Spectrophotometric Study On Interaction Of Some Small Molecules With Bovine Serum Albumin With The Aid Of Chemometrics

In this work,the research work of interactions between small molecules and serum albumin in recent years has been reviewed and the interaction between some drugs with bovine serum albumin(BSA) was studied.Spectroscopic techniques are widely used in analytical chemistry, however,the spectroscopic investigation combined with chemometrics,particularly for the interaction of small molecules and macromolecules,are relatively few.The application of chemometrics method-parallel factor analysis(PARAFAC) and multivariate curve resolutionalternating least squares(MCR-ALS) in processing of spectral data also has been developed. And the application of chemometrics in complicated biochemical systems can be used to solve some problems and the equilibrium concentration and pure spectra of each component can be obtained.This thesis is divided into five chapters:ChapterⅠIn this chapter,the definition,structure,function and nature of proteins were introduced briefly. Then the research methods and existent problems of interaction between small molecules and proteins were reviewed in turns.The application of chemometrics in the field of protein research was discussed.At last,the development trends were forecasted and exploiting new probes,using various techniques simultaneously and the use of chemometrics were chosen as the emphasis of this thesis.ChapterⅡThe binding interaction of the pesticide isoprocarb and its degradation product,sodium 2-isopropylphenate,with BSA was studied by spectrofluorimetry.Both isoprocarb and sodium 2-isopropylphenate quenched the intrinsic fluorescence of BSA.This quenching proceeded via a static mechanism.The binding of isoprocarb to BSA involved hydrogen bonds and that of sodium 2-isopropylphenate to BSA involved hydrophobic and electrostatic interactions. Synchronous fluorescence spectroscopy of the interaction of BSA with either isoprocarb or sodium 2-isopropylphenate showed that the molecular structure of the BSA was changed significantly,which is consistent with the known toxicity of the pesticide.The resolution of the three-way excitation-emission fluorescence spectra by the PARAFAC method extracted the relative concentration profiles of BSA.These profiles showed that the degradation product, sodium 2-isopropylphenate,displaced the pesticide in a competitive reaction with the BSA protein and the binding site of sodium 2-isopropylphenate and isoprocarb on BSA is the same one.ChapterⅢIn this part,the binding interaction of the terazosin hydrochloride and prazosin hydrochloride with BSA was studied by spectrofluorimetry under simulated physiological conditions.Both terazosin hydrochloride and prazosin hydrochloride quenched the intrinsic fluorescence of BSA. This quenching proceeded via a static mechanism.The thermodynamic parameters(ΔH~0,ΔS~0 andΔG~0) obtained from the fluorescence data measured at two different temperatures showed that the binding of terazosin hydrochloride to BSA involved hydrogen bonds and that of prazosin hydrochloride to BSA involved hydrophobic and electrostatic interactions.In this work,the competitive interaction of the terazosin hydrochloride(prazosin hydrochloride) and site marker (warfarin or diazepam) with BSA was studied by three-way excitation-emission fluorescence with the aid of PARAFAC.The estimated relative concentration profiles of the components showed that the binding site of the terazosin hydrochloride and prazosin hydrochloride on BSA is site I.ChapterⅣThe interaction between bisoprolol frumarate(BSP) and BSA was studied by fluorescence and UV-vis absorbance spectroscopy with the aid of MCR-ALS.The spectrum of each component in the BSP-BSA system showed overlapping between each other.Moreover,the analysis of individual data matrices by the MCR-ALS method might exist possible unresolved underlying factor analysis ambiguities.Therefore,the whole set of spectroscopic data matrices were augmented and analyzed by the MCR-ALS method simultaneously.This procedure allowed the recovery of the concentration profiles and pure spectra for each species and the apparent equilibrium constant.The effect of bisoprolol frumarate on the conformation of BSA was also analyzed using synchronous fluorescence and UV-vis absorbance spectrometry.ChapterⅤThe interaction between umbelliferone and bovine serum albumin(BSA) was studied by fluorescence spectroscopy and MCR-ALS in this part.The interaction between umbelliferone and BSA was studied by taking two different kinds of titration modes.The two spectroscopic data matrices were augmented,and then the information of experiment data was increased.The augmented experiment data matrix was analyzed by the MCR-ALS method.This procedure allowed the recovery of the concentration profiles and pure spectra for each species and the apparent equilibrium constant.The effect of umbelliferone on the conformation of BSA was also analyzed using synchronous fluorescence spectrometry.