Study On Interaction Of Organic Small Molecules Substances With Human Serum Albumin

Human Serum Albumin (HSA) is the most abundant carrier protein in blood circulation. It has many important physiological and pharmacological functions, which can bind many exogenous and endogenous ligands in blood, and realize transport and distribution of many molecules and metabolites, such as fatty acids, amino acids, bilirubin, hormones and many diverse drugs. Therefore, it has been one of the most extensively studied of all proteins. Investigating the binding mechanism of toxic materials and drugs with HSA has many importances in toxicology and pharmacokinetics. Thus, it has been an interesting research field of life sciences, chemistry and clinical medicine. In this dissertation, on the basis of the previous research, the fluorescence spectroscopy combined with UV-visible absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and computational modeling were used to investigate the interaction of several organic small molecule substances with HSA. The following major innovative works were carried out:1. Several methods were associated to study the interaction of diphacinone and four components of Chinese herb medicine (glycyrrhetinic acid, sophoricoside, 5,7,4'-trihydroxy-6,3',5'-trimethoxy flavone and eupatilin) with HSA. The binding properties including the fluorescence quenching mechanisms, binding constants and the number of binding sites were investigated in detail, thermodynamic parameters were calculated, and the main interaction force between drugs and proteins was discussed.2. The cause of departure from the Stern-Volmer plots at different conditions was analyzed.3. The effects of drugs on HSA second structure were investigated with FT-IR techniques.4. The computational modeling method was used to study the drug-HSA interaction and the study results were in consistent with the experimental results.This dissertation consists of five chapters:Chapter 1: The structures, functions and natures of proteins were briefly introduced. The contents and methods of interaction of different kind of small ligands with protein were reviewed.Chapter 2: The interaction of diphacinone with HSA was studied by the methods of fluorescence and FT-IR spectroscopy under simulative physiological conditions. The results of spectroscopic measurements suggested that the Stern-Volmer plots and Scatchard plots both has two regression curves intersecting at Cdiphatinone/CHSA ~l-2 with diphacinone concentrations S.OxlO'M.SxlO"6 mol/L and 1.8><10'6~3.2xl0"6 mol/L, and the Stern-Volmer plots deflected to Y axes. When CdiPhacmone/CHSA was lower than 1.2, the numbers of binding sites were near 1.1, and CdiPhacinone/CHSA was higher than 1.2, the numbers of binding sites were approximately 1.8. The FT-IR spectra proved that the secondary structure of HSA changed after interacting with diphacinone in aqueous solution. On the basis of fluorescence experimental results and the molecular modelling study, it is considered that diphacinone binds to the subdomain IIA (site I) mainly by hydrophobic interaction.Chapter 3: Fluorescence spectroscopy, FT-IR and molecular modeling methods were employed to analyze the binding of glycyrrhetinic acid (GEA) toHSA with GEA concentrations from 4.0X10"6 to 4.5xlO"5 mol/L. The results indicated that the Stern-Volmer plots deflected to X axes. The binding of GEA to HSA was via two types of sites: the numbers of binding site for the first type was near 0.45 and for the second type was approximately 0.75. And the binding constants of the second type binding site were lower than of the first type binding site at corresponding temperatures. Thermodynamic analysis showed that van der Waals interactions was the mainly binding force in the first type of binding site, and electrostatic interactions might play a main role in the second type of binding sites. The study of computational modeling implied the GEA was only partially bound to HSA which was in agreement with the fluorescence experimental results. The FT-IR spectra evidence showed that the protein secondary structure changed with reduction of a-helices about 26.2% atthe drug to protein molar ratio of 3.Chapter 4: The interaction of sophoricoside with HSA has been investigated by UV-absorption, fluorescence spectroscopy, FT-IR spectroscopy and molecular modelling methods at simulative physiological pH. The results of spectroscopic measurements suggested that the intrinsic fluorescence of HSA was quenched by sophoricoside through static quenching mechanism and the interaction was via a single class of binding site. Secondary structure of HSA changed with reduction of a-helices after interaction of sophoricoside with HSA. The results of fluorescence spectroscopy and molecular modelling method suggested that sophoricoside can bind within the subdomain IIA of the HSA (site I) and hydrophobic interaction was the predominant binding force between sophoricoside and HSA.Chapter 5: The binding of 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone and eupatilin with HSA have been studied using UV absorption spectroscopy, fluorescence spectroscopy, FT-IR spectra and molecular modelling methods, respectively. The binding constants, numbers of binding site, mainly intermolecular force and the changes of HSA secondary structure induced by drugs binding were obtained from the fluorescence spectroscopy, FT-IR spectra. The research results indicated that the two drugs can bind to HSA by only one class of binding sites, respectively. The binding constants decreased with increasing temperature, hydrophobic interaction can play a main role in the binding process. The primary binding site of the two drugs was located in site I of HSA.