Interaction Of Some Small Molecules With Bovine Serum Albumin Using Spectroscopy

The reaction mechanism of small molecules with Protein was studied by fluorescence spectroscopy (FS) and UV spectroscopy (UV) and interaction of several organic molecules with bovine serum albumin (BSA) were investigated. There are two parts in this work:1. the recent developments and its significance of interaction between organic molecules and serum albumin were summarized; 2. the binding interaction of several organic molecules with BSA were studied by kinds of spectroscopy methods under simulated physiological conditions and the spectral data were resolved by chemometrics methods in turns. 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 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.In the first section, the definition, structure, function and nature of proteins were introduced briefly. Furthermore, 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, forecasting the development trends and exploiting new probes, applying various techniques simultaneously and the use of chemometrics were chosen as the emphasis of this thesis.In the second section, the binding interaction of Alpinetin (APT) with BSA was studied by fluorescence, UV-visible and synchronous fluorescence spectroscopy (SFS). The measured complex spectra were resolved by multivariate curve resolution-alternating least squares (MCR-ALS), which yielded a host of data and information, which otherwise would have been impossible to obtain. The extracted profiles corresponded to the spectra of the single species in the APT/BSA mixture. In addition, the presence of the APT-BSA complex was demonstrated, and it was shown that the associated quenching of the fluorescence from the BSA protein resulted from the formation of APT-BSA complex via a static mechanism. The binding constant (Ka(ave)= 2.34×106 L mol-1) and the number of sites (n=1) were obtained by fluorescence methods as were the thermodynamic parameters (ΔH0,ΔS0 andΔG0). This work suggested that the principal binding between APT to BSA was facilitated by hydrophobic interactions. The thermodynamic parameters for APT were compared to those from the structurally similar Chrysin and Wogonin molecules. It appeared that the entropy parameters were relatively more affected by the small structural changes. SFS from the interaction of BSA and APT showed that the ligand affected the conformation of BSA. The competitive interaction of APT and site makers with BSA indicated site I as the binding area of APT in BSA.In the third section, the association of brodifacoum (BDF) with BSA was monitored with the using of fluorescence spectroscopy. The measured spectra were resolved by SIMPLISMA, which was the first application to the system concerned with the interaction of BSA with small molecules, and the extracted profiles corresponded to the spectra of the single species in the BDF/BSA mixture. In addition, the presence of the BDF-BSA complex was demonstrated, and it was shown that the associated quenching of the fluorescence from the BSA protein resulted from the formation of BDF-BSA complex via a static mechanism. Fluorescence sensitization of BSA to BDF also was studied by adding different concentration of BSA into different concentration of BDF. The results showed that the higher concentration corresponds the more quantum efficiency resulting in stronger sensitization. Synchronous fluorescence spectra three-dimensional fluorescence spectroscopy was used to study the conformation change of BSA invoked by BDF. As a result, BDF could lead to the conformation change of BSA apparently and the polarity around the Trp residues on BSA increased and the hydrophobicity decreased, while the interaction of BSA with BDF did not have a distinct effect on the conformation of the microenvironment around Tyr. The binding sites of BDF on BSA were studied by using Eosin Y which is a new site marker and has been found to bind to site I. The conclusion was obtained that the two molecules, BDF and Eosin Y, did not readily share a common binding site on the BSA protein rather than the BDF been replaced by EY absolutely.In the forth section, The binding interaction of carbofuran (CF) and its degradation product, 2,3-dihydro-2,2-dimethyl-7-hydroxy benzofuran (carbofuranphenol, CFP), with BSA was studied by spectrofluorimetry using chemometrics approaches. It was shown that both CF and CFP quenched the intrinsic fluorescence of BSA via a static mechanism. By making use of the chemometrics techniques, SVD and MCR-ALS, to solve the data obtained from the fluorescence spectrum of the interaction of either CF or CFP with BSA, we can draw a conclusion that there were three components exiting in the mixture solutions of BSA and either carbofuran or its degradation product, in other words, they interacted each other and gave birth to a complex. Synchronous and three-dimensional fluorescence spectroscopy of the interaction of BSA with either CF or CFP showed that the molecular structure of the BSA was changed significantly, which is consistent with the known toxicity of the pesticide i.e., the protein is denatured. The value of the binding constant K of BSA with its degradation product is bigger than that of BSA with CF which illuminated its degradation product is more toxic than its parent, CF. In addition, the effect of some metal ions on the binding constants of either CF or CFP with BSA was also investigated.In the fifth section, the binding interaction of the 10-hydroxycamptothecin (10-HCPT) with BSA, using warfarin as a fluorescence probe, was studied by spectrofluorimetry and UV-vis spectroscopy with the aid of chemometrics. The result indicated that the endogenetic fluorescence quenching of BSA by 10-HCPT is static quenching. In this work, the competitive interaction of the 10-HCPT and warfarin with BSA was also studied by three-way excitation-emission fluorescence by parallel factor analysis (PARAFAC). The estimated relative concentration profiles of the components showed that the binding site of the 10-HCPT on BSA is site I. The effect of 10-HCPT on the conformation of BSA was studied by synchronous fluorescence technique, and the results indicated that the conformation of BSA was changed when 10-HCPT was added, and the hydrophobic properties of the environment of residues in BSA decreased.