Study On Interaction Of Active Components In Chinese Traditional Medicines With Human Serum Albumin

The molecular interactions between molecule and biological molecule have been important for chemistry biology investigation. Now, the interactions between protein and drug have been widely studied in this field. Serum albumin, the most abundant protein in blood plasma, has many important physiological functions. It can bind many exogenous and endogenous compounds, and realizes transport and distribution of many molecules and metabolites. Protein-drug interactions play an important role in a variety of biological processes. The studies on this aspect may provide information of the structural features that determine the therapeutic effectiveness of drug, and became of an important research field in life sciences,chemistry and clinical medicine. With development of molecule biology and apparatus technology, the investigation of interaction between small molecule and biological molecule make a great progress in this field. A series of study methods which can not research in this field begin to exert important affection. On the basis of the previous research, the following major innovative work was carried out in this dissertation.(1) Several methods including fluorescence spectra, CD and FT-IR specta methods were used to study the interaction of isoquinoline alkaloid,isoflavones and Rhein with serum albumin, and the results from every method was in good agreement;(2) The binding constants,the numbers of binding site,the binding region and the interaction force involving in the interaction of drugs and serum albumin were confirmed by Scatchard equation and Van't Hoff equation. The binding mechanism of isoflavones with serum albumin were investigated in detail using fluorescence polarization;(3) The secondary structure compositions of protein and their drugs complexs were estimated by qualitative and quantitative analysis using FT-IR spectrogram;(4) The molecular modeling method was used to study the drug-protein interaction and the study results were in consistent with the experimental results;(5) A novel flow injection analysis method with Rayleigh light scattering detection was developed for the determination of protein concentration in human blood plasma. This dissertation consists of five chapters:Chapter 1: The structures,functions and natures of proteins were briefly introduced firstly. The developments of interaction of drugs with protein were reviewed, and the methods used to study the interaction of drugs with protein were summarized.Chapter 2: The binding of Jatrorrhizine and Berberine with human serum albumin was investigated by spectroscopy and the molecular modelling study. The studied results indicated that Jatrorrhizine and Berberine can strongly bind to human serum albumin. The binding constant were 7.278×10⁴ M^-1 and 4.071×10⁴ M^-1 at 296K. The interaction of Jatrorrhizine and Berberine with serum albumin can induce the changes of secondary structure of serum albumin with reduction ofα-helices. On the basis of all the experimental results and the molecular modelling study, it is considered that Jatrorrhizine and Berberine binds to the site I and site II mainly by hydrophobic interaction and there are hydrogen bond and electrostatic interactions.Chapter 3: Fluorescence anisotropy and fluorescence spectroscopy were used to characterize optical properties of the drug-HSA complex and to gain information on the binding mechanism at molecular level. Binding of Daidzein and Formononetin with HSA leads to dramatic enhancement in the fluorescence intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. Fourier transform infrared measurements shown that the secondary structures of the protein have been changed by the interaction of drug with HSA. Computational mapping of possible binding sites of drug revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA, and hydrophobic force plays a main role in the binding process.Chapter 4: The binding of Rhein with human serum albumin (HSA) has been studied in detail by Spectroscopic method including circular dichroism (CD), fourier transformation infrared spectra (FT-IR), fluorescence spectra. The binding parameters for the reaction have been calculated according to Scatchard equation at different temperatures. The plots indicated that the binding of HSA to Rhein at 303K is characterized by one binding site with the affinity constant K at 4.93×10⁵ M^-1. The secondary structure compositions of free HSA and its Rhein complexes were estimated by the FT-IR spectra. FT-IR and curve-fitted results of amide I band are in good agreement with the analyses of CD spectra. Computational mapping of possible binding sites of drug revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA, and hydrophobic force plays a main role in the binding process.Chapter 5: A novel flow injection analysis (FIA) method with Rayleigh light scattering (RLS) detection was developed for the determination of protein concentrations. This method is based on the weak intensity of RLS of Amide Black 10B and Eriochrome Black T which can be enhanced by addition of protein in weakly acidic solution. It has proved that the application of this method to quantify the proteins by using human serum albumin was available in real samples. In addition, this method is very sensitive, simple, rapid and tolerance of most interfering substances. The FIA-RLS method was more stabile than the general RLS method and the average RSD value of FIA-RLS less than general RLS. The effects of different interfering substances will be also examined. The amount of proteins in human serum sample was determined.