Study On Interaction Mechanism Of Drug With BSA By Spectrometry Techniques

This thesis is divided into four parts. The research actuality of interaction between small moleculeswith serum albumin in recent years has been reviewed. The application of chemometrics in analysis ofspectral data also had been developed. And the application of chemometrics in complicated biochemicalsystem can solve some problems and obtain the equilibrium concentration and pure spectra of eachcomponent.PartⅠIn this section, the chemical and biological characters of serum albumin were introduced briefly first,then the research methods and existent problems of interaction between small molecules and proteinswere reviewed in turns. Serum albumin is the most abundant of all proteins in blood plasma of manyspecies, and study on it has become an important research field of life science. Along with more attentionhas been given on the research of the interaction between serum albumin and small molecules, themechanisms of the action of some drugs and origins of some diseases have generally been understood.Moreover, the investigation based on serum albumin interactions with small molecular compounds alsohas great significance to design new protein-targeted drugs and to screen these drugs in vitro. At last, thedevelopment trend was forecasted and exploiting new probes, using various techniques simultaneouslyand the use of chemometrics were chosen as the emphasis of this thesis.PartⅡIn part two, the interactions of salicylic acid (SL) and two different site markers (warfarin for siteⅠand ibuprofen for siteⅡ) with bovine serum albumin (BSA) in pH 7.4 Tris-HCl buffer have beeninvestigated with the use of spectrofluorimetry. An equilibrium solution of BSA and SL was titratedseparately with the two markers. This initial work showed that the binding of SL with BSA could be quitecomplex, and that there was probably a competitive interaction occurring between ibuprofen and SL.However, the spectral results were difficult to interpret clearly for the interaction of warfarin and SL insimilar circumstances.To extract more information from the resolution of fluorescence excitation-emission spectra, thecontour plots of the fluorescence spectra indicated that the optimal excitation wavelengths for BSA, SL,warfarin and ibuprofen were different, and were found to be at 278, 295, 306 and 218 nm, respectively.The spectral information was arranged into three-way EEM stack arrays, and was submitted for analysisby the PARAFAC algorithm. Firstly, it was demonstrated that the estimated excitation and emissionspectral responses for SL, BSA and the site markers, warfarin and ibuprofen, agreed well with themeasured spectra. Then, the interpretation of the plots of simultaneously extracted (by PARAFAC)equilibrium concentrations for the above four reactants, showed that:ⅰ) the SL primarily appears to bindin siteⅠbut at a different location from the high-affinity binding site (HAS) for warfarin, and theinteraction partially overlaps with the low-affinity binding site (LAS) for warfarin,ⅱ) the SL may havetwo LAS, -one in siteⅡwhere the HAS for ibuprofen is located, and the other in siteⅠat the LAS foribuprofen.PartⅢIn part three, the interaction between fluoroquinolones (FQ, here, ofloxacin and enrofloxacin) andBSA was investigated by fluorescence and UV-vis absorbance spectroscopy. The Stern-Volmerquenching constant Ksv, the binding constant K, the number of binding sites n and corresponding thermodynamic parameters AH, AS and AG were calculated. The distance r between donor (BSA) andacceptor (FQ) was obtained according to the theory of F(o|¨)rster's nonradiative energy transfer. The effectof FQ on the conformation of BSA has been analyzed by means of both UV-vis absorbance spectra andsynchronous fluorescence spectroscopy. It was proved that the fluorescence quenching of BSA by FQ ismainly a result of the formation of FQ-BSA complex and hydrogen bonds and van der Waals play majorrole in the reaction. All the results showed that the mechanism of the interaction between ofloxacin withthe BSA is similar to that of enrofloxacin.PartⅣIn part four, the interaction between berberine chloride (BC) and BSA was studied by fluorescenceand UV-vis absorbance spectroscopy in pH 7.4 Tris-HC1 buffer at 37℃. The spectra of the mixturesolutions varied in evidence through the process of the interaction, but the spectrum of each component inthe BC-BSA system showed significant overlapping between each other. Moreover, the analysis ofindividual data matrices by the MCR-ALS method might exist possible unresolved underlying factoranalysis ambiguities. Therefore, the whole set of spectroscopic data matrices was simultaneouslyanalyzed by the MCR-ALS method. This procedure allowed the recovery of the concentration profilesand pure spectra for each species and the calculation of the BSA:BC ration in the complex and theapparent equilibrium constant.