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Study of the interaction of Na9[SbW9O33]·19.5H2O with bovine serum albumin: Spectroscopic and voltammetric methods
ARTICLE IN PRESS Journal of Luminescence 129 (2009) 1320–1325
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Study of the interaction of Na9[SbW9O33] 19.5H2O with bovine serum albumin: Spectroscopic and voltammetric methods Su-Zhen Hao, Si-Dong Liu, Xiao-Hong Wang, Xiu-Jun Cui, Li-Ping Guo Faculty of Chemistry, Northeast Normal University, Changchun 130024, People’s Republic of China
a r t i c l e in f o
a b s t r a c t
Article history: Received 3 October 2008 Received in revised form 16 June 2009 Accepted 23 June 2009 Available online 30 June 2009
The interaction of Na9[SbW9O33] 19.5H2O (SbW) with bovine serum albumin (BSA) is studied by spectroscopic and voltammetric methods. Absorption spectroscopy of BSA and the linear sweep voltammetry of SbW proved the formation of ground-state SbW–BSA complex. Fluorescence quenching of serum albumin by SbW is also found to be a static quenching process. The binding constant Ka is 4.13 104 L mol1 for SbW–BSA at pH 7.40 Tris–HCl buffer at 295 K. The number of binding sites and the apparent binding constants at different temperatures are obtained from the analysis of the fluorescence quenching data. The thermodynamic parameters determined by the Van’t Hoff analysis of the binding constants (DH ¼ 80.01 kJ mol1 and DS ¼ 182.85 J mol1 K1) clearly show that the binding is absolutely entropy driven. Hydrogen bonding and van der Waals interaction force play major role in stabilizing the complex. The effect of SbW on the conformation of BSA is analyzed using synchronous fluorescence spectroscopy. & 2009 Elsevier B.V. All rights reserved.
Keywords: Heteropolyanions Bovine serum albumin Fluorescence quenching Linear sweep voltammetry Binding constant Conformation
1. Introduction Serum albumin is the most abundant soluble protein in the circulatory system and has high affinity to many endogenous and exogenous compounds. It serves as a solubilizer and transporter for drugs and other organic molecules to their targets. When drugs come into organism, they bind the target proteins first, which constitutes the first step in the complex mechanism of their biological action. Thus, the affinity between drug and serum albumin plays a crucial role in determining the bioavailability of many bioactive compounds. A detailed description of the structural and energetic aspects of these processes is expected to provide a rational basis for the fundamental understanding of the interaction and the development of efficient therapeutic agents [1]. Bovine serum albumin (BSA) is widely used in this kind of experiment because it has comparability with human serum albumin (HSA) and is more accessible than HSA. BSA is a major circulatory protein of well-known structure. It is made up of three homologous domains (I, II, III), which are divided into nine loops (L1–L9) by 17 disulfide bonds. The loops in each domain are made up of a sequence of large–small–large loops forming a triplet. Each domain in turn is the product of two sub-domains [2]. BSA molecule has two tryptophan residues that possess intrinsic fluorescence: Trp134 in the first sub-domain IB of the albumin molecule and Trp212 in sub-domain IIA. Trp212 is located within
Corresponding author. Tel.: +86 43185099762; fax: +86 413 85099762.
E-mail address: [email protected] (L.-P. Guo). 0022-2313/$ - see front matter & 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2009.06.020
a hydrophobic binding pocket of the protein and Trp134 is located on the surface of the albumin molecule [3,4]. Na9[SbW9O33] 19.5H2O (SbW) belonging to the heteropoly compounds, is sort of inorganic acid radical anion. Much attention has been paid to these compounds due to their unique combination of physical and chemical properties. Heteropoly compounds have been widely used in analytical and clinical chemistry, catalysis (including photocatalysis), medicine (antitumonal, antiviral and even anti-HIV activity), biochemistry (electron transport inhibition) and solid-state devices [5]. SbW with the typical keggin structure is a highly-efficient catalyst for selective oxidation of alcohols [6] and epoxidation of alkenes with H2O2 [7]. In the present investigation, interaction of SbW with BSA has been examined using steady state fluorescence and linear sweep voltammetry (LSV) measurements. This study assumes the importance in the context of understanding the carrier role of serum albumin for SbW in blood under physiological conditions. The parameters such as mode of interaction, association constant and number of binding sites are studied. In addition, the conformational change of BSA is discussed on the basis of synchronous fluorescence spectra.
2. Materials and methods 2.1. Materials BSA (approximately 96%) was purchased from Sigma (St. Louis, MO, USA) and used without further purification. Na2WO4 2H2O
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and Sb2O3 were obtained from technology company (Beijing, China). SbW was synthesized and purified according to the procedures described previously [8]. The Tris (99.7%) was purchased from SeaskyBio technology company (Beijing, China), and NaCl, HCl, etc. were all of analytical purity. NaCl (1 mol L1) solution was used to maintain the ionic strength at 0.1. Tris (0.1 mol L1)–HCl (0.1 mol L1) buffer solution containing NaCl (0.1 mol L1) was used to keep the pH of the solution at 7.40. BSA solution (5.0 mmol L1) and SbW solution (1.0 104 mol L1) were prepared in pH 7.40 Tris–HCl buffer solution.
2.2. Equipments and spectral measurements 2.2.1. Spectral methods UV–vis absorption spectra were recorded using Cary 500 UV–vis spectrophotometer (Varian Australia) at room temperature in the range 200–500 nm. The fluorescence quenching measurements and synchronous spectra were carried out in a Cary Eclipse fluorescence spectrophotometer (Varian Australia) attached with 1.0 cm quartz cells and a thermostated circulating water bath. The excitation wavelength was 280 nm and the emission spectra were recorded between 300 and 450 nm. The excitation and emission slit width (each 5 nm), scan rate (600 nm/ min) were constantly maintained for all the experiments. The range of synchronous scanning were Dl ¼ 15 and 60 nm. Appropriate blanks corresponding to the buffer were subtracted to correct background of fluorescence. Fluorometric titration experiments: a 2.0 mL solution, containing appropriate concentration of BSA, was titrated by successive additions of a 1.0 104 mol L1 stock solution of SbW. Titrations were done manually by using micro-injector. The fluorescence spectra were then measured at two temperatures (295, 309 K). The data obtained were analyzed to calculate the binding constants and the number of binding sites. Synchronous fluorescence spectra of BSA in the absence and presence of increasing amount of SbW were recorded to determine the conformation change of BSA in the presence of SbW.
2.2.2. Electrochemical method Electrochemical experiments were performed on a CHI 610a Electrochemical Analyzer (Chenhua Instruments, China) in a conventional three-electrode cell. The working electrode used was glassy carbon electrode (Model CHI104, 3 mm diameter). A platinum electrode was taken as the counter electrode and an Ag/ AgCl (in saturated KCl solution) electrode served as reference electrode. The SbW with concentration of 1.0 104 mol L1 was titrated by the increasing amount of BSA solution at room temperature and the data were collected.
3. Results and discussion 3.1. Absorption spectra UV–vis absorption measurement is a very simple method and applicable to explore the structural change and to know the complex formation. Hence, the absorption spectra of BSA in the presence and absence of SbW are shown in Fig. 1. The absorption of BSA is characterized by a strong band at 278 nm. SbW has no absorption band in the range 250–350 nm. The absorption of BSA increases with the addition of SbW and the peak has a blue-shift. These observations can be rationalized in terms of interactions between SbW and BSA in the ground state, and formation of a ground-state complex.
Fig. 1. The UV–vis absorption of 5.0 mmol L1 BSA in the absence and presence of 5.0 mmol L1 SbW.
Fig. 2. The change of BSA emission spectrum with the addition of SbW (pH ¼ 7.4, T ¼ 295 K, lex ¼ 280 nm). Fix BSA concentration at 5.0 mmol L1, curves from (a) to (j) respond to 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 22.5 mmol L1 SbW, respectively.
3.2. The fluorescence quenching mechanism BSA contains luminophores and presents strong fluorescence emissions in aqueous solution. The intrinsic fluorescence of proteins is sensitive to its local environment. Changes in the emission spectra are common in response to protein conformational transitions, subunit association, substrate binding or denaturation [9]. Consequently, the fluorescence quenching is an effective method to study the protein folding and association reactions, which can determine the accessibility of ligand to BSA and offer some information such as the binding mechanism, binding mode, binding constants, binding sites. To determine the nature of interaction between SbW and BSA, the spectra of BSA with various amount of SbW were recorded in the range 300–450 nm upon excitation at 280 nm (as shown in Fig. 2). As the Fig. 2 shows, the fluorescence intensity of BSA can be decreased regularly by SbW, such decrease in intensity is called fluorescence quenching. Generally speaking, fluorescence quenching is the decrease of the quantum yield of fluorophore induced by a variety of molecular interactions with quencher molecule, such
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as excited-state reaction, molecules’ rearrangement, energy transfer, ground-state complex formation and collision quenching [10]. We may conclude that there is a static mechanism between BSA and SbW because the maxima emission and shape of peaks have not been changed. In order to confirm this view, the process is assumed to be dynamic quenching. It can be described by the Stern–Volmer equation [11] F0 ¼ 1 þ Kqt0 ½Q ¼ 1 þ KSV ½Q F
ð1Þ
where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. Kq is the bimolecular quenching constant, t0 the average lifetime of the bimolecular without quencher (t0 ¼ 108 s), [Q] the concentration of the quencher and KSV is the Stern–Volmer dynamic quenching constant. The KSV value is obtained from the slope of the Stern–Volmer plot (Fig. 3). By using Eq. (1), the quenching constants Kq at different temperatures are calculated (as shown in Table 1). Fig. 3 shows that the curves have good linear relationships. The linear Stern–Volmer plots may either reveal the occurrence of just a binding site for quencher in the proximity of the fluorophore, or indicate the existence of a single type of quenching (dynamic or static). The obtained Kq values are in the range of 1012 L mol1 s1, which far exceed the diffusion controlled rate constant in aqueous solution 2.0 1010 L mol1 s1 [12], confirming that quenching does not involve the dynamic diffusion process but occurs statically in the BSA–SbW complex. In addition, the slopes (KSV) decrease with the rise in temperature, which also indicates the occurrence of static quenching interaction between SbW and BSA. Since higher temperatures result in larger diffusion coefficients, the bimolecular quenching constants are expected to increase with increasing temperature in dynamic quenching. The fluorescence data were further examined using modified Stern–Volmer equation [13]
Fig. 4. Modified Stern–Volmer plots for the quenching of BSA by SbW at different temperatures.
Table 2 Modified Stern–Volmer equations and association constants Ka at different temperatures. pH
T/K
7.40
295 309
Regression equation 5
1
F0/(F0F) ¼ 1.94+4.7 10 [Q] F0/(F0F) ¼ 0.78+8.28 105[Q]1
F0 1 1 1 ¼ þ F0 F fa Ka ½Q fa
Ka(L mol1)
R
4.13 104 9.42 103
0.9997 0.9969
ð2Þ
Ka is the effective quenching constant for the accessible fluorophores, and fa the fraction of accessible fluorescence. Fig. 4 displays the modified Stern–Volmer plots and the corresponding results of Ka values at different temperatures are shown in Table 2. The decreasing trend of Ka with increasing temperature is in accordance with KSV’s dependence on temperature as mentioned above. The Ka values show that the binding between SbW and BSA is moderate. Thus, SbW can be stored and carried by protein. 3.3. Binding constant and the binding sites When small molecules bind independently to a set of equivalent sites on a macromolecule, the apparent binding constant K and binding sites n can be obtained from Eq. [14] log
Fig. 3. Stern–Volmer polts for the steady fluorescence quenching of BSA by SbW at different temperatures.
Table 1 Stern–Volmer quenching constants for the interaction of SbW with BSA at different temperatures. pH 7.40
T/K 295 309
KSV (L mol1)
Regression equation 4
F0/F ¼ 1.00+1.61 10 [Q] F0/F ¼ 1.00+1.25 104[Q]
4
1.61 10 1.25 104
Kq (L mol1 s1) 12
1.61 10 1.25 1012
R 0.9980 0.9985
F0 F ¼ log K þ n log½Q F
ð3Þ
where F0 and F are the fluorescence intensities before and after the addition of the quencher, [Q] the total quencher concentration. Fig. 5 is the plots of log(F0F)/F versus log[Q] for the SbW–BSA system at different temperatures obtained from the fluorometric titration. It can be seen from Fig. 5 that the plots of log(F0F)/F versus log[Q] have good linear relationship in the range of concentration of SbW studied. The values of n at the experimental temperature are approximately equal to 1, which indicates that there maybe a single class-binding site in the neighborhood of the tryptophan residues. In BSA, the tryptophan residues involved in binding could be either Trp134 or Trp212 of both tryptophans. Trp134 is more exposed to a hydrophilic environment, whereas Trp212 is deeply buried in the hydrophobic loop. From the value of n, it is proposed that SbW most likely binds to the hydrophobic pocket
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located in sub-domain IIA; that is to say, Trp212 is near or within the binding site [15].
3.4. Effects of ionic strength and pH on binding of SbW to BSA In order to investigate the influence of the ionic strength on the binding process, the fluorescence experiments were carried out in the presence of different concentrations of NaCl (0, 0.1, 1.0 mol L1) (Fig. 6a). For NaCl, which is not an anionic quencher, its influence on the BSA fluorescence intensity only comes from the ionic strength [16]. Fig. 6a shows the modified Stern–Volmer plots in the solution with different ionic strength. The slopes of plots are actually related to the binding constants of the tryptophans to the aqueous quencher (SbW). As can be seen in Fig. 6a, increasing the concentration of NaCl from 0.1 to 1.0 mol L1, there is no significant change in the slope, which implies that the binding ability of SbW to BSA is not changed and the electrostatic interaction does not work in this range. The binding constant obtained is decreased from 3.74 105 to 4.13 104 L mol1 when concentration of NaCl increases from 0 to 0.1 mol L1. When ionic strength of the solution increases in this range, the selfaggregation of the lipophilic group occurs so the steric effect of
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BSA core becomes serious. It is unfavorable for insertion of SbW into BSA. The fact that the quenching is not favored by the increase of the ionic strength from 0 to 0.1 mol L1, leads to the idea that the electrostatic interactions should be part of the binding forces in this range. We also investigate the quenching experiments of BSA in the presence of SbW at different pH separately. The results are displayed in Fig. 6b and dealt with modified Stern–Volmer equation. From the Fig. 6b, it is favorable for the binding of SbW with BSA at pH 3.0 and 8.9, which is induced by the different conformation of BSA at different pH. On the basis of Fullerton’s conclusion [17], in the region pH 1–3, there is strong electrostatic repulsion between the positive charges on the surface of the protein. This denaturing results in the extending of the length, and BSA molecule becomes fully uncoiled within the limits of its disulfide bonds. In the region pH 3–8, as the pH approaches the isoelectric point of BSA (pH 5.4), the charges on the surface of the protein are insufficient to cause a strong repulsive force. This results in a compact protein structure. In the region pH 8–11, the negative charges on the protein surface repel each other leading to expansion of the protein. And the unfolding of BSA in alkaline solution is not extreme as in acid since aggregation is found above pH 9. So at pH 3 and 8.9, BSA exists in the extendible structure comparing with the compact structure at neutral solution. As the protein unfolds, the Trp212 located within hydrophobic pocket will be exposed, allowing a better interaction between Trp and SbW.
3.5. Thermodynamic parameters and nature of the binding forces
Fig. 5. Plots of log[(F0F)/F]] versus log[Q].
In the past decade, a complete thermodynamic description of the self-association of many proteins and their interactions with small molecular substrates has become available [18]. The interaction forces between drug and host may involve hydrophobic forces, electrostatic interactions, van der Waals forces, Hbonds, etc. Depending on the extent of enthalpy change (DH) and entropy change (DS), different binding models for the interaction between drug and biomolecule have been proposed [19]. To obtain such information, the temperature dependence of the binding constant is studied. Experiments are carried out at 295 and 309 K, since BSA does not undergo any gross structural change in this temperature range. The thermodynamic parameters of BSA–SbW complex were calculated according to the Van’t Hoff equation. Assuming that the temperature does not vary
Fig. 6. The modified Stern–Volmer plots of BSA in the presence of SbW at different concentration of NaCl (a) and different pH (b).
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significantly, the enthalpy change DH can be regarded as constant. ðKa Þ2 DH 1 1 ln ð4Þ ¼ ðKa Þ1 R T1 T2
DG ¼ RT ln Ka DS ¼
ð5Þ
DH DG
ð6Þ
T
where R is the universal gas constant, T the experimental temperature (in Kelvin) and Ka the analogous to the effective quenching constant at the corresponding temperature. Then, DH and DS can be calculated. The results are displayed in Table 3. Table 3 The binding site n at different temperatures and relative thermodynamic parameters of the BSA–SbW system. T/K
DH (kJ mol1)
DS (J mol1 K1)
DG (kJ mol1)
n
295 309
80.01
182.85 182.85
26.07 23.51
0.89 1.00
As DH and DS are both negative, thus the key binding forces in this case are van der Waals interaction and hydrogen bonding [18]. The negative values of DG and DH mean that the binding process is spontaneous and the formation of BSA–SbW complex is an exothermic reaction. At pH 7.4 solutions, the serum albumin bears negative charge because of the ionization of amino acid residues, and SbW is negatively charged in aqueous environment, so electrostatic interaction are not likely to occur. 3.6. Electrochemical behavior of SbW By the electrochemical method, we can make sure that there is interaction between SbW and BSA, and the binding reaction results in non-electroactive complexes. The LSV curves of 1.0 104 mol L1 SbW in the absence (curve a) and presence (curves b, c, d, e and f) of BSA are shown in Fig. 7. We can see that SbW has a reduction peak at 1.58 V without BSA, and this peak current exhibits a distinct decrease with the addition of BSA. The decrease in the peak current is attributed to the formation of SbW–BSA, a non-electroactive complex, which results in the decrease of equilibrium concentration of SbW in solution. On the other hand, the addition of BSA makes the reduction peak potential shifts in a positive direction, which can be attributed to the changes of the molecular environment of SbW as a result of its interaction with BSA. 3.7. Conformation investigation
Fig. 7. LSV curves of 1.0 104 mol L1 SbW with increasing concentrations of BSA in solution. Curves from (a) to (f) correspond to 0, 1.0, 3.0, 5.0, 7.0, 9.0 mmol L1 BSA, respectively. Curve g: the blank solution consists of Tris–HCl buffer and 0.1 mol L1 NaCl.
It is well known that the fluorescence of BSA comes from the tyrosine, tryptophan and phenylalanine residues. The spectrum of BSA is sensitive to the micro-environment of these chromophores and it allows non-intrusive measurements of protein in low concentration under physiological conditions. The synchronous fluorescence spectra can give information about the molecular environment in a vicinity of the chromophores and have several advantages, such as sensitivity, spectral simplification, spectral bandwidth reduction and avoiding different perturbing effects [20]. When the D-value (Dl) (the difference between excitation wavelength and emission wavelength) is set at 15 or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine or tryptophan residues. A useful method to study the environment of amino acid residues has been reported by Yuan et al. [21], which by measuring the possible shift in maximum emission wavelength lmax. The shift in position of lmax corresponds to the changes of the polarity around the chromophore
Fig. 8. Synchronous fluorescence spectra of BSA (5.0 mmol L1) with Dl ¼ 60 nm (a) and 15 nm (b) in the absence and presence of SbW (up to down): 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 mmol L1, pH ¼ 7.40, T ¼ 295 K.
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molecule [22]. The dominant fluorophore in BSA is the indole group of tryptophan. Indole absorbs near 280 nm and emits near 340 nm. The emission of indole may be blue-shifted if the group is buried within a native protein, and its emission may shift to longer wavelengths (red-shift) when protein is unfolded [23]. When considering the effect of SbW on the fluorescence spectra of BSA, there is no shift in position of lmax when Dl ¼ 60 nm (Fig. 8a). This suggests that there is no change in the immediate environment of the tryptophan residues. It is apparent from Fig. 8b that the maximum emission wavelength has no significant shift either at the investigated concentration range when Dl ¼ 15 nm, which indicates that the polarity around the tyrosine residues has not been changed. So the interaction of SbW with BSA does not obviously affect the conformation of tryptophan and tyrosine micro-region.
4. Conclusions Interaction between SbW and BSA is investigated by spectral and electrochemical methods. It is found that SbW is a strong quencher of BSA fluorescence and the nature of quenching is static. The binding constants at different temperatures are evaluated. The thermodynamic parameters of the binding interaction are determined and the values suggest that van der Waals interaction and hydrogen bonding are the key binding forces. The effects of pH and ionic strength on the binding constants between SbW and BSA are also studied. In the conformational investigation, the synchronous fluorescence spectra reveal that the conformation of BSA is not changed in the presence of SbW. The binding study of inorganic drug with protein is of great importance in pharmacy, pharmacology and biochemistry. This study is expected to provide important insight into the interactions of the
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physiologically important protein BSA with inorganic ions keggin shaped.
Acknowledgement The authors gratefully acknowledge the financial support by the Analysis and Testing Foundation of Northeast Normal University. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
G.J. Zhang, B. Keita, J. Phys. Chem. B 111 (2007) 11253. A. Papadopoulou, R.J. Green, R.A. Frazier, J. Agric. Food Chem. 53 (2005) 158. U. Kragh-Hansen, Pharmacol. Rev. 33 (1981) 17. T. Peter, Adv. Protein Chem. 37 (1985) 161. X.G. Sang, Q.G. Wu, W.Q. Pang, Mater. Chem. Phys. 82 (2003) 405. Rohit H. Ingle, N.K. Kala Raj, P. Manikandan, J. Mol. Catal. A: Chem. 262 (2007) 52. Rohit H. Ingle, N.K. Kala Raj, J. Mol. Catal. A: Chem. 294 (2008) 8. ¨ Michael Bosing, Ina Loose, Heinrich Pohlmann, Bernt Krebs, J. Chem. Eur. 3 (1997) 1232. A. Sulkowska, J. Mol. Struct. 614 (2002) 227. Y.J. Hu, Y. Liu, L.X. Zhang, J. Mol. Struct. 750 (2005) 174. J.R. Lakowicz, G. Weber, Biochemistry 12 (1973) 4171. W.R. Ware, J. Phys. Chem. 66 (1962) 455. S.S. Lehrer, Biochemistry 10 (1971) 3254. M. Alain, B. Michel, D. Michel, J. Chem. Educ. 63 (1986) 365. A. Sulkowska, J. Mol. Struct. 614 (2002) 227. G. Prativel, J. Bernadou, B. Meunier, Angew. Chem. Int. Ed. Engl. 34 (1995) 746. Gary D. Fullerton, Kalpana M. Kanal, Ivan L. Cameron, Cell Biol. Int. 30 (2006) 86. P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096. D.T. Haynie, Biological Thermodynamics, Cambridge University Press, Cambridge, 2001, pp. 73–117. J.N. Miller, Proc. Anal. Div. Chem. Soc. 16 (1979) 203. T. Yuan, A.M. Weljie, H.J. Vogel, Biochemistry 37 (1998) 3187. B.F. Pan, F. Gao, L.M. Ao, J. Magn. Magn. Mater. 293 (2005) 252. W.C. Abert, W.M. Gregory, G.S. Allan, Anal. Biochem. 213 (1993) 407.
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Study of the interaction of Na9[SbW9O33] 19.5H2O with bovine serum albumin: Spectroscopic and voltammetric methods Su-Zhen Hao, Si-Dong Liu, Xiao-Hong Wang, Xiu-Jun Cui, Li-Ping Guo Faculty of Chemistry, Northeast Normal University, Changchun 130024, People’s Republic of China
a r t i c l e in f o
a b s t r a c t
Article history: Received 3 October 2008 Received in revised form 16 June 2009 Accepted 23 June 2009 Available online 30 June 2009
The interaction of Na9[SbW9O33] 19.5H2O (SbW) with bovine serum albumin (BSA) is studied by spectroscopic and voltammetric methods. Absorption spectroscopy of BSA and the linear sweep voltammetry of SbW proved the formation of ground-state SbW–BSA complex. Fluorescence quenching of serum albumin by SbW is also found to be a static quenching process. The binding constant Ka is 4.13 104 L mol1 for SbW–BSA at pH 7.40 Tris–HCl buffer at 295 K. The number of binding sites and the apparent binding constants at different temperatures are obtained from the analysis of the fluorescence quenching data. The thermodynamic parameters determined by the Van’t Hoff analysis of the binding constants (DH ¼ 80.01 kJ mol1 and DS ¼ 182.85 J mol1 K1) clearly show that the binding is absolutely entropy driven. Hydrogen bonding and van der Waals interaction force play major role in stabilizing the complex. The effect of SbW on the conformation of BSA is analyzed using synchronous fluorescence spectroscopy. & 2009 Elsevier B.V. All rights reserved.
Keywords: Heteropolyanions Bovine serum albumin Fluorescence quenching Linear sweep voltammetry Binding constant Conformation
1. Introduction Serum albumin is the most abundant soluble protein in the circulatory system and has high affinity to many endogenous and exogenous compounds. It serves as a solubilizer and transporter for drugs and other organic molecules to their targets. When drugs come into organism, they bind the target proteins first, which constitutes the first step in the complex mechanism of their biological action. Thus, the affinity between drug and serum albumin plays a crucial role in determining the bioavailability of many bioactive compounds. A detailed description of the structural and energetic aspects of these processes is expected to provide a rational basis for the fundamental understanding of the interaction and the development of efficient therapeutic agents [1]. Bovine serum albumin (BSA) is widely used in this kind of experiment because it has comparability with human serum albumin (HSA) and is more accessible than HSA. BSA is a major circulatory protein of well-known structure. It is made up of three homologous domains (I, II, III), which are divided into nine loops (L1–L9) by 17 disulfide bonds. The loops in each domain are made up of a sequence of large–small–large loops forming a triplet. Each domain in turn is the product of two sub-domains [2]. BSA molecule has two tryptophan residues that possess intrinsic fluorescence: Trp134 in the first sub-domain IB of the albumin molecule and Trp212 in sub-domain IIA. Trp212 is located within
Corresponding author. Tel.: +86 43185099762; fax: +86 413 85099762.
E-mail address: [email protected] (L.-P. Guo). 0022-2313/$ - see front matter & 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2009.06.020
a hydrophobic binding pocket of the protein and Trp134 is located on the surface of the albumin molecule [3,4]. Na9[SbW9O33] 19.5H2O (SbW) belonging to the heteropoly compounds, is sort of inorganic acid radical anion. Much attention has been paid to these compounds due to their unique combination of physical and chemical properties. Heteropoly compounds have been widely used in analytical and clinical chemistry, catalysis (including photocatalysis), medicine (antitumonal, antiviral and even anti-HIV activity), biochemistry (electron transport inhibition) and solid-state devices [5]. SbW with the typical keggin structure is a highly-efficient catalyst for selective oxidation of alcohols [6] and epoxidation of alkenes with H2O2 [7]. In the present investigation, interaction of SbW with BSA has been examined using steady state fluorescence and linear sweep voltammetry (LSV) measurements. This study assumes the importance in the context of understanding the carrier role of serum albumin for SbW in blood under physiological conditions. The parameters such as mode of interaction, association constant and number of binding sites are studied. In addition, the conformational change of BSA is discussed on the basis of synchronous fluorescence spectra.
2. Materials and methods 2.1. Materials BSA (approximately 96%) was purchased from Sigma (St. Louis, MO, USA) and used without further purification. Na2WO4 2H2O
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and Sb2O3 were obtained from technology company (Beijing, China). SbW was synthesized and purified according to the procedures described previously [8]. The Tris (99.7%) was purchased from SeaskyBio technology company (Beijing, China), and NaCl, HCl, etc. were all of analytical purity. NaCl (1 mol L1) solution was used to maintain the ionic strength at 0.1. Tris (0.1 mol L1)–HCl (0.1 mol L1) buffer solution containing NaCl (0.1 mol L1) was used to keep the pH of the solution at 7.40. BSA solution (5.0 mmol L1) and SbW solution (1.0 104 mol L1) were prepared in pH 7.40 Tris–HCl buffer solution.
2.2. Equipments and spectral measurements 2.2.1. Spectral methods UV–vis absorption spectra were recorded using Cary 500 UV–vis spectrophotometer (Varian Australia) at room temperature in the range 200–500 nm. The fluorescence quenching measurements and synchronous spectra were carried out in a Cary Eclipse fluorescence spectrophotometer (Varian Australia) attached with 1.0 cm quartz cells and a thermostated circulating water bath. The excitation wavelength was 280 nm and the emission spectra were recorded between 300 and 450 nm. The excitation and emission slit width (each 5 nm), scan rate (600 nm/ min) were constantly maintained for all the experiments. The range of synchronous scanning were Dl ¼ 15 and 60 nm. Appropriate blanks corresponding to the buffer were subtracted to correct background of fluorescence. Fluorometric titration experiments: a 2.0 mL solution, containing appropriate concentration of BSA, was titrated by successive additions of a 1.0 104 mol L1 stock solution of SbW. Titrations were done manually by using micro-injector. The fluorescence spectra were then measured at two temperatures (295, 309 K). The data obtained were analyzed to calculate the binding constants and the number of binding sites. Synchronous fluorescence spectra of BSA in the absence and presence of increasing amount of SbW were recorded to determine the conformation change of BSA in the presence of SbW.
2.2.2. Electrochemical method Electrochemical experiments were performed on a CHI 610a Electrochemical Analyzer (Chenhua Instruments, China) in a conventional three-electrode cell. The working electrode used was glassy carbon electrode (Model CHI104, 3 mm diameter). A platinum electrode was taken as the counter electrode and an Ag/ AgCl (in saturated KCl solution) electrode served as reference electrode. The SbW with concentration of 1.0 104 mol L1 was titrated by the increasing amount of BSA solution at room temperature and the data were collected.
3. Results and discussion 3.1. Absorption spectra UV–vis absorption measurement is a very simple method and applicable to explore the structural change and to know the complex formation. Hence, the absorption spectra of BSA in the presence and absence of SbW are shown in Fig. 1. The absorption of BSA is characterized by a strong band at 278 nm. SbW has no absorption band in the range 250–350 nm. The absorption of BSA increases with the addition of SbW and the peak has a blue-shift. These observations can be rationalized in terms of interactions between SbW and BSA in the ground state, and formation of a ground-state complex.
Fig. 1. The UV–vis absorption of 5.0 mmol L1 BSA in the absence and presence of 5.0 mmol L1 SbW.
Fig. 2. The change of BSA emission spectrum with the addition of SbW (pH ¼ 7.4, T ¼ 295 K, lex ¼ 280 nm). Fix BSA concentration at 5.0 mmol L1, curves from (a) to (j) respond to 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 22.5 mmol L1 SbW, respectively.
3.2. The fluorescence quenching mechanism BSA contains luminophores and presents strong fluorescence emissions in aqueous solution. The intrinsic fluorescence of proteins is sensitive to its local environment. Changes in the emission spectra are common in response to protein conformational transitions, subunit association, substrate binding or denaturation [9]. Consequently, the fluorescence quenching is an effective method to study the protein folding and association reactions, which can determine the accessibility of ligand to BSA and offer some information such as the binding mechanism, binding mode, binding constants, binding sites. To determine the nature of interaction between SbW and BSA, the spectra of BSA with various amount of SbW were recorded in the range 300–450 nm upon excitation at 280 nm (as shown in Fig. 2). As the Fig. 2 shows, the fluorescence intensity of BSA can be decreased regularly by SbW, such decrease in intensity is called fluorescence quenching. Generally speaking, fluorescence quenching is the decrease of the quantum yield of fluorophore induced by a variety of molecular interactions with quencher molecule, such
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as excited-state reaction, molecules’ rearrangement, energy transfer, ground-state complex formation and collision quenching [10]. We may conclude that there is a static mechanism between BSA and SbW because the maxima emission and shape of peaks have not been changed. In order to confirm this view, the process is assumed to be dynamic quenching. It can be described by the Stern–Volmer equation [11] F0 ¼ 1 þ Kqt0 ½Q ¼ 1 þ KSV ½Q F
ð1Þ
where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. Kq is the bimolecular quenching constant, t0 the average lifetime of the bimolecular without quencher (t0 ¼ 108 s), [Q] the concentration of the quencher and KSV is the Stern–Volmer dynamic quenching constant. The KSV value is obtained from the slope of the Stern–Volmer plot (Fig. 3). By using Eq. (1), the quenching constants Kq at different temperatures are calculated (as shown in Table 1). Fig. 3 shows that the curves have good linear relationships. The linear Stern–Volmer plots may either reveal the occurrence of just a binding site for quencher in the proximity of the fluorophore, or indicate the existence of a single type of quenching (dynamic or static). The obtained Kq values are in the range of 1012 L mol1 s1, which far exceed the diffusion controlled rate constant in aqueous solution 2.0 1010 L mol1 s1 [12], confirming that quenching does not involve the dynamic diffusion process but occurs statically in the BSA–SbW complex. In addition, the slopes (KSV) decrease with the rise in temperature, which also indicates the occurrence of static quenching interaction between SbW and BSA. Since higher temperatures result in larger diffusion coefficients, the bimolecular quenching constants are expected to increase with increasing temperature in dynamic quenching. The fluorescence data were further examined using modified Stern–Volmer equation [13]
Fig. 4. Modified Stern–Volmer plots for the quenching of BSA by SbW at different temperatures.
Table 2 Modified Stern–Volmer equations and association constants Ka at different temperatures. pH
T/K
7.40
295 309
Regression equation 5
1
F0/(F0F) ¼ 1.94+4.7 10 [Q] F0/(F0F) ¼ 0.78+8.28 105[Q]1
F0 1 1 1 ¼ þ F0 F fa Ka ½Q fa
Ka(L mol1)
R
4.13 104 9.42 103
0.9997 0.9969
ð2Þ
Ka is the effective quenching constant for the accessible fluorophores, and fa the fraction of accessible fluorescence. Fig. 4 displays the modified Stern–Volmer plots and the corresponding results of Ka values at different temperatures are shown in Table 2. The decreasing trend of Ka with increasing temperature is in accordance with KSV’s dependence on temperature as mentioned above. The Ka values show that the binding between SbW and BSA is moderate. Thus, SbW can be stored and carried by protein. 3.3. Binding constant and the binding sites When small molecules bind independently to a set of equivalent sites on a macromolecule, the apparent binding constant K and binding sites n can be obtained from Eq. [14] log
Fig. 3. Stern–Volmer polts for the steady fluorescence quenching of BSA by SbW at different temperatures.
Table 1 Stern–Volmer quenching constants for the interaction of SbW with BSA at different temperatures. pH 7.40
T/K 295 309
KSV (L mol1)
Regression equation 4
F0/F ¼ 1.00+1.61 10 [Q] F0/F ¼ 1.00+1.25 104[Q]
4
1.61 10 1.25 104
Kq (L mol1 s1) 12
1.61 10 1.25 1012
R 0.9980 0.9985
F0 F ¼ log K þ n log½Q F
ð3Þ
where F0 and F are the fluorescence intensities before and after the addition of the quencher, [Q] the total quencher concentration. Fig. 5 is the plots of log(F0F)/F versus log[Q] for the SbW–BSA system at different temperatures obtained from the fluorometric titration. It can be seen from Fig. 5 that the plots of log(F0F)/F versus log[Q] have good linear relationship in the range of concentration of SbW studied. The values of n at the experimental temperature are approximately equal to 1, which indicates that there maybe a single class-binding site in the neighborhood of the tryptophan residues. In BSA, the tryptophan residues involved in binding could be either Trp134 or Trp212 of both tryptophans. Trp134 is more exposed to a hydrophilic environment, whereas Trp212 is deeply buried in the hydrophobic loop. From the value of n, it is proposed that SbW most likely binds to the hydrophobic pocket
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located in sub-domain IIA; that is to say, Trp212 is near or within the binding site [15].
3.4. Effects of ionic strength and pH on binding of SbW to BSA In order to investigate the influence of the ionic strength on the binding process, the fluorescence experiments were carried out in the presence of different concentrations of NaCl (0, 0.1, 1.0 mol L1) (Fig. 6a). For NaCl, which is not an anionic quencher, its influence on the BSA fluorescence intensity only comes from the ionic strength [16]. Fig. 6a shows the modified Stern–Volmer plots in the solution with different ionic strength. The slopes of plots are actually related to the binding constants of the tryptophans to the aqueous quencher (SbW). As can be seen in Fig. 6a, increasing the concentration of NaCl from 0.1 to 1.0 mol L1, there is no significant change in the slope, which implies that the binding ability of SbW to BSA is not changed and the electrostatic interaction does not work in this range. The binding constant obtained is decreased from 3.74 105 to 4.13 104 L mol1 when concentration of NaCl increases from 0 to 0.1 mol L1. When ionic strength of the solution increases in this range, the selfaggregation of the lipophilic group occurs so the steric effect of
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BSA core becomes serious. It is unfavorable for insertion of SbW into BSA. The fact that the quenching is not favored by the increase of the ionic strength from 0 to 0.1 mol L1, leads to the idea that the electrostatic interactions should be part of the binding forces in this range. We also investigate the quenching experiments of BSA in the presence of SbW at different pH separately. The results are displayed in Fig. 6b and dealt with modified Stern–Volmer equation. From the Fig. 6b, it is favorable for the binding of SbW with BSA at pH 3.0 and 8.9, which is induced by the different conformation of BSA at different pH. On the basis of Fullerton’s conclusion [17], in the region pH 1–3, there is strong electrostatic repulsion between the positive charges on the surface of the protein. This denaturing results in the extending of the length, and BSA molecule becomes fully uncoiled within the limits of its disulfide bonds. In the region pH 3–8, as the pH approaches the isoelectric point of BSA (pH 5.4), the charges on the surface of the protein are insufficient to cause a strong repulsive force. This results in a compact protein structure. In the region pH 8–11, the negative charges on the protein surface repel each other leading to expansion of the protein. And the unfolding of BSA in alkaline solution is not extreme as in acid since aggregation is found above pH 9. So at pH 3 and 8.9, BSA exists in the extendible structure comparing with the compact structure at neutral solution. As the protein unfolds, the Trp212 located within hydrophobic pocket will be exposed, allowing a better interaction between Trp and SbW.
3.5. Thermodynamic parameters and nature of the binding forces
Fig. 5. Plots of log[(F0F)/F]] versus log[Q].
In the past decade, a complete thermodynamic description of the self-association of many proteins and their interactions with small molecular substrates has become available [18]. The interaction forces between drug and host may involve hydrophobic forces, electrostatic interactions, van der Waals forces, Hbonds, etc. Depending on the extent of enthalpy change (DH) and entropy change (DS), different binding models for the interaction between drug and biomolecule have been proposed [19]. To obtain such information, the temperature dependence of the binding constant is studied. Experiments are carried out at 295 and 309 K, since BSA does not undergo any gross structural change in this temperature range. The thermodynamic parameters of BSA–SbW complex were calculated according to the Van’t Hoff equation. Assuming that the temperature does not vary
Fig. 6. The modified Stern–Volmer plots of BSA in the presence of SbW at different concentration of NaCl (a) and different pH (b).
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significantly, the enthalpy change DH can be regarded as constant. ðKa Þ2 DH 1 1 ln ð4Þ ¼ ðKa Þ1 R T1 T2
DG ¼ RT ln Ka DS ¼
ð5Þ
DH DG
ð6Þ
T
where R is the universal gas constant, T the experimental temperature (in Kelvin) and Ka the analogous to the effective quenching constant at the corresponding temperature. Then, DH and DS can be calculated. The results are displayed in Table 3. Table 3 The binding site n at different temperatures and relative thermodynamic parameters of the BSA–SbW system. T/K
DH (kJ mol1)
DS (J mol1 K1)
DG (kJ mol1)
n
295 309
80.01
182.85 182.85
26.07 23.51
0.89 1.00
As DH and DS are both negative, thus the key binding forces in this case are van der Waals interaction and hydrogen bonding [18]. The negative values of DG and DH mean that the binding process is spontaneous and the formation of BSA–SbW complex is an exothermic reaction. At pH 7.4 solutions, the serum albumin bears negative charge because of the ionization of amino acid residues, and SbW is negatively charged in aqueous environment, so electrostatic interaction are not likely to occur. 3.6. Electrochemical behavior of SbW By the electrochemical method, we can make sure that there is interaction between SbW and BSA, and the binding reaction results in non-electroactive complexes. The LSV curves of 1.0 104 mol L1 SbW in the absence (curve a) and presence (curves b, c, d, e and f) of BSA are shown in Fig. 7. We can see that SbW has a reduction peak at 1.58 V without BSA, and this peak current exhibits a distinct decrease with the addition of BSA. The decrease in the peak current is attributed to the formation of SbW–BSA, a non-electroactive complex, which results in the decrease of equilibrium concentration of SbW in solution. On the other hand, the addition of BSA makes the reduction peak potential shifts in a positive direction, which can be attributed to the changes of the molecular environment of SbW as a result of its interaction with BSA. 3.7. Conformation investigation
Fig. 7. LSV curves of 1.0 104 mol L1 SbW with increasing concentrations of BSA in solution. Curves from (a) to (f) correspond to 0, 1.0, 3.0, 5.0, 7.0, 9.0 mmol L1 BSA, respectively. Curve g: the blank solution consists of Tris–HCl buffer and 0.1 mol L1 NaCl.
It is well known that the fluorescence of BSA comes from the tyrosine, tryptophan and phenylalanine residues. The spectrum of BSA is sensitive to the micro-environment of these chromophores and it allows non-intrusive measurements of protein in low concentration under physiological conditions. The synchronous fluorescence spectra can give information about the molecular environment in a vicinity of the chromophores and have several advantages, such as sensitivity, spectral simplification, spectral bandwidth reduction and avoiding different perturbing effects [20]. When the D-value (Dl) (the difference between excitation wavelength and emission wavelength) is set at 15 or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine or tryptophan residues. A useful method to study the environment of amino acid residues has been reported by Yuan et al. [21], which by measuring the possible shift in maximum emission wavelength lmax. The shift in position of lmax corresponds to the changes of the polarity around the chromophore
Fig. 8. Synchronous fluorescence spectra of BSA (5.0 mmol L1) with Dl ¼ 60 nm (a) and 15 nm (b) in the absence and presence of SbW (up to down): 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 mmol L1, pH ¼ 7.40, T ¼ 295 K.
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molecule [22]. The dominant fluorophore in BSA is the indole group of tryptophan. Indole absorbs near 280 nm and emits near 340 nm. The emission of indole may be blue-shifted if the group is buried within a native protein, and its emission may shift to longer wavelengths (red-shift) when protein is unfolded [23]. When considering the effect of SbW on the fluorescence spectra of BSA, there is no shift in position of lmax when Dl ¼ 60 nm (Fig. 8a). This suggests that there is no change in the immediate environment of the tryptophan residues. It is apparent from Fig. 8b that the maximum emission wavelength has no significant shift either at the investigated concentration range when Dl ¼ 15 nm, which indicates that the polarity around the tyrosine residues has not been changed. So the interaction of SbW with BSA does not obviously affect the conformation of tryptophan and tyrosine micro-region.
4. Conclusions Interaction between SbW and BSA is investigated by spectral and electrochemical methods. It is found that SbW is a strong quencher of BSA fluorescence and the nature of quenching is static. The binding constants at different temperatures are evaluated. The thermodynamic parameters of the binding interaction are determined and the values suggest that van der Waals interaction and hydrogen bonding are the key binding forces. The effects of pH and ionic strength on the binding constants between SbW and BSA are also studied. In the conformational investigation, the synchronous fluorescence spectra reveal that the conformation of BSA is not changed in the presence of SbW. The binding study of inorganic drug with protein is of great importance in pharmacy, pharmacology and biochemistry. This study is expected to provide important insight into the interactions of the
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physiologically important protein BSA with inorganic ions keggin shaped.
Acknowledgement The authors gratefully acknowledge the financial support by the Analysis and Testing Foundation of Northeast Normal University. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
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