Spectroscopy Studys On The Mechanism Of Cefepime With Bovine Serum Albumin And Fluorescence Properties Of Sarafloxacin

At present,methods of traditional fluorescence quenching and synchronous fluorescence are widely applied to study the interaction between proteins and small molecule drugs.This article is mainly about cefepime hydrochloride(CH)as the representative of small molecule drugs,and a variety methods of fluorescence spectrometry is used to study the reaction mechanism of the interaction.Moreover,researches on the microenvironment changes of amino acid residues in the process are further studied.Also,fluorescence characteristics of small molecular drugs itself are studied in the meanwhile.This research contents include the following five parts:Chapter one: Research methods of studying the interaction between proteins and small molecule drugs are summarized.Hereby,development in studying interaction between proteins with small molecules by spectroscopy is introduced systematicly.And based on above,the research contents for study of the reaction mechanism between small molecules and proteins are established.Chapter two: The fluorescence spectra of sarafloxacin(SAR)in different pH conditions are studied in order to determine its structural changes due to protonation with pH changes.When pH<1.02,SAR exists of H3L2+ form of which maximum fluorescence emission wavelength is about 455 nm.When pH is 1.87~4.94,SAR exists of H2L+ form that H3L2+ loses one proton which 1-position of nitrogen of quinoline ring.When pH is 7.14~9.30,the maximum emission wavelengths are gradually blue shifted to 430 nm with the increase of pH.SAR exists in the form of bipolar ion HL that H2L+ loses proton of carboxyl.When pH>11.6,HL transforms into anionic L-that HL loses one proton of piperazine ring,leading to adecrease in the fluorescence intensity,and the maximum emission wavelength is red shifted to approximately 466 nm.The two-step dissociation constant pKa of SAR is calculated,the pKa1 is 6.06±0.37 and the pKa2 of SAR is 10.53±0.19.In the buffer solution of pH 3.62,with quinine sulfate as reference,the fluorescence quantum yield of SAR in the maximum excitation wavelength of 276 nm is 0.09.Chapter three: Under simulated physiological conditions,the interaction of colistinsulfate,cefpirome sulfate,cefpiramide sodium with BSA are investigated by fluorescence quenching,synchronous fluorescence,resonance light scattering and UV/vis absorption spectroscopy at 298 K.The results show that the binding constants of three systems obtained from four methods are in the same order of magnitude.The values of Kq obtained from this experiment are all larger than 2×1010 L·mol-1·s-1.The values of n approximately equal to 1.The values of Hill’s coefficients are approximately equal to 1.The experimental results obtained from four methods can show that the methods are feasible.Chapter four: Under simulated physiological conditions,the reaction mechanism between CH and bovine serum albumin at different temperatures is investigated by fluorescence quenching method and synchronous fluorescence,respectively.The results indicate that the fluorescence intensity and synchronous fluorescence intensity of BSA decreased regularly with the addition of CH.In addition,the quenching mechanism,binding constants,the number of binding site,type of interaction force and energy transfer parameters of CH with BSA obtained from two methods by the same equation are consistent,which indicates synchronous fluorescence spectrometry can be applied in the study of the binding mechanism.It is a useful supplement to the conventional method.Chapter five: Changes in the microenvironmental polarity of aromatic amino acid residues during the interaction between BSA and CH are studied using fourth-derivative ultraviolet spectroscopy,intrinsic fluorescence spectroscopy,synchronous fluorescence spectroscopy,near-UV circular dichroic spectroscopy and site reagent.CH molecules first gather near the Trp-213 in structure domain IIA with CH concentration increasing,which enhances the microenvironmental polarity of Trp amino acid residue.BSA unfolds from domain IIA and more aromatic amino acid are exposed,causing the changes of microenvironment polarity enhancement of Phe and Tyr residues.When increasing to a certain concentration,microenvironment polarities remain constant and the BSA conformation remain unchanged.