1 cm were used at a scanning speed of 500 nm/min. Each spectrum was the average of three successive scans. The data were ex-pressed in terms of mean residue ellipticity (MRE) in (°)•cm2•dmol－1. An appropriate buffer solution run un-der the same conditions was taken as a blank and sub-tracted from the sample spectra.

Fluorescence anisotropy measurements

The anisotropy (r) is defined as the difference be-tween the fluorescence intensity emitted parallel and perpendicular (I|| and I⊥) divided by the total intensity. Fluorescence anisotropy was calculated from fluores-cence intensity measurement employing a vertical exci-tation polarizer and vertical and horizontal emission polarizers according to Eq. (1):11

||

|| 2

I GIr

I GI⊥

⊥

－＝

＋ (1)

where I|| is the intensity of emitted light measured in the direction parallel to excitation, I⊥ is the intensity of emitted light measured in the direction perpendicular to excitation and G＝I⊥/I|| is the instrument grating correc-tion factor. Fluorescence anisotropy was measured by using the automatic polarization device of a LS55 spec-trofluorometer (Perkin-Elmer, USA) equipped with a polarization. Excitation and emission bandwidths were all adjusted to 5 nm. Each titration point of the sample of equilibration at least four times with an integration time of 1 min was collected.

Interaction of Caffeine with Bovine Serum Albumin

Chin. J. Chem. 2011, 29, 433—440 © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cjc.wiley-vch.de 435

Results and discussion

Fluorescence quenching mechanism and binding constants

Fluorescence quenching efficiency and aspects of the quenching mechanism of the BSA by caffeine were studied by steady-state fluorescence measurements. The quenching of fluorescence is known to occur mainly by a collisional process (dynamic quenching) and/or for-mation of a complex between quencher and fluorophore (static quenching). The effect of caffeine on BSA fluorescence intensity is shown in Figure 2. The ob-served spectra (curves A—K) display a single broad band centered at 342 nm. It can be clearly seen that the presence of caffeine induces a decrease in fluorescence intensity without a shift in the wavelength of the emis-sion maximum. Curve N (dashed line) shows the emis-sion spectrum of caffeine only, which indicates that the emission of caffeine in the investigated concentration range is negligible.

Figure 2 Emission spectra of BSA in the presence of various concentrations of caffeine (T＝298 K, λex＝290 nm). c(BSA)＝2.0×10－6 mol•L－1; c(caffeine)/(10－5 mol•L－1), A—K: 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, respectively; curve N shows the emission spectrum of caffeine only, c(caffeine)＝1.0×10－5 mol•L－1.

Fluorescence quenching is described by the well- known Stern-Volmer equation:12

0q 0 SV1 [Q] 1 [Q]

Fk K

Fτ＝＋＝＋ (2)

where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, kq is the biomolecular quenching constant, τ0 is the life time of the fluorescence in absence of quencher, [Q] is the con-centration of quencher, and KSV is the Stern-Volmer quenching constant. Hence, Eq. (2) was applied to de-termine KSV by a linear regression of the plot of F0/F against [Q] at different temperatures.

Figure 3 displays the Stern-Volmer plots for the quenching of the fluorescence of BSA tryptophan resi-dues by caffeine at different temperatures, and the cor-responding Stern-Volmer quenching constants are

shown in Table 1. The results show the Stern-Volmer quenching constant KSV is inversely correlated with temperature, which indicates that the probable quench-ing mechanism of fluorescence of BSA by caffeine is not initiated by dynamic collision but from complex formation. Moreover, the UV-vis absorption spectra of BSA and the difference absorption spectrum between BSA-caffeine and caffeine at the same concentration could not be superposed within experimental error (Fig-ure 4). This result reconfirms that the probable quench-ing mechanism of fluorescence of BSA by caffeine is mainly a static quenching procedure.12

Figure 3 Stern-Volmer plots for the quenching of BSA fluo-rescence by caffeine at three different temperatures.

Table 1 Stern-Volmer quenching constants of the caffeine-BSA system at different temperatures

pH T/K KSV/(103 L•mol－1) Ra S.D.b

298 7.964 0.9981 0.0085

7.4 304 7.465 0.9978 0.0086

310 6.875 0.9959 0.0109 a R is the correlation coefficient; b S.D. is standard deviation.

Figure 4 UV-vis absorption spectra of BSA in the absence and presence of caffeine. A: The absorption spectrum of caffeine only; B: The absorption spectrum of BSA only; C: The difference ab-sorption spectrum between BSA-caffeine and caffeine at the same concentration, c(BSA)＝c(caffeine)＝2.0×10－6 mol•L－1.

Therefore, the quenching data were analyzed ac-cording to the modified Stern-Volmer equation:13

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0

a a a

1 1 1

[Q]

F

F f K f∆＝＋ (3)

In the present case, ∆F is the difference in fluores-cence intensity in the absence and presence of the quencher at concentration [Q], ƒa is the fraction of ac-cessible fluorescence, and Ka is the effective quenching constant for the accessible fluorophores. The modified Stern-Volmer plots are shown in Figure 5.

Figure 5 Modified Stern-Volmer plots for the quenching of BSA by caffeine at three different temperatures.

The dependence of F0/∆F on the reciprocal value of the quencher concentration [Q]－1 is linear with slope equal to the value of (faKa)

－1. The quotient of the ordi-nate fa

－1 and the slope (ƒaKa)－1 is equal to the value of

Ka. The binding constants at different temperatures were shown in Table 2. The decreasing trend of Ka with in-creasing temperature is in accordance with KSV’s de-pendence on temperature as mentioned above, which coincides with the static type of quenching mecha-nism.12

Determination of the force acting between caffeine and BSA

Analysis of Stern-Volmer plots in this system yields equilibrium expressions for static quenching, Ka, which are analogous to associative binding constants for the quencher-acceptor system.14 The interaction forces be-tween drugs and biomolecules may include hydrophobic forces, electrostatic interactions, van der Waals interac-tions, hydrogen bonds, etc.15 In order to elucidate the interaction of caffeine with BSA, the thermodynamic parameters were calculated. If the enthalpy change (∆H) does not vary significantly over the temperature range

studied, then it can be regarded as a constant and its value and that of the entropy change (∆S) can be deter-mined from the van’t Hoff equation:

lnH S

KRT R

∆ ∆＝－＋ (4)

where association constant K is analogous to the effec-tive quenching constant Ka at the corresponding tem-perature and R is the gas constant. The free energy change (∆G) is estimated from the following relation-ship:

∆G＝∆H－T∆S (5)

Figure 6, by fitting the data of Table 2, shows that assumption of near constant ∆H is justified. According to the binding constants Ka at the three temperatures (298, 304 and 310 K), the thermodynamic parameters can be obtained as mentioned above and the corre-sponding results were presented in Table 2. The nega-tive value of free energy (∆G) supports the assertion that the binding process is spontaneous. The negative en-thalpy (∆H) and entropy (∆S) values of the interaction of caffeine and BSA indicate that the binding is mainly enthalpy-driven, and van der Waals interactions and hydrogen bonds played major roles in the reaction.16

Figure 6 van’t Hoff plot, pH＝7.4, c(BSA)＝2.0×10－6 mol•L－1.

Energy transfer between caffeine and BSA

According to Förster’s non-radioactive energy trans-fer theory, a transfer of energy can take place through a direct electrodynamic interaction between the primarily excited molecule and its neighbors.17 The distance r of binding between caffeine and BSA can be calculated by the equation:18

Table 2 Modified Stern-Volmer constants Ka and relative thermodynamic parameters

T/K Ka/(103 L•mol－1) Ra ∆H/(kJ•mol－1) ∆G/(kJ•mol－1) ∆S/(J•mol－1•K－1) Rb

298 11.53 0.9988 －23.16

304 8.828 0.9928 －36.02 －22.91 －43.14 0.9971

310 6.553 0.9921 －22.65 a R is the correlation coefficient for the Ka values. b R is the correlation coefficient for the van’t Hoff plot.



60

6 60 0

1F R

EF R r

＝－＝＋

(6)

where E denotes the efficiency of energy transfer be-tween the donor and acceptor, and R0, the critical dis-tance at which the transfer efficiency equals 50%, and is given by the following equation:

6 25 2 40 8.79 10R K n Jφ－－

＝ × (7)

In Eq. (7), K2 is the space factor of orientation; n is the refracted index of medium; φ is the fluorescence quantum yield of the donor; J expresses the degree of spectral overlap between the donor emission spectrum and the acceptor absorption spectrum, which can be calculated by the equation:

4

0

0

( ) ( )

( )

F λ ε λ λ dλJ

F λ dλ

∞

∞∫

∫＝

(8)

where, F(λ) is the fluorescence intensity of the donor in the wavelength range λ to λ＋∆λ; and ε(λ) is the extinc-tion coefficient of the acceptor at λ.

The overlap of the absorption spectra of the caffeine with the fluorescence emission spectra of BSA was shown in Figure 7. In the present case, K2

＝2/3, n＝1.36, φ＝0.15.19 According to the Eqs. (6)—(8), we can ob-tain that R0＝3.56 nm and r＝5.96 nm. The average distance between a donor fluorophore and acceptor fluorophore is on the 2—8 nm scale,20 which indicate that the energy transfer from BSA to caffeine occurs with high probability.

Figure 7 Spectral overlap of caffeine absorption (a) with BSA fluorescence (b). c(BSA)＝c(caffeine)＝2.0×10－6 mol•L－1 .

Conformational change of BSA induced by caffeine

Synchronous fluorescence spectroscopy can give in-formation about the molecular environment in the vicin-ity of the chromosphere molecules at low concentrations under physiological condition. It involves the simulta-

neous scanning of the excitation and the fluorescence monochromators of a fluorimeter, while maintaining a fixed wavelength difference (∆λ) between them. When the D-value (∆λ) between excitation and emission wavelength is stabilized at 15 or 60 nm, the synchronous fluorescence gives the characteristic information of ty-rosine or tryptophan residues.21

To investigate the conformational changes of BSA by addition of caffeine, we measured the synchronous fluorescence spectroscopy (Figure 8) of BSA by adding various amounts of caffeine. It can be seen from Figure 8(a) that when ∆λ was set at 15 nm, the maximum emis-sion wavelength undergoes a slight red-shift of 3 nm (from 288 to 291 nm) in the investigated concentration range; and when ∆λ＝60 nm [Figure 8(b)], the maxi-mum emission wavelength red-shifts 6 nm (from 282 to 288 nm ), which suggested the conformation of BSA being changed and a more polar (or less hydrophobic) environment of both residues.22

Figure 8 Synchronous fluorescence spectra of BSA. (a) ∆λ＝15 nm; (b) ∆λ＝60 nm. c(BSA)＝2.0×10－6 mol•L－1; c(caffeine)/ (10－5 mol•L－1), A—K: 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, respectively.

For secondary-structure analysis of protein, CD spectroscopy is a sensitive technique which has been widely used. The CD spectra of BSA exhibit two nega-tive bands in the far-UV region at 208 and 222 nm, which is characteristic of the α-helix structure of protein (Figure 9, curve A). The reasonable explanation is that

Wu et al.FULL PAPER


the negative peaks between 208 and 222 nm both con-tribute to n→π* transfer for the peptide bond of the α-helix.23 As can be seen from Figure 9, the binding of caffeine to BSA decreases both of these bands, clearly indicating the decrease of the α-helical content in pro-tein. In order to quantify the different types of secondary structures content, the CD spectra have been analyzed by the algorithm SELCON3, with 43 mode proteins with known precise secondary structures used as the reference set.24,25 The fraction contents of different sec-ondary structures for BSA in the absence and presence of caffeine are shown in Table 3. A decreasing tendency of the α-helices content and an increasing tendency of β-strands, turn, and unordered structure contents were observed with the increasing concentration of caffeine. A decrease in α-helical content (the total content of regular and distorted α-helix) from 54.5% to 48.7% in-dicated that caffeine bound with the amino acid residues of the main polypeptide chain of protein and destroyed their hydrogen bonding networks.26 The conformation changes here implied that the serum albumin would adopt a more incompact conformation state and resulted in the decrease of the hydrophobicity. This conclusion agrees with the results of synchronous fluorescence spectra experiment.

Figure 9 CD spectra of BSA in the presence of caffeine at room temperature. c(BSA)＝2.0×10－6 mol•L－1; c(caffeine)/(10－6 mol• L－1), A—C: 0, 10.0, 20.0, respectively.

Table 3 Fractions of different secondary structures determined by SELCON3a

Molar ratio [BSA]∶[caffeine]

H(r)/% H(d)/% S(r)/% S(d)/% Trn/% Unrd/%

1∶0 30.5 24.0 2.1 5.8 19.7 22.6

1∶5 25.2 26.2 5.4 4.7 18.6 22.9

1∶10 21.0 27.7 11.1 7.0 18.2 13.7 a H(r): regular α-helix; H(d): distorted α-helix; S(r): regular β-strand; S(d): distorted β-strand; Trn: turns; Unrd: unordered structure.

Identification of the binding site

If the binding reaction in the serum albumin mole-

cule happens for the static quenching interaction, there are similar and independent binding sites in the serum albumin. The apparent binding constant Kb and binding sites n can be found from equation:27

0blg lg lg[Q]

F FK n

F

－＋＝ (6)

According to the relationship between 0lgF F

F

－

and lg[Q], the fit to the fluorescence data using Eq. (6) for the system of caffeine and BSA was found by setting n＝1.041. The value of n was approximately equal to 1, which shows the interaction of caffeine with BSA seems to be the presence of one class of binding site.

Crystal structure of BSA shows that BSA is a heart-shaped helical monomer composed of three ho-mologous domains named I, II and III, and each domain includes two sub-domains called A and B to form a cyl-inder. The principal regions of ligand-binding sites on albumin are located in sub-domains IIA and IIIA, which exhibit similar chemistry properties.28 The binding cavi-ties associated with sub-domains IIA and IIIA are also referred to as site I and site II according to the termi-nology proposed by Sudlow et al.29

Competitive binding of probes to serum albumin, such as warfarin (site I) or ibuprofen (site II) is often used to probe an unknown ligand of the binding region on serum albumin. Most reports observed that both warfarin and ibuprofen possess one high-affinity site (HAS) and several low-affinity binding sites (LAS).30 Moreover, HAS and LAS are both located in site I for warfarin, but HAS and LAS for ibuprofen lie in site II and site I, respectively. Generally, at a ligand/protein molar ratio not larger than 1, the ligand is mainly bound in its HAS but otherwise, it begins to bind in its LAS.31

To identify the binding site location of caffeine on the region of BSA, the competitive ligand binding to BSA is employed. As a result, the site marker of the displacement experiment was carried out, caffeine was added to solutions of BSA and site markers held in equimolar concentrations (1.0×10－5 mol•L－1). In the presence of warfarin, the fluorescence intensity of the caffeine-BSA complex was significantly lower than that in the absence of warfarin (Figure 10). By contrast, the occupancy by ibuprofen of their respective HAS did not prevent the binding of caffeine in its usual binding loca-tion. It seems clear that the behavior of caffeine in binding to BSA is much closer to typical site I markers than to site II markers. This is an indication that caffeine actually bind BSA within site I (IIA subdomain).

Measurement of fluorescence anisotropy has a great impact for its tremendous potential in biochemical re-search because any factor which affects size, shape, or segmental flexibility of a molecule will affect the ob-served anisotropy. An increase in the rigidity of the sur-rounding environment of a fluorophore results in an in-crease in the fluorescence anisotropy, i.e., fluorescence



Figure 10 Caffeine binding to BSA in the absence (■) and presence of selected site markers at 342 nm (T＝298 K, λex＝290 nm): (●) warfarin, (▲) ibuprofen. BSA and site marker concen-trations set at 1.0×10－5 mol•L－1.

anisotropy reflects the extent of restriction imposed by the microenvironment on the dynamic properties of the fluorophore, thus it can be exploited for finding out the probable location of the probe in the constrained media.32

Furthermore, we considered caffeine as a probe to compete with the binary mixture of warfarin-BSA. We examined the changes of the fluorescence anisotropy when caffeine was added to the binary mixture of 1.0×10－5 mol•L－1 warfarin with 1.0×10－5 mol•L－1 BSA (the excitation and emission wavelengths: 320 and 380 nm, the wavelengths of maxima absortion and emission for warfarin). Figure 11 reveals that with increasing caffeine concentrations fluorescence anisotropy (r) in-creases rapidly at the beginning and then levels off gradually. The gradual increase in the fluorescence ani-sotropy value implies an imposed motional restriction on warfarin introduced by caffeine in the protein me-dium. It suggests that the binding location of caffeine with BSA could be the same as the warfarin site I of BSA.33 With the addition of caffeine, attainment of the plateau in Figure 11 implies saturation in the binding

Figure 11 Correct fluorescence anistropy values of war-farin-BSA complex when different aliquots of caffeine were added. λex＝320 nm, λem＝380 nm, T＝298 K, c(BSA)＝1.0×

10－5 mol•L－1.

interaction between caffeine and BSA. The turning point of the anisotropy is approximately the equimolar of the ternary mixture, indicating that warfarin cannot be dis-placed by caffeine when it has bound to BSA. Warfarin competing with caffeine in the binding site I of BSA is possible to occur, because the binding constant of war-farin to BSA was higher than that of caffeine.34 In fact, site I may simultaneously accommodate two ligands.35 Our results reflect this multiple binding ability with formation of the ternary warfarin-caffeine-BSA complex. Taking the results discussed above, we can put forward that caffeine does bind at the region of site I (subdomain IIA), under the condition used in this work, and only binds to one, or predominately to one site on the BSA.

Conclusion

The interaction of caffeine with BSA under physio-logical condition was studied by spectral methods. The results indicated that the probable mechanism of caf-feine interaction with BSA is a static quenching process. The binding process was enthalpy driven, spontaneous and, as indicated by the thermodynamic parameters analyzed, van der Waals interactions and hydrogen bonds played major role in the reaction. The distance r between the donor (BSA) and acceptor (caffeine) was calculated to be 5.96 nm according to Förster’s energy transfer theory. The conformational changes of BSA induced by caffeine have been analyzed by means of CD and synchronous fluorescence spectra. These changes here implied that the serum albumin would adopt a more incompact conformation state and resulted in the de-crease of the hydrophobicity. Emission and anisotropy data show that warfarin and caffeine share a common binding site I corresponding to the subdomain IIA of BSA.

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