Study On The Interaction Between Plant Active Components And Serum Albumin By Spectroscopic Method

In this thesis, the binding modes between some plant active components with serum albumin were studied using different spectroscopic techniques viz., fluorescence, UV-vis absorption, Fourier transform infrared spectroscopy and circular dichroism. The application of chemometrics in analysis of three-dimensional data also had been developed, and it can solve some problems and obtain the equilibrium concentration and pure spectra of each component in complicated biochemical system.The thesis consists of five parts and the main conclusions are summarized as following.1. The status quo of drug molecules and protein interaction was summarized briefly. The some methods in this research field were summarized including fluorescence, UV-vis absorption, Fourier transform infrared spectroscopy and circular dichroism.2. The binding interaction between alpinetin and bovine serum albumin (BSA) in physiological buffer solution (pH 7.4) was investigated by fluorescence, UV-vis spectroscopy and fourier transform infrared (FT-IR) spectroscopy. It was proved from fluorescence spectra that the fluorescence quenching of BSA by alpinetin was probably a result of the formation of BSA-alpinetin complexes. The thermodynamic parameters, enthalpy change (△H) and entropy change (△S), were calculated to be 22.10 kJ-mol-1 and 166.04 J·mol-1·K11, respectively, which indicated that the interaction between alpinetin and BSA was driven mainly by hydrophobic interaction. Moreover, the competitive experiments of site markers suggested that the binding site of alpinetin to BSA was located in the region of subdomain IIA (sudlow site I). In addition, the results of synchronous fluorescence and FT-IR spectra demonstrated that the microenvironment and the secondary structure of BSA were changed in the presence of alpinetin.3. The interaction between chrysin or salidroside and human serum albumin (HSA) was studied by fluorescence and UV-vis absorption spectroscopy in physiological buffer solution. There was a strong fluorescence quenching reaction of chrysin to HSA. However, the fluorescence enhancement of salidroside was observed. The thermodynamic parameters of the interactions indicated that the interaction between chrysin and HSA was driven mainly by electrostatic force, whereas the interaction between salidroside and HSA was driven mainly by hydrogen bond and van der Waals forces. The results of synchronous fluorescence spectra and three-dimensional showed that binding of chrysin or salidroside to HSA induced conformational changes in HSA.4. The interaction between vitexin and HSA has been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, FT-IR spectroscopy and circular dichroism (CD). Fluorescence results revealed the presence of static type of quenching mechanism in the binding of vitexin to HSA. The thermodynamic parameters indicated that the interaction of vitexin with HSA was driven mainly by hydrogen bond and van der Waals forces. It was found that vitexin was located near the tryptophan residue region of subdomainⅡA (sudlow siteⅠ) in HSA by competing binding experiments. The results of synchronous fluorescence, FT-IR and CD spectra demonstrated that the microenvironment and the secondary structure of HSA were changed in the presence of vitexin. In addition, the effect of vitamin C (VC) or vitamin B2 (VB2) on the interaction between vitexin and HSA was investigated.5. The interaction of puerarin with human serum albumin (HSA) in pH 7.4 Tris-HCl buffer has been investigated by fluorescence spectroscopy. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of puerarin to HSA. It was found that puerarin was located near the tryptophan residue region of subdomainⅡA (sudlow siteⅠ) in HSA by competing binding experiments using three different site markers (warfarin for siteⅠ, ibuprofen for siteⅡand digitoxin for siteⅢ). Furthermore, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way synchronous fluorescence spectra data of the competitive interaction between puerarin and warfarin with HSA, and the result provided simultaneously the concentration information for the three reaction components, warfarin, puerarin-HSA and puerarin, for the system at equilibrium. The PARAFAC analysis indicated the binding sites of puerarin to HSA located within subdomain IIA (sudlow site I).