Studies On The Interaction Of Drugs With Bovine Serum Albumin By Synchronous Fluorescence Spectroscopy And Improved Fluorescence Spectroscopy

At present, utilizing fluorescence analysis method to study the interaction of protein with small molecules drug has widely used on many fields, such as biological, chemical, pharmacology. Bovine Serum Albumin was considered as an object in this paper, the reaction between protein and drugs were investigated by fluorescence spectroscopy. In addition, there is a need to further improve the research methods. The research content has been divided into five chapters:Chapter one: Summarizes research methods of the interaction between proteins and small molecules drug. Hereby, this development in studying interaction between proteins with small molecules by spectroscopy has been reviewed, based on this, the research contents for study of the reaction mechanism between small molecules and protein were established,with 57 literatures quoted.Chapter two: Under simulated physiological conditions(p H=7.4), the reaction mechanism between cefixime and bovine serum albumin at different temperatures(293 K, 303 K, 310 K) was investigated by fluorescence quenching method and synchronous fluorescence method, respectively. The results indicated that the fluorescence intensity and synchronous fluorescence intensity of bovine serum albumin decreased regularly with the addition of cefixime, in addition, the quenching mechanism, binding constants, the number of binding site, type of interaction force and energy transfer parameters of cefixime with bovine serum albumin obtained from two methods by the same equation were consistent, which indicated synchronous fluorescence spectrometry studied the binding mechanism between drug and protein, and it was a useful supplement to the conventional method.Chapter three: The reaction mechanism between cefixime and bovine serum albumin at different temperature(293 K, 303 K, 310 K) was investigated by the classical fluorescence spectroscopy with focus on the fluorescence change of protein, as well as the elastic scattering fluorescence spectroscopy with focus on the fluorescence change of drug. The results indicated that cefixime could quench the intrinsic fluorescence of bovine serum albumin strongly, and the quenching mechanism was a static quenching process. The electrostatic force played an important role on the conjugation reaction between cefixime and bovine serum albumin, the number of binding site(n) in the binary system was approximately equal to 1; Besides, the values of Hill’s coefficients were less than 1, which indicated very weakly negative cooperativity in bovine serum albumin-cefixime system. The binding constant obtained from elastic scattering fluorescence spectroscopy was larger than the one obtained from classical fluorescence spectroscopy by two orders of magnitude for the cefixime-bovine serum albumin system. This indicated that the elastic scattering fluorescence spectroscopy with focus on the fluorescence change of drug was a more accurate and more reasonable method. At last the correctness of elastic scattering fluorescence spectroscopy method was verified by UV-vis absorption spectroscopy. And which also speculated that "point to side" interaction between drugs and peptides was existed.Chapter four: The interaction of aspirin with bovine serum albumin was investigated at different temperatures by utilizing fluorescence quenching method and synchronous fluorescence method, respectively. The results indicated that aspirin could quench the intrinsic fluorescence of bovine serum albumin through a static quenching process; the electrostatic force played an important role on the conjugation reaction between aspirin and bovine serum albumin. The order of magnitude of binding constants was 103, and the primary binding for aspirin was located at the structure domain IIA of bovine serum albumin. The results obtained by the two methods were consistent, which indicated synchronous fluorescence spectrometry was a new method of studying the binding mechanism between drug and protein, and it was a useful supplement to the conventional method.Chapter five: The reaction mechanism between aspirin and bovine serum albumin at different temperatures was investigated by the fluorescence spectroscopy with focus on the fluorescence change of protein, as well as the fluorescence spectroscopy with focus on the fluorescence change of drug. The results indicated that the electrostatic force played an important role on the conjugation reaction between aspirin and bovine serum albumin. The binding constant obtained from the fluorescence spectroscopy with focus on the fluorescence change of drug was larger than the one obtained from the fluorescence spectroscopy with focus on the fluorescence change of protein by two orders of magnitude for the aspirin-bovine serum albumin system. This indicated that the fluorescence spectroscopy with focus on the fluorescence change of drug with focus on the fluorescence change of drug was a more accurate and more reasonable method. At last the correctness of the fluorescence spectroscopy with focus on the fluorescence change of drug was verified by UV-vis absorption spectroscopy and synchronous fluorescence spectroscopy with focus on the fluorescence change of drug.