Spectroscopic studies on the interaction of 3,3'-diselenadibenzoic acid with human serum albumin

Sun Shaofa^a,b , Hou Hannab, Wu Minghua, Ding Xinliangb, Chen Xusheng ^b
(^a Department of Chemistry and Life Sciences, Xianning College, Xianning 437005;^b College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072,  China)

Supported by the Natural Science Foundation of Hubei Province(No.2006ABA333), and Science Foundation of Hubei Provincial Educational Department (D200528003)

Abstract  The interaction between 3,3'-diselenadibenzoic and Human serum albumin (HSA) was investigated by means of fluorescence and absorption spectroscopy. The quenching mechanism of fluorescence of HSA by 3,3'-diselenadibenzoic acid was discussed. It is proved that the fluorescence quenching of HSA by 3,3'-diselenadibenzoic acid is a result of the formation of HSA-3,3'-diselenadibenzoic acid complex. The binding sites number (n), apparent binding constant (K) and corresponding thermodynamic parameters, such as , , andetc., were calculated. Results indicate that the electrostatic interactions forces played major role in the reactions.
Keywords Human serum albumin (HSA), 3, 3'-diselenadibenzoic acid, Fluorescence quenching, Thermodynamic parameters

1. INTRODUCTION
Serum albumin is the most abundant proteins in the circulatory system of a wide variety of organisms, being the major macromolecule contributing to the osmotic blood pressure;1 they can play a dominant role in drug disposition and efficacy.2 Many drugs and other bioactivity small molecules bind reversibly to albumin and other serum components that then function as carriers. Serum albumin often increases the apparent solubility of hydrophobic drugs in plasma and modulates their delivery to cells in-vivo and in-vitro. Consequently, it is important to know the affinity of a drug for serum albumin even if it is not the only factor predictive of serum concentrations of the free drug.
    Fluorescence spectroscopy is a powerful tool for the study of the reactivity of chemical and biological systems. Quenching measurement of albumin fluorescence is an important method to study interactions of several substances with this protein.3
    Selenium, as an essential trace element, is of fundamental importance to human health. It appears to be a key nutrient in counteracting the development of virulence and inhibiting HIV progression to AIDS.4 Therefore many organoselenium compounds have been synthesized and their bioactivities have been studied.5 Also our previous studies showed that the anti-microbial activity of organoselenium compounds is many times higher than that of sulfur and oxygen analogs having isosteric elements.6
    In this work, we studied in vitro interaction of 3,3'-diselenadibenzoic acid (Fig.1) with HSA using the quenching of intrinsic fluorescence of tryptophan residues. The aim of our work was to estimate the interaction mechanism, and to investigate the thermodynamics of their interaction.

Figure 1. Molecular structure of 3, 3'-diselenadibenzoic acid

2. EXPERIMENTAL
2.1 Materials
3,3'-diselenadibenzoic acid was synthesized and characterized by our group. Human serum albumin, being electrophoresis grade reagents, were obtained from Sigma. The buffer Tris had a purity of no less than 99.5% and NaCl, HCl, etc. were all of analytical purity. The samples were dissolved in Tris-HCl buffer solution (0.05 mol·dm-1 Tris, 0.10 mol·dm-1 NaCl, pH = 7.4). The solutions of albumin were prepared 30 minutes before experiment. All solutions were used with doubly distilled water.
2.2 Apparatus and Measurements
Fluorescence spectra were recorded on F-2500 Spectrofluorimeter (Hitachi, Japan) with 1×1×4cm cell. Addtionally the circulating water bath was used to maintain temperature, the fluorescence measurements were performed at different temperatures (298, 302, 306 and 310K). Excitation wavelength was 285 nm. The excitation and emission slit widths were set at 2.5 nm. Appropriate blanks corresponding to the buffer were subtracted to correct background of fluorescence.
    TU-1901 spectrophotometer ( PuXi Ltd of Beijing, China ) was used to measure absorption spectra. The absorption spectra of HSA, 3, 3'-diselenadibenzoic acid and their mixture were performed at room temperature.
    The mass of the sample was accurately weighted using a microbalance (Sartorius, ME215S) with a resolution of 0.1 mg.

3. RESULTS AND DISCUSSION
3.1 Fluorescence spectra and quenching mechanism
The concentrations of HSA were stabilized at 1.0×10-5 mol·L-1, and the content of 3, 3'-diselenadibenzoic acid varied from 0 to 1.2×10-5 mol·L-1 at the step of 0.2×10-5 mol·L-1. The effects of 3, 3’-diselenadibenzoic acid on HSA fluorescence intensity are shown in Fig. 2.

Figure 2. Emission spectra of HSA in the presence of various concentrations of 3, 3'-diselenadibenzoic acid
T=298K, lex=285nm; c (HSA)= 1.0×10-5 mol·L-1; c (3,3'-diselenadibenzoic acid) / (10-5 mol·L-1), A-G: 0; 0.2; 0.4; 0.6; 0.8; 1.0; 1.2; curve K shows the emission spectrum of 3,3'-diselenadibenzoic acid only( c = 1.0×10-5 mol·L-1).

    From Fig. 2, it is apparent that the fluorescence intensity of HSA decreased regularly with the increasing of 3,3'-diselenadibenzoic acid concentration.
    Quenching can occur by different mechanisms, which usually classified as dynamic quenching and static quenching. Dynamic and static quenching can be distinguished by their differing dependence on temperature and viscosity. Higher temperatures result in faster diffusion and hence larger amounts of collisional quenching. Higher temperatures will typically result in the dissociation of weekly bound complexes, and hence smaller amounts of static quenching. 7
    In order to confirm the quenching mechanism, the fluorescence quenching data disposed by us was assumed to be dynamic quenching. For dynamic quenching, the decrease in intensity is described by the well-known Stern-Volmer equation3, 8:
(1)
    Where, F0 and F are the steady-state fluorescence intensities in the absence and in the presence of quencher (3,3'-diselenadibenzoic acid), is the Stern-Volmer quenching constant, Kq is the bimolecular quenching constant, is the unquenched lifetime, and is the concentration of quencher.

Figure 3. The line relation of (F0/F-1) and [Q] at different temperatures

    Figure 3 displays the Stern-Volmer plots of the quenching of HSA tryptophan residues fluorescence by 3,3'-diselenadibenzoic acid at different temperatures. From the corresponding Stern-Volmer quenching constant and the linear relation at different temperatures, we conclude the probable quenching mechanism of fluorescence of HSA by 3,3'-diselenadibenzoic acid are not initiated by dynamic collision but by formation of complex.
    For static quenching, the decrease in intensity is described by the Lineweaver-Burk equation: 9
(2)
    In the present case, KLB is the formation constant. The corresponding Lineweaver-Burk formation constant and the linear relation at different temperatures are shown in Table 1. It shows that the binding constant between 3,3'-diselenadibenzoic acid and HSA is great and the effect of temperature is small. Thus, 3,3'-diselenadibenzoic acid can be stored and carried by protein in the body.

Table 1 Lineweaver-Burk quenching constant and the linear relation of KLB and T at different temperatures

aR is the linear correlated coefficients

3.2 Analysis of binding equilibria                     
When small molecules bind independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound molecules is given by the equation:8
(3)
    Where, in the present case, K is the binding constant to a site, and n is the number of binding sites per serum albumin. According to the equation (3), the fit of the fluorescence date for the system of 3,3’- diselenadibenzoic acid and HSA show that n is almost a constant at four different temperature, we can conclude that 3,3’-diselenadibenzoic acid molecule formed 1:1 complex with HSA molecule. Figure 4 is the fit between log((F0-F)/F) and log[Q] at four different temperatures. From figure 4, it can be seen that the value of KLB and n decreased slightly with the temperatures rising, which may indicates that there is molecular binding with HSA and forming an unstable compound. The unstable compound would be partly decomposed when the temperature rising, therefore, the values of K and n decreased slightly with the temperatures rising.

Figure 4. The fit between log((F0-F)/F) versus log[Q] at four different temperatures

3.3 Thermodynamic parameters
In order to elucidate the interaction of 3,3'-diselenadibenzoic acid with HSA, the thermodynamic parameters were calculated from the Van't Hoff plots. If the enthalpy change () does not vary significantly over the temperature range studied, then its value and that of entropy change () can be determined from the Van't Hoff equation:

(4)
    Where K is the binding constant (KLB) at the corresponding temperature and R is the gas constant. The temperatures used were 298, 302, 306 and 310 K. The enthalpy change () is calculated from the slope of the Van’t Hoff relationship. The free energy change () is estimated from the following relationship:
(5)
    Fig.5, by fitting the data of Table 1, shows that assumption of near constant is justified. The values of and obtained for the binding sites from the slopes and ordinates at the origin of the fitted lines are shown inTable 2. From Table2, it can be seen that the negative sign for free energy () means that the binding process is spontaneous. The negative enthalpy () and positive entropy () values of the interaction of 3,3’-diselenadibenzoic acid and BSA indicate that the electrostatic interactions forces played major role in the reactions.9

Figure 5. Van't Hoff plot, pH 7.40; c(HSA)= 1.0×10-5 mol·L-1

Table 2. Binding constant (KLB )and relative thermodynamic parameters

3.4 Energy transfer from HSA to 3, 3'-diselenadibenzoic acid                    
The Förster theory of molecular resonance energy transfer10 point out: in addition to radiation and reabsorption, a transfer of energy could also take place through direct electrodynamic interaction between the primarily excited molecule and its neighbors. According to this theory, the distance r of binding between 3,3'-diselenadibenzoic acid and serum albumins could be calculated by the equation.11
(6)
    Where E is the efficiency of transfer between the donor and the acceptor, R0 is the critical distance when the efficiency of transfer is 50%.
(7)
    In equation (7), K2 is the space factor of orientation; N is the refracted index of medium; f is the fluorescence quantum yield of the donor; J is the effect of the spectral overlap between the emission spectrum (b) of the donor and the absorption spectrum (a) of the acceptor (Fig. 6), which could be calculated by the equation:
(8)
    Where, F(l) is the corrected fluorescence intensity of the donor in the wavelength rangel tol+Dl; e(l) is the extinction coefficient of the acceptor at l. The efficiency of transfer (E) could be obtained by the equation:
(9)
    In the present case, K²= 2/3, N =1.36, f= 0.15, 12according to the equation (6) - (9), we could calculate that R₀ = 3.64 nm; E = 0.47 and r = 3.71 nm. The distance r﹤8 nm,13, 14 indicate that the energy transfer from BSA to 3,3'-diselenadibenzoic acid occurs with high probability.

Figure 6. Spectral overlap of the fluorescence of HSA(b) and the absorption of 3,3'-diselenadibenzoic acid(a)(CHSA=C(3,3'-diselenadibenzoic acid)=1.0×10-5 mol·L-1))

4. CONCLUSIONS
The interaction of 3,3'-diselenadibenzoic acid with HSA was studied by Spectroscopic methods including fluorescence spectroscopy and UV-visible absorption spectroscopy. The experimental results also indicate that the probable quenching mechanism of fluorescence of HSA by 3,3'-diselenadibenzoic acid is a constant quenching procedure. From the number binding sites we can conclude that 3,3'-diselenadibenzoic acid molecule formed 1:1 complex with HSA molecule. The enthalpy change () and the entropy change () were also calculated. From the two parameters we know that the electrostatic interactions forces played major role in the reaction. The main purpose of this research is to study the binding properties between HSA and 3,3'-diselenadibenzoic acid for the great importance in pharmacy, pharmacology and biochemistry. As a kind of pharmaceutical that is not used orally, this experiment can supply some important information to clinically research.

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光谱法研究3, 3’-二硒苯二甲酸与人血清白蛋白的相互作用
孙绍发^a,b,吴鸣虎^a,候汉娜^b
(^a咸宁学院化学与生命科学系, 湖北咸宁, 437005; ^b武汉大学化学与分子科学学院, 湖北武汉, 430072)
摘要 光谱法研究3, 3’-二硒苯二甲酸与人清白蛋白的相互作用，实验发现3, 3’-二硒苯二甲酸能强烈猝灭牛血清白蛋白的荧光，其荧光猝灭机理为静态猝灭。分析并处理实验数据，计算了二者相互作用的结合常数、结合位点数及热力学参数等。结果表明3, 3’-二硒苯二甲酸与HSA 1:1 结合，其反应主要是熵趋动的，相互作用的主要作用力为静电力。根据F?rster无辐射能量转移理论计算了给体（HSA）与 授体（3, 3’-二硒苯二甲酸）之间的结合距离。为有机硒化合物的开发与应用提供了重要信息。
关键词 荧光猝灭，3, 3’-二硒苯二甲酸，人血清白蛋白，热力学参数