Study On Interaction Of Some Active Components In Chinese Traditional Medicines With Globulins

Chinese traditional medicines have been widely used in multi-component form. The pharmacological effect of active component plays a key role in that of Chinese herbs. It is very important for advancing and realizing Chinese herb modernization to elucidate the substance basic and the mechanism of active component. Protein serves as a transport carrier for drugs, and the binding of drugs with protein has a great influence not only upon the distribution of the drugs in the body but also upon their patterns of metabolism and excretion. Further, from the standpoint of drug efficacy, only that portion of a drug that is not bound with plasma protein is generally bioactive. Thus, the study of the binding characteristics of medical drugs to protein is an important field in drug research. In a series of study methods concerning the interaction of drugs and protein, fluorescence techniques are great aids in the study of interactions between drugs and serum albumin because of their high sensitivity, rapidity and ease of implementation. Fourier-transform infrared (FTIR) spectroscopy is a useful tool for examining the secondary structure of proteins. The most important advantage of FTIR spectroscopy for biological studies is that the spectra of almost any biological system can be obtained in a wide variety of environments. Circular dichroism (CD) spectroscopy is another frequently used technique for secondary-structure analysis of protein. On the basis of the previous research, the following major innovative works were carried out in this dissertation:1. Several active components of Chinese herbs possessed similar or analogicalstructure were selective as research targets. In combination with severalspectroscopic methods and the molecular modeling technique, the interactions ofthese active components with different proteins have been investigated fully.2. Three globulins (including human serum albumin, human gammaglobulin andlysozyme) were used as model protein to study the interactions with different drugs and their change of secondary structure.3. The secondary structure compositions of three globulins and their drugs complexs were estimated by qualitative and quantitative analysis and the results were in good agreement with CD experimental data.4. The effect of fluorescence enhancement was used to study the interaction between Chinese herb guaiacol and globulins. The fluorescence anisotropy methods indicated the structure change of proteins in molecular level.5. The information from the synchronous fluorescence spectra were used to determine the different binding modes for the different drug-protein systems.6. It is the first time to investigate several parameters of physical chemistry for the different drug-protein systems including charge density, dissociation constants (pKa) and quantum yield.7. The effect of common ions on the binding constants of the different drug-protein complexes was discussed comparatively.This dissertation consists of six chapters:Chapter 1: In this chapter, the actuality and the development of Chinese herbs were briefly introduced firstly. Then the structures, functions and natures of proteins, especially globulins were described. In succession, the principles and methods of fluorescence, UV-visible, CD, FTIR and the molecular modeling techniques for studying the interaction of drugs and proteins were described and the applications of these techniques in investigating the binding of drugs to proteins were reviewed in detail.Chapter 2: Two active components of Chinese herbs possessed similar or analogical structure (Alpinetin and Cardamonin) were selective as research targets. A combination of intrinsic fluorescence, CD, FTIR and the molecular modeling techniques has been used to characterize the binding between two flavones and three globulins (human serum albumin HSA;human gammagobulin, IgG;lysozyme, LYSO) under physiological conditions. The results from fluorescence quenching dataindicated that the two drugs could interact with three proteins strongly. The binding constants for the different dug-protein systems were different under different temperatures, and the binding modes were also different. The results of molecular model study revealed the different binding locations for drug-HSA or drug-IgG complexs. The secondary structure compositions of three globulins and their drugs complexs were estimated by qualitative and quantitative analysis from FT-IR and CD experimental data.Chapter 3: The interactions between two coumarins (psoralen and isopsoralen) and two globulins (HSA and IgG) were investigated by spectroscopy methods and the molecular modeling techniques. The results showed that the two drugs can bind with two proteins strongly and quench the fluorescence of proteins. The results from FT-IR and CD spectra indicated the different degree on the changes of the secondary structure compositions of two globulins and their drugs complexs. The efficiency of FSrster energy transfer provided different distances between tryophan and drugs binding site.Chapter 4: This study was designed to examine the interaction between shikonin and two globulins (HSA and LYSO) under simulated physiological conditions. Fluorescence quenching methods in combination with CD and FTIR technique were used to determine the drug-binding modes, the binding constants and protein-structure changes in the presence of shikonin in aqueous solution. In addition, the different binding mode between shikonin and proteins was explored through Silicon Graphics Ocatane2 workstation. The research results indicated that shikonin bound to protein with high binding constant, and the distances between the binding location and tryptophan residue. Shikonin binds within the subdomain IIA of HSA, and hydrophobic force plays a main role in the binding force.Chapter 5: The fluorogenic enhancement effect of guaiacol was exploited for the first time to analyze the interaction with target protein as a probe by molecule modeling, fluorescence, Fourier transform infrared spectroscopy (FT-IR), and circular dichroism (CD) spectroscopy. The different binding constants were obtained from the experimental results. In addition, from the high value of fluorescence anisotropy(r=0.40) it is argued that the probe molecule is located in motionally restricted environment of the protein. The thermodynamic functions enthalpy (AH) and entropy (AS) for the reaction were calculated according to Vant's Hoff equation. The binding modes were also determined combining the molecular modeling. The effects of guaiacol on protein second structure were studied using CD and FTIR techniques.Under the conditions studied, the values of the negative charge densitys ( S) of guaiacoK the dissociation constants (Ka) and Quantum yield were calculated.