Study On The Interaction Mechanism Between Tofu Glucoside And Gastrodin And Protease

In this thesis,the respective interactions of Helicid and Gastrodin with Pepsin and Trypsin have been studied by fluorescence spectroscopy,UV-vis absorption and circular dichroism(CD)techniques under physiological pH condition,along with computer molecular simulation.The mechanism of the interactions between drugs and proteins,including quenching process,binding site,binding constant,binding force type and the change of protein structure was investigated.Concretely,this thesis mainly consists of the following points:(1)The structure,biological function and properties of Helicid,Gastrodin,pepsin and trypsin were reviewed respectively,the techniques and methods of interaction between small molecular drugs and proteins were introduced,and then the content and significance of this thesis were proposed.(2)The conformation changes of pepsin induced by Helicid and Gastrodin respectively were investigated using the synchronous fluorescence spectrum,UV-visible spectra and circular dichroism,the bonding behavior of Helicid and Gastrodin to pepsin respectively were then explored by fluorescence spectroscopy.The experimental results show that the two drugs can quench the endogenous fluorescence of pepsin via a combination process of both static and dynamic quenching,namely the formation of the drug-protein complex results in the fluorescence quenching.Pepsin has only one Helicid or Gastrodin binding site.The thermodynamic analysis suggests that the interaction of Helicid with Pepsin is driven by enthalpy,and hydrogen bond and Van der waals' force are the main forces between Helicid and Pepsin,while hydrophobic interaction is the main force maintaining the complex of Pepsin with Gastrodin,and the interaction of Pepsin with Gastrodin is an entropy-driven process.Both the reactions are spontaneous thermodynamics reactions.(3)The conformation changes of trypsin induced by Helicid and Gastrodin,and the bonding behaviors of Helicid and Gastrodin to trypsin were examined respectively using the same methods as above.The experimental results show thatthe two drugs can quench the endogenous fluorescence of trypsin,and the endogenous fluorescence quenching type is also a combination of static and dynamic quenching,duo to the formation of the drug-protein complex.Trypsin has only one Helicid or Gastrodin binding site,whereas the binding of Gastrodin to trypsin is much weaker than that of Helicid to trypsin.The thermodynamic analysis suggests that the hydrogen bond and van der Waals force are the main forces maintaining both the complexes of trypsin with Helicid and Gastrodin respectively,and both the spontaneous interactions are driven by enthalpy.(4)The above experimental results were comparatively analyzed and the respective interactions of the two drugs with the two proteases were simulated through molecular docking using softwares SYBYL and MOE.Taken together of the results from experiments and simulations,the possible resources for the different binding behaviors of the same protease with different drugs are proposed,which should be valuable to the design of relative drugs.