Microcalorimetric Study Of Displacment Adsorption Enthalpies Of Denatured α-Amylase On The Hydrophobic Surface

α-Amylase was used as a research object and the appropriate denaturing agent guanidine hydrochloride was selected in this paper. The Micro DSCⅢcalorimeter was used to measure the displacement adsorption enthalpy ofα-Amylase in different concentrations of guanidine hydrochloride degeneration conditions on the hydrophobic surface according with the same condition that for refolding and separation at the same time withα-Amylase by HPLC. Adsorption isotherms ofα-Amylase were determined under different conditions using vibrator, the adsorption equilibrium time and adsorbed amounts ofα-Amylase were also discussed,and the energy changes during the adsorption-folding process were calculated in accordance with the stoichiometric displacement theory of adsorption (SDT-A) and its thermodynamics. Besides, combined with microcalorimetric study ofα-Amylase, the thermal effect of protein folding on liquid/solid interface and the thermal method of free energy were determined perfectly, thus, the thermal effect of protein folding on hydrophobic surface was gained, thereby, the folding free energy of protein was further obtained.On the basis of adsorption experiments, the Sapphire DSC was used to study the effect of various concentrations of GuHCl, (NH4)2SO4 and the surface coverage on thermal stability forα-Amylase during calefactive process in accordance with heat scanning of nature or denaturedα-Amylase adsorbed on the PEG-600 surface at 25℃. And, compared with the results of Fourier-transformation infrared spectroscopy for adsorbedα-Amylase under the same condition, the rule of its conformational changes was further discussed.Meanwhile, the retention behavior forα-Amylase denatured by different concentrations of GuHCl in hydrophobic interaction chromatography(HIC) was investigated in the temperature ranging from 20℃to 35℃. The results have been found that the retention behaviors forα-Amylase with or without being denatured by GuHCl were all well followed the non-linear Van't Hoff equation. The calculated retention thermodynamic parameters (ΔΗ~0,ΔS~0,ΔC_P~0 andΔG~0) ofα-Amylase showed that during the experimental temperature, the retention ofα-Amylase in HIC was driven by entropy,ΔΗ~0,ΔS~0 andΔC_P~0 correlate linearly with the reciprocal of absolute temperature. Simultaneity, calculated refolding free energiesΔΔG_F of partially denaturedα-Amylase on the surface of immobile phase in HIC are much higher than that in solution, and for theα-Amylase denatured by the same concentration of GuHCl, the highest folding free energy was at the temperature of 25℃. And, the effect of guanidine hydrochloride in the mobile phase on the retention behavior for natureα-Amylase in HIC at 25℃was further investigated, the Z value of the stoichiometric displacement theory of retention(SDT-R) was used to characterize the changes in the molecular conformations ofα-Amylase denatured by guanidine hydrochloride in HIC, thus, it is significant to understand the hydrophobic interaction between life and the thermal denaturation of protein from the molecular level.