Fluorescence Spectroscopic Studies On The Interaction Mechanism Of Cefpirome, Cefepime And Trichazol With Lysozyme

In recent years, with the increase in the incidence of human diseases, the drug has been inseparable from people's daily lives. So, to study the interaction between drug molecules and proteins has been a hot topic. In this paper, lysozyme(LYSO) was considered as research carrier, and the reaction mechanism of cefpirome sulfate(CPS), cefepime hydrochloride(CPH) and trichazol(TRI) to lysozyme(LYSO) was investigated using synchronous fluorescence spectroscopy and improved spectroscopy which was based on the traditional fluorescence spectrometry. The research content has been divided into five chapters:Chapter one: The combination of protein and drugs greatly affects the absorption, distribution, metabolism and other physiological activities of drugs in vivo. Moreover, it has an important influence on the drug's efficacy and changes in the structure and function of the protein. And then, the research methods of interaction between drug molecules and protein were reviewed, with 51 literatures quoted.Chapter two: The reaction mechanism of cefpirome sulfate(CPS) to lysozyme(LYSO) at different temperatures was investigated using fluorescence quenching and synchronous fluorescence spectroscopy under simulative physiological conditions. The results clearly demonstrated that CPS caused strong quenching of the fluorescence of LYSO by a static quenching mechanism. The binding constants(Ka) obtained from above method were of the same order of magnitude and very similar. The static electricity forces attraction played a key role in the interaction between CPS and LYSO, the number of binding site(n) in the interaction was closed to 1. The values of Hill's coefficients(nH) were more than 1, it indicated that drugs or proteins had a very weakly positive cooperativity in the system. In addition, the conclusions obtained from two methods using the same equation were consistent. It indicated that the synchronous fluorescence spectrometry could be used to study the binding mechanism between drugs and protein. Meanwhile, the effect of CPS on the secondary structure of LYSO was analyzed using circular dichroism(CD) spectroscopy.Chapter three: The interaction between cefepime hydrochloride(CPH) and lysozyme(LYSO) at different temperatures was investigated using fluorescence quenching and synchronous fluorescence spectroscopy. The results demonstrated that the quenching mechanism, binding constants, the number of binding site, type of interaction force obtained from two methods were consistent. It indicated that the synchronous fluorescence spectrometry could be used to study the binding mechanism between drugs and protein, and was a useful supplement to the fluorescence quenching method.Chapter four: The interaction mechanism of trichazol(TRI) to lysozyme(LYSO) at different temperatures was investigated using fluorescence spectroscopy under simulative physiological conditions. The results clearly demonstrated that TRI caused strong quenching of the fluorescence of LYSO by a static quenching mechanism. The binding constants(Ka) were order of magnitude of 104 and the number of binding site(n) in the interaction was closed to 1. The thermodynamic parameters of LYSO-TRI system binding were determined as follows: negative ?H and positive ?S, which indicated that electrostatic interaction played a major role in the binding reaction. The effect of cooperative binding of TRI was quantified by Hill's coefficient(nH) and the value of nH were slightly more than 1 in the systems, which indicated weakly positive cooperativeness in the interaction of TRI with LYSO. The binding distance(r) was obtained between TRI and LYSO based on the theory of F?rster nonradioactive resonance energy transfer and r were less than 7 nm. Furthermore, the effect of TRI on the conformation of LYSO was analyzed by using the synchronous fluorescence spectroscopy.Chapter five: To resolve the deficiencies in the interaction analysis by fluorescence quenching spectroscopy between drugs and proteins, elastic scattering fluorescence spectrometry with drugs as research object has been applied to study the reaction mechanism between trichazol(TRI) and lysozyme(LYSO) under human physiological conditions at different temperatures. The results clearly demonstrated that the reaction mechanism obtained by elastic scattering fluorescence spectrometry in LYSO-TRI system was consistent with fluorescence quenching spectroscopy that LYSO was used as investigation object. The binding constants(Ka) that obtained by elastic scattering fluorescence spectrometry was much larger than the one obtained by fluorescence quenching spectroscopy, which indicated that it was more accurate and comprehensive when regarding the drug as the investigation object. At last, the scientificalness of the new method based on elastic scattering fluorescence spectrometry was verified by ultraviolet(UV) spectroscopy.