Spectroscopy Studies On The Reaction Mechanism Of Cefoxitin Sodium,Pioglitazone Hydrochloride And Other Drugs With Protein

Protein-drug is a form of temporary storage of drug when the drug is taken by human being, and is main way to control the drug release to the receptor and avoids drug fast metabolism. After drug is absorbed into the blood circulation, only free molecule can transfer from the blood to the tissue exerting pharmacological effect. It is important significance to study the interaction between biological macromolecules such as protein and small molecular. The research is useful for the study of biological effect of small molecule, promoting the research and development of proteomics. This article regards bovine serum albumin and lysozyme as the research object, and studies the interaction between drug and protein by fluorescence spectrum method and some improved research methods. The research content has been divided into seven chapters.Chapter one: Summarizes the important role of proteins in life activity. The process of drugs in blood plasma and the importance of protein were reviewed. Commonly used methods for studying the interaction between protein and drugs were introduced comprehensively, as a starting point, the research contents for study the interaction of drugs with protein were established, with 51 literatures quoted.Chapter two: The reaction mechanism of cefoxitin sodium with bovine serum albumin was investigated using fluorescence spectroscopy and synchronous fluorescence spectroscopy at different temperatures. The results showed that the change of binding constant of synchronous fluorescence method with increasing temperature could be used to estimate the types of quenching mechanism of drug with protein and was consistent with the one of fluorescence quenching method. In addition, the number of binding sites, type of interaction force, cooperativity between dugs and protein and energy-transfer parameters of cefoxitin sodium and bovine serum albumin obtained from two methods using the same equation were consistent. Electrostatic force played a major role on the conjugation reaction between bovine serum albumin and cefoxitin sodium, and the type of quenching was static quenching. Primary binding site for cefoxitin sodium was sub-hydrophobic domain IIA, and the number of binding sites was 1. The value of Hill’s coefficients(nH) was approximately equal to 1, which suggested no cooperativity in bovine serum albumin-cefoxitin sodium system. The donor-to-acceptor distance r <7 nm indicated that the static fluorescence quenching of bovine serum albumin by cefoxitin sodium was also a non-radiation energy transfer process. The results indicated that synchronous fluorescence spectrometry could be used to study the reaction mechanism between drug and protein, and was a useful supplement to the conventional method.Chapter three: The reaction mechanism of cefoxitin sodium with bovine serum albumin was investigated using 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 at different temperatures. The results indicated that cefoxitin sodium could quench the intrinsic fluorescence of bovine serum albumin strongly by a static quenching process. The results of two methods were consistent. In addition, the binding constant obtained from elastic scattering fluorescence spectroscopy was larger than the one obtained from classical fluorescence spectroscopy. At last the correctness of elastic scattering fluorescence spectroscopy method was verified by UV-visible absorption spectroscopy. It is also speculated that "point to surface" interaction between drugs and peptides was existed.Chapter four: The reaction mechanism between pioglitazone hydrochloride and bovine serum albumin was studied by fluorescence quenching method and synchronous fluorescence method under different temperatures. The results show that the conclusions of two methods are identical on the quenching mechanism, type of interaction force, binding constants, binding location, synergistic effect, energy transfer parameters of pioglitazone hydrochloride with bovine serum albumin, etc. These indicate that the synchronous fluorescence spectroscopy can be used to replace the traditional fluorescence spectroscopy to study the reaction mechanism of drugs with protein.Chapter five: Under simulated physiological conditions, the reaction mechanism between pioglitazone hydrochloride and bovine serum albumin at different temperatures 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 pioglitazone hydrochloride could quench the intrinsic fluorescence of bovine serum albumin strongly by a static quenching process. The electrostatic force played an important role on the conjugation reaction of pioglitazone hydrochloride with bovine serum albumin, and the number of binding site(n) in the binary system was approximately equal to 1. The value of Hill’s coefficients(nH) was approximately equal to 1, which suggested no cooperativity in bovine serum albuminpioglitazone hydrochloride system. In addition, the binding constant obtained from elastic scattering fluorescence spectroscopy was larger than the one obtained from classical fluorescence spectroscopy with two orders of magnitude for the bovine serum albuminpioglitazone hydrochloride system. The results show that the research is more accurate and reasonable with focus on the fluorescence change of drug and is also speculated that "point to side" interaction between drugs and proteins is existed.Chapter six: The reaction mechanism of rhodamine B with bovine serum albumin was investigated using fluorescence spectroscopy and synchronous fluorescence spectroscopy at different temperatures. The results showed that electrostatic force played a major role on the conjugation reaction between bovine serum albumin and rhodamine B, and the type of quenching was static quenching. Primary binding site for rhodamine B was sub-hydrophobic domain IIA, and the number of binding sites was 1. The order of magnitude of binding constants(Ka) was 104. The value of Hill’s coefficients(nH) was approximately equal to 1, which suggested no cooperativity in bovine serum albumin-rhodamine B system. The donor-to-acceptor distance r <7 nm indicated that the static fluorescence quenching of bovine serum albumin by rhodamine B was also a non-radiation energy transfer process. The results of two methods were consistent that showed the synchronous fluorescence spectroscopy could be used to study the reaction mechanism between drug and protein, and was a useful supplement to the conventional fluorescence quenching method.Chapter seven: Under simulated physiological conditions, the interaction between Tylosin Tartrate and Lysozyme was investigated at pH = 7.40 by fluorescence spectroscopy. The results indicated that Tylosin Tartrate could strongly quench the intrinsic fluorescence of Lysozyme. By determining the quenching constants of the reaction between Tylosin Tartrate and Lysozyme at different temperatures, the quenching mechanism was proven to be a static quenching process. The thermodynamic parameters(ΔH, ΔS) of the reaction between Tylosin Tartrate and Lysozyme were obtained by the Vant’s-Hoff equation, and were 27.80 KJ/mol and 166.28 J/mol·K, respectively. The results showed that hydrophobic interaction between Tylosin Tartrate and Lysozyme was dominant. Synchronous fluorescence spectra revealed that the conformation of Lysozyme was changed. This method could be applied to measure the content of Tylosin Tartrate.