Spectroscopic Studies On The Interaction Of Three Kinds Of Cephalosporin Drugs With Trypsin And Pepsin

Protein is an important nutrient body and has a special status in the human.Cephalosporins are widely used as a wide variety of antibiotics.They have a strong selective effect on bacteria and have little toxicity to humans.Drugs that enter the body through oral administration are bound to come into contact with trypsin and pepsin.Studying the combination of protein and drug helps to understand the detailed process of drug efficacy and provides a theoretical basis for clinical drug use and pharmacokinetics.The interaction of three kinds of cephalosporin drugs with trypsin and pepsin has been investigated by fluorescence spectra,synchronous fluorescence spectra,UV-vis absorption spectra,circular dichroism spectra and molecular docking method at different temperatures.The research content has been divided into five sections:Chapter one:In this chapter,the commonly used method for the system of proteins and drugs was introduced,and made a brief summary of trypsin and pepsin,with 44 literatures quoted.Chapter two:The binding of ceftazidime(CFD)with trypsin(TRP)was investigated by spectroscopic and molecular docking methods under different temperatures conditions(298,303 and 310 K).The results demonstrated that the interaction between CFD and TRP was taking place via static quenching with 1:1 binding ratio.The fluorescence datas were treated by using the double logarithmic equation,and the binding constants K_a of the interaction of TRP-CFD system and the number of binding sites n were obtained.The thermodynamic parameters of TRP-CFD system under different temperatures were obtained by the thermodynamic equation.The experimental data show that the interactions between them were mainly electrostatic force,and with the molecular docking results are consistent.Chapter three:In the Tris-HCl buffer solution with pH=7.4,the reaction mechanism of cefonicid sodium(CFS)with trypsin(TRP)was investigated at different temperatures.The results showed that the quenching of TRP fluorescence by CFS was a static process,the number of binding sites in this system was approximately equal to 1.The quenching curves indicated that the tyrosine and tryptophan residues were both involved in the reaction.The distance(r)between TRP and CFS was less than 7 nm,which indicated that the energy transfer from TRP to CFS was non-radiative energy transfer.The binding of TRP-CFS system was investigated by molecular docking methods under simulated physiological conditions.The docking result is the same as the experimental result,the force of the system is mainly electrostatic force.Chapter four:The interaction of Cefetamet Pivoxil(CFP)and Pepsin(PEP)has been investigated by fluorescence spectra,synchronous fluorescence spectra,UV-vis absorption spectra,circular dichroism(CD)spectra and molecular docking method at 298,303 and 310K.The results indicated that CFP mainly uses the static quenching method of nonradiative energy transfer to cause the fluorescence quenching of PEP,which is mainly combined by static electricity forces.The binding rate was 74.73%~92.13%at 310 K.The effect of CFP on PEP structure was studied by synchronous and circular dichroism(CD).The results showed that the binding of CFP and PEP induced the conformational change of PEP,and quenched the endogenous fluorescence in PEP.The results of molecular docking revealed that CFP were located in the catalytic active site of PEP,and the results of docking is consistent with that of experimental calculation.It is confirmed that the addition of CFP leads to the gradual quenching of PEP fluorescence.At the same time,the fluorescence quenching reaction of CFP to PEP can be used to determine the content of CFP in the medicine.Chapter five:The reaction mechanism of CFP with PEP was investigated using the classical fluorescence spectroscopy with focus on the fluorescence change of protein,as well as the resonance light scattering spectroscopy with focus on the fluorescence change of drug at different temperatures.The results indicated that CFP could quench the intrinsic fluorescence of PEP strongly by a static quenching process.The value of n was approximately 1,indicating there was only a single class of binding sites on PEP-CFP system.The value of Hill's coefficients was approximately equal to 1,which suggested no cooperativity in PEP-CFP system.The binding constants that obtained by resonance light scattering 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.What's more,the scientificalness of the new method based on resonance light scattering spectrometry was verified by ultraviolet absorption spectroscopy.