Interaction Between Gn10 And α-ct And Regulation In Conformation And Activity Of α-CT

The interaction of surfactant with enzyme is widely used in all aspects of life, such as the designation of drugs, cosmetics industry, detergent and so on. There are a lot of reports about the interaction between surfactants and enzyme. Most of surfactants employed in previous literatures was the traditional single-chain surfactants, such as cationic alkyl trimethyl ammonium bromide, anionic sodium di-(2-ethylhexyl) sulfosuccinate(AOT) and sodium dodecyl sulfate(SDS), non-ionic polyoxyethylene. There are a few reports on the mixed system of gemini surfactants and enzymes. In recent years, gemini surfactants have been applied in some biotechnical applications, promoting strongly the researcher’s interesting in enzyme-surfactant systems and the effect of surfactants on protein structure and stability.In this work, α-chymotrypsin(α-CT) was selected as a model enzyme whose structure and catalysis mechanism are well definite in much previous works as a representative of hydrolase protein. The hydrophobicity of the headgroups of the positively charged gemini surfactant can be controled by adjusting the alkyl chain length of the spacer. We selected a gemini surfactant C12C10C12Br2(GN10) as an additive and activator, referencing the results that the more hydrophobic tributyl hexadecyl phosphonium bromide(CTBAB) activates α-CT. This thesis mainly includes the following contents:1. The interaction between cationic gemini surfactant(GN10) and α-chymotrypsin(α-CT) was studied by UV-vis, fluorescence spectroscopy and isothermal titration calorimetry(ITC). The absorbance and fluorescence spectra gave about information in the tertiary structure of α-CT and its change in the surfactant solution. By ITC measurement we can understand the interaction in the terms of thermodynamics.2. α-CT catalyzing hydrolysis of 2-Naphthyl-acetate(2-NA) in phosphate buffer solution was monitored by UV-vis measurement in the presence or absence of GN10. The effect of GN10 on catalysis activity of α-CT changes with the incubation duration. In the concentration range of GN10 below cmc α-CT was activated but its residual activity lowered with time after one day faster than that in the absence of GN10.3. The interaction between GN10 and α-CT was also characterized by denatural temperature by using differential scanning calorimeter(DSC). In the presence of GN10 the denatural temperature shifts onto lower temperature and a shoulder peak in high temperature side occurs, indicating probably a different denatural mechanism. In addition, we measured the activity in the presence of GN10 at different temperature.4. For comparison, the interaction between anionic surfactant sodium n-dodecyl sulfate(SDS) and α-CT also was researched by UV-vis and ITC.