Study On The Interaction Between Four Cephalosporins And Papain By Fluorescence Spectroscopy

In recent years,people have paid more and more attention to the interaction between small drug molecules and protein macromolecules.In this thesis,cefepime,cefoperazone sodium,cephalexin sodium,cefpirome sulfate,and papain were used as the main research subjects.The drug-papain system was studied by fluorescence spectroscopy and synchronous fluorescence spectroscopy.The research content was divided into the following sections:Chapter one:The properties and uses of papain were reviewed.And the fluorescence spectrometry and synchronous fluorescence spectroscopy were used to describe and explain the drug-protein system.Chapter two:The reaction mechanism of cefepime（CEF）with papain（PAPA）was investigated by both fluorescence quenching and synchronous fluorescence spectroscopy in different temperature.The results showed that the fluorescence intensity PAPA was decreased considerably upon the addition of CEF through a static quenching mechanism.Thermodynamic parameters revealed that electrostatic interaction played major roles in the interaction.The order of magnitudes of binding constant was 10⁴,and the binding ratio of cefepime to papain was found to be 1:1.The cefepime-papain system showed zero synergy.The results obtained by the two methods were consistent.Synchronous fluorescence showed that both tryptophan residues（Trp）and tyrosine residues（Tyr）of PAPA were involved in the binding reaction between CEF and PAPA.The binding rate and model of the binding reaction were obtained when the excitation wavelength was at 280 nm.At the same time,there was a linear relationship betweenΔI and the concentrations of CEF in the range of6.0×10^-67.0×10^-55 mol/L at 318 K.The detection limit of the method was 3.78×10^-66 mol/L（n=10）.The rapid determination of CEF content in real drugs,which could be achieved by using fluorescence quenching reaction of CEF to PAPA.Chapter three:Using PAPA as the test object,the combined behavior between cefoperazone sodium（CFZ）and PAPA was investigated by fluorescence spectroscopy and synchronous fluorescence spectroscopy at three temperatures.The results demonstrated that the fluorescence of PAPA and the fluorescence of tyrosine（Trp）residues and tryptophan（Tyr）residues of PAPA were all quenched upon the addition of CFZ by static quenching.At the same time,the parameters such as binding constant（K_a）,binding site（n）,thermodynamic parameters,binding force type,and hill coefficient were obtained.Synchronous fluorescence spectra also showed that the combination of CFZ and PAPA could change the conformation of PAPA,while both Trp and Tyr of PAPA participated in the binding reaction.And the fluorescence quenching ratio of Trp and Tyr residues of PAPA were calculated.In addition,the binding rate and binding model of the binding reaction were obtained when the excitation wavelength was at 295 nm.There was a linear relationship betweenΔI and the concentrations of CFZ in the range of 4.0×10^-61.0×10^-44 mol/L at 293 K.The detection limit of the method was 2.43×10^-66 mol/L（n=10）,indicating that the reaction could be used to determinate the content of CFZ in actual drugs rapidly.Chapter four:This study aimed to investigate the interaction between cefonicid sodium（CFS）and PAPA using fluorescence spectroscopies and synchronous fluorescence spectroscopy.The results showed that the fluorescence intensity of PAPA was decreased considerably upon the addition of CFS through a static quenching mechanism.The CFS and PAPA binded 1:1 by electrostatic attraction and had a zero synergy.The fluorescence quenching method and the synchronous fluorescence method had the same conclusions.Synchronous fluorescence spectra also showed that the combination of CFS and PAPA could change the conformation of PAPA,while both Trp and Tyr of PAPA participated in the binding reaction.The fluorescence quenching ratio of Trp and Tyr residues of PAPA,the binding rate and binding model of the binding reaction were calculated.At the same time,there was a good linear relationship betweenΔI and the concentrations of CFS in the range of6.0×10^-61.0×10^-44 mol/L at 293 K.The detection limit of the method was 3.05×10^-66 mol/L（n=10）.The rapid determination of CFS content in actual drugs,which could be achieved by using fluorescence quenching reaction of CFS to PAPA.Chapter five:Using PAPA as the test object,the interaction between cefpirome sulfate（CPS）and PAPA was studied by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy.The parameters such as the binding constants（K_a）,the number of binding sites（n）,the quenching type,binding force type,and hill coefficient of CPS-PAPA,CPS-PAPA_（Trp）and CPS-PAPA_（Tyr）were obtained at different temperatures.The two methods had the same conclusion.At the same time,Synchronous fluorescence spectra also showed that the combination of CPS and PAPA could change the conformation of PAPA,while both Trp and Tyr of PAPA participated in the binding reaction.The fluorescence quenching ratio of Trp and Tyr residues of PAPA,the binding rate and binding model of the binding reaction were obtained.The study had a certain guiding significance for clinical drug use.