Sun Shaofa, Xiang Guangya
(Department of Chemistry and Life Science, Xianning College, Xianning 437000; School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430015)
AbstractThe interaction between a kind of NO-releasing oxaprozin(Oxaprozin-P)and bovine serum albumin (BSA) was studied by spectroscopic methods including fluorescence and UV-vis absorption spectroscopy. The quenching mechanism of fluorescence of BSA by Oxaprozin-P was discussed to be a dynamic quenching procedure. The number of binding sites n and apparent binding constant was measured by fluorescence quenching method. The thermodynamic parameters, such as and etc., were calculated. The results indicate that the binding reaction was mainly entropy-driven and hydrophobic forces played major role in the reaction. The distance r between donor (BSA) and acceptor (Oxaprozin-P) was obtained according to Förster theory of non-radioactive energy transfer.
Nitric oxide (NO) is an important messenger moleculer and has wide biological activity . Furoxans are widely used as nitric oxide donor in new drugs development. Oxaprozin  is one of the most widely used Nonsteroidal antiinflammatory drugs (NSAIDs) in clinic, but as most of the NSAIDs, its side effect, especially gastric or intestinal ulceration, limits its application. According to symbiotic approach principle, the hybrids by coupling nitric oxide-releasing group Furoxan with Oxaprozin may greatly reduce its side effects. Therefore, Oxaprozin-P (Fig.1) have been synthesized by our group.
It has been shown that the distribution, free concentration and the metabolism of various drugs may be strongly affected by drug-protein interactions in the blood stream. This type of interaction can also influence the drug stability and toxicity during the chemotherapeutic process. Serum albumins are the most abundant proteins in plasma. As the major soluble protein constituents of the circulatory system, they have many physiological functions. They contribute to colloid osmotic blood pressure and are chiefly responsible for the maintenance of blood pH , So they can play a dominant role in drug disposition and efficacy . Many drugs and other small bioactivity 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 to serum albumin, even if it is not the only factor to predict serum concentrations of the free drug.
Fluorescence and UV-vis absorption spectroscopy are powerful tool for the study of the reactivity of chemical and biological systems since it allows non-intrusive measurements of substances in low concentration under physiological conditions. In the present work, the interaction between Oxaprozin-P and BSA by fluorescence and UV-vis absorption spectroscopy was demonstrated. The effect of the energy transfer was studied according to fluorescence resonance energy transfer (FRET). The aim of this work is to determine the affinity of Oxaprozin-P to BSA, and to investigate the thermodynamics of their interaction. To resolve this problem, the UV-vis and fluorescent properties of Oxaprozin-P, as well as BSA were investigated.
Figure 1. Molecular structure of Oxaprozin-P
Oxaprozin-P was synthesized and characterized by the Group of Organic Synthesis, Pharmacy School of Tongji Medical College, Huazhong University of Science and Technology, P. R. China. BSA, electrophoresis grade reagents, was obtained from Sigma. Tris-Base 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·L-1 Tris-Base, 0.10 mol·L-1 NaCl, pH = 7.4). The bovine serum albumin solutions were prepared 30 minutes before experiment. All solutions were used with doubly distilled water.
All fluorescence spectra were recorded on F-2500 Spectrofluorimeter in the ratio mode with temperature maintained by circulating bath (Hitachi, Japan); TU-1901 spectrophotometer (Puxi Ltd. of Beijing, China) was used for scanning UV Cvis spectra; the mass of the sample was accurately weighed using a microbalance (Sartorius, ME215S) with a resolution capacity of 0.1 mg.
2.3. Spectroscopic measurements
The absorption spectra of BSA, Oxaprozin-P and their mixture were performed at room temperature. The fluorescence measurements were performed at different temperatures (298, 302 and 310 K). 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 fluorescence.
3. RESULTS AND DISCUSSIONS
3.1 Fluorescence characteristics of BSA and Oxaprozin-P
Fluorescence quenching refers to any process which decreases the fluorescence intensity of a sample. A variety of molecular interactions can result in quenching. These include excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching.
For collisional quenching, the decrease in intensity is described by the well-known Stern-Volmer equation as follows 
Where, and are the steady-state fluorescence intensities in the absence and in the presence of quencher, SV is the Stern-Volmer quenching constant, and is the concentration of quencher respectively.
Figure 2 shows the emission spectra of BSA in the presence of various concentrations of Oxaprozin-P. It is apparent that the fluorescence intensity of BSA decreased regularly with the increasing of Oxaprozin-P concentration. The experiment was carried out within the linear part of Stern-Volmer dependence ( against ), and stabilized the concentrations of BSA at 1.0×10-5 mol·L-1, the concentration of Oxaprozin-P varied from 0 to 4.0×10-5 mol·L-1 at the step of 0. 5×10-5 mol·L-1
Figure 2. Emission spectra of BSA in the presence of various concentrations of Oxaprozin-P (=298K)
[BSA]= 1.0×10-5 mol·L-1; [Oxaprozin-P] / (10-5 mol·L-1), A-I: 0; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0.
curve K shows the emission spectrum of Oxaprozin-P only(ex=370nm), [Oxaprozin-P] = 1.0×10-5 mol·L-1
Quenching can occur through different mechanisms, which usually classified as dynamic quenching and static quenching. Dynamic and static quenching can be distinguished by their different 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 weakly bound complexes, and hence smaller amounts of static quenching.
Figure 3 displays the Stern-Volmer plots of the quenching of BSA tryptophan residues fluorescence by Oxaprozin-P at different temperatures. The corresponding Stern-Volmer quenching constant at different temperatures are shown in Table 1.
These results indicate that the probable quenching mechanism of fluorescence of BSA by Oxaprozin-P is a dynamic quenching procedure, because the SV increased with the temperature rising.
Figure 3. Stern-Volmer plot at three different temperatures
Table 1. Stern-Volmer quenching constant SV and relative thermodynamic parameters
R is the linear correlated coefficient; SD is standard deviation.
3.2 Thermodynamic parameters and nature of the binding forces
The interactions forces between a drug and biomolecule may include hydrophobic force, electrostatic interactions, van der Waals interactions, hydrogen bonds, etc. The Stern-Volmer quenching constants of BSA were measured at three different temperatures (298, 302 and 310 K). The slope of a plot of the logarithum of bimolecular quenching constant vs. 1/ (, absolute temperature) are linear within experimental error, which allows one to calculate the energy change for the quenching process . 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:
Where constants are analogous to the Stern-Volmer quenching constants SV at the corresponding temperature and R is the gas constant  (the temperatures used were 298, 302 and 310 K). The free energy change () is estimated from the following relationship:
Figure 4, by fitting the data of Table 1, shows that the assumption of near constant is justified. Table 1 shows the values of and obtained for the binding site from the slopes and ordinates at the origin of the fitted lines.
Figure 4. Van’t Hoff plot, pH 7.40, [BSA]=1.0×10-5 mol·L-1
From Table 1, it can be seen that the negative sign for free energy () means that the interaction process is spontaneous. The positive enthalpy and entropy values of the interaction of Oxaprozin-P and BSA, indicate that the binding is mainly entropy-driven and the enthalpy is unfavorable for it, the hydrophobic forces played major role in the reaction [7,8]
3.3 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 below 
Where, in the present case, is the binding constant to a site, and n is the number of binding sites per BSA 
Table 2 gives the results at different temperatures analyzed in this way for BSA. The linear correlated coefficients (R) are larger than 0.999, and the standard deviation is less than 0.02, indicating that the assumptions underlying the derivation of equation (4) are satisfied. Table 2 shows the results of decreased slightly with the temperatures rising, which may indicates that there is Oxaprozin-P molecular binding with BSA and forming an unstable compound. The unstable compound would be partly decomposed when the temperature rising, therefore, the values of decreased slightly with the temperatures rising. Table 2 shows the number of binding sites n approach 1，we can conclude that Oxaprozin-P molecule formed 1:1 compound with BSA molecule.
Table 2. Binding constant and binding sites n at different temperatures
is the linear correlated coefficient; SD is standard deviation
3.4 Energy transfer between BSA and Oxaprozin-P
The Förster theory of molecular resonance energy transfer  points 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 efficiency of energy transfer between the donor and acceptor, E, could be calculated by the following equation 
Where r is the distance between the donor and acceptor , and is the critical distance when the efficiency of transfer is 50%.
In equation (6), is the orientation factor related to the geometry of the donor and acceptor of dipoles and = 2/3  for random orientation as in fluid solution; is the refracted index of medium;is the quantum yield of the donor in the absence of acceptor; expresses the degree of spectral overlap between the donor emission and the acceptor absorption (Fig. 6), which could be calculated by the equation(7):
Where, ) is the corrected fluorescence intensity of the donor in the wavelength rangeto+De(is the extinction coefficient of the acceptor at. In the present case, = 1.36,= 0.15 , according to the equation (5) ~ (7), we could calculate that = 3.49 nm; = 0.11 and = 4.94 nm. The donor-to-acceptor distance 8 nm , and 0.5 < < 1.5, indicate that the energy transfer from BSA to Oxaprozin-P occurs with high possibility.
Figure 5. Spectral overlap of Oxaprozin-P absorption (a) with BSA fluorescence (b)
[BSA] = [Oxaprozin-P] =1.0×10-5 mol·L-1
The main purpose of this research is to study the binding properties between BSA and Oxaprozin-P for the great importance in pharmacy, pharmacology and biochemistry. The interaction of Oxaprozin-P with BSA 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 BSA by Oxaprozin-P is a dynamic quenching procedure, and the binding reaction is mainly entropy-driven and hydrophobic interactions played major role in the reaction.
ACKNOWLEDGEMENT The authors gratefully acknowledge financial support of Natural Science Foundation of Hubei Province(No.2006ABA333) and Science Foundation of Hubei Provincial Educational Department (No.D200528003)