Determination Of Protein Folding Free Energy Of Protein Renaturation Of Liquid Chromatography And Liquid-solid Interface

The thesis is composed of three parts as follows: 1. Studies on the unfolding kinetics of urea-denatured ?-amylase and ?-chymotrypsin by hydrophobic interaction chromatographyHigh performance hydrophobic interaction chromatography (HPHIC) was applied to study the kinetic behavior of ?-amylase (?-Amy) and ?-chymotrypsin (?-Chy) originally denatured by 8.0mol/L urea. It was found both of ?-Amy and ?-Chy to have different unfolding kinetics. The unfolding of ?-Amy is a gradually changed process, while the process for ?-Chy is fast. The intermediates of ?-Amy are more than that of ?-Chy. Different unfolding kinetics may be due to different contents of ?-helix and the stability of the proteins.2. Studies on the Renaturation of ?-Chy by Liquid Chromatography HPHIC, ion exchange chromatography (IEC) and size exclusion chromatography (SEC) were applied to study the renaturation of ?-Chy originally unfolded by different concentrations of urea. The low efficiency of renaturation was mainly attributed to the loss of bioactivity of ?-Chy in presence of salt of the mobile phase employed and the irreversible adsorption of ?-Chy to the stationary phase. The higher the ionic strength of the mobile phase, the greater the bioactivity loss was. After the correcting for the mobile phase's negative contribution to the bioactivity and its mass loss, we found that the stationary phases of HPHIC and SEC have positive contributions to the renaturation of ?-Chy originally unfolded by urea, while that for the contribution by using IEC was not evident. Three kinds of HPHIC column with different hydrophobic strengths were used to make a comparative study on the renaturation of ?-Chy, the refolding ability of the stationary phase was found to relate to its hydrophobic strength. SEC was also used to make the comparative study with dialysis method for the renaturation of ?-Chy denatured by both of urea and guanidine hydrochloride. SEC method was found to be better than that of usual dialysis method. The optimum concentration of salt was in the range from 0.05 to 0.5 mol/L in the mobile phase employed, and the type of the salt was found to have a little effect on the renaturation of unfolded ?-Chy.3. Determination of Free Energy of Protein Refolding on Liquid-Solid InterfaceAccording to the principle and method of determination of free energy of protein refolding (??GF) on liquid-solid interface, the free energy on liquid-solid interface of four kinds of proteins ?-Chy, ?-Amy, lysozyme (Lys) and ribonuclease-A (RNase-A) in the system as urea to be denaturing agent and two kinds of proteins ?-Chy and ?-Amy in the presence of guanidine hydrochloride were determined. The fact that the maximum relative average deviation of two parallel tests was only less than 4.30% indicates that the method used had satisfactory reproducibility and reliability. The free energy of protein refolding on liquid-solid interface was found to be much higher than that reported from solution and increased with the increase of the concentration of denaturing agent employed. With the plot of ??GF versus the concentration of denaturing agent of either urea or guanidine hydrochloride, it was further confirmed that energy barriers and energy wells on the refolding pathway of ?-chymotrypsin and ?-amylase really exist.