Spectroscopic Studies On The Interaction Of Quinolones With Proteins

The interactions between drugs and biological molecule have been important for chemistry biology investigation.Now,the interactions between protein and drug have been widely studied in this field.Thereinto,Human serum albumin(HSA)is the most abundant carrier protein in blood circulation,which can bind many endogenous and exogenous substances,such as metabolites,drugs and other biologically active compounds present in blood,and realize transport and distribution of them.Therefore, the investigation of the interaction of HSA with drugs contributes to understand disposition,transportation,metabolism and efficacy of drugs.Lysozyme(Lys)is a small monomeric globular protein as a bacteriolytic agent having an ability to hydrolyze bacterial cell walls.Catalase is a key antioxidant which detoxifies H₂O₂ by reducing this molecule into molecular oxygen(O₂)and water(H₂O).Lysozyme and catalase also can bind many small molecule substances such as drugs,which has effect on metabolism and efficacy of drugs and enzymic activity.Thus,it has been an interesting research field of life sciences,chemistry and clinical medicine.With development of molecule biological and apparatus technology,the investigation of interaction betwwen small molecule and biological molecule make a great progress in this field.A series of study methods which can not research in this field begin to exert important affection.On the basis of the previous research,the following major innovative works were carried out in this dissertation:1.Several methods including fluorescence spectra,UV-visible absorption spectra and circular dichroism spectra methods were used to study the interaction of drugs (Pazufloxacin mesilate and Conggo red)with several proteins(Human serum albumin, Lysozyme and Catalase),and the results from every method was in good agreement.2.The fluorescence quenching mechisms,binding constants,the number of binding sites and the interaction forces involving in the interaction of drugs with several proteins were confirmed by Stern-volmer equation and Van't Hoff equation.3.The cause of using synehornous fluorescence spectroscopy in the interaction pazufloxacin mesilate with proteins was analyzed.The binding mechanism of pazufloxacin mesilate with catalase was investigated in detail using fluorescence polarization.The contour plot of the fluorescence spectra provided the optimal excitation wavelengths for lysozyme and congo red.The data of resonance light scanning fluorescence indicated the changes of microenvironment of lysozyme induced by the binding congo red. 4.The effects of drugs on proteins secondary structure were investigated with circular dichroism spectra techniques.This dissertation consists of four chapters:Chapter 1:The structure,functions and properties of proteins were briefly introduced.The developments of interaction of drugs with protein were reviewed,and the methods used to study the interaction between drugs with protein were summarized.Chapter 2:The binding of pazufloxacin mesilate with human serum albumin or lysozyme has been studied using synchomous fluorescence spectra,UV-visible absorption spectra and circular dichroism spectra methods under simulative physiological conditions.The studied results indicated that the intrinsic fluorescence of human serum albumin or lysozyme was quenched by pazufloxacin mesilate through a formation of complex mechanism.The binding constants,numbers of binding site,and mainly intermolecular force induced by drug binding were obtained from the fluorescence spectroscopy.The effects of pazufloxacin mesilate on protein secondary structure were investigated by circular dichroism spectroscopy. Chapter 3:Fluorescence anisotropy,synchornous fluorescence spectroscopy and circular dichroism spectroscopy were used to characterize optical properties of pazufloxacin mesilate-catalase complex and to gain information on the binding mechanism at molecular level.Binding of pazufloxacin mesilate with catalase leads to dramatic decrease in the fluorescence intensity at 280 nm(catalase)and increase in anisotropy values.The binding constants,number of binding sites,and the effect of catalase secondary structure induced by drug binding were obtained from the fluorescence spectroscopy,circular dichroism spectroscopy.The thermodynamic analysis indicated both hydrogen bonds and electrostatic interactions can play a major role in the binding process.Chapter 4:The binding of congo red with lysozyme has been studied using excitation-emission matrix fluorescence,steady fluorescence,resonance light scanning fluorescence and circular dichroism measurements under simulative physiological conditions.The contour plot of the fluorescence spectra provided the optimal excitation wavelengths for lysozyme and congo red at 280 and 320 nm, respectively.The results of the fluorescence spectra showed that both static and dynamic quenching occurred together with complex formation.Thermodynamic parameters for binding suggested that the interactions are primarily through hydrophobic interactions.The results of circular dichroism spectrum indicated that the secondary conformational changes of lysozyme upon interaction with congo red.In addition,the data of resonance light scanning fluorescence also indicated the changes of microenvironment of lysozyme induced by the binding congo red.