胍变及脲变α-淀粉酶的研究 Ⅰ.用高效疏水色谱法研究变性机理和复性效率

用疏水性强弱不同的两种色谱柱对７．０ｍｏｌ／Ｌ盐酸胍及８．０ｍｏｌ／Ｌ脲变性的α－淀粉酶变体和在疏水色谱介质表面上折叠的中间体进行了分离和复性。通过研究和比较发现，两者的变性机理和形成折叠中间体的个数以及复性效率均不相同。在用疏水性较弱的疏水色谱柱对脲变α－淀粉酶的折叠中间体进行分离时，得到了疏水性接近连续的、数目很多的中间体。用疏水性较强的疏水色谱柱对胍变α－淀粉酶进行复性的效果较好。还研究了柱温变化对其折叠、分离效果和复性效率的影响。

Investigation of alpha-amylase denatured by urea and guanidine hydrochloride. I. Studies on denaturation mechanism and renaturation efficiency by high-performance hydrophobic interaction chromatography]

The varieties of alpha-amylase denatured with 8.0 mol/L urea and 7.0 mol/L guanidine hydrochloride (GuHCl) solutions and their refolded intermediates were separated and renatured through high performance hydrophobic interaction chromatography (HPHIC). With investigating and comparing the numbers of the refolded intermediates and the bioactivity recovery of renaturation by means of HPHIC, it was found that these are quite different. The results can be attributed to the different denaturation mechanism of alpha-amylase with the two denaturing agents. The numbers of the refolded intermediates of urea-unfolded alpha-amylase were found to be more than that denatured by GuHCl, because GuHCl may make the changes in the surface of alpha-amylase molecules, by contrast, urea may do the changes not only in the surface of the protein, but also in the hydrophobic packet interior of the molecules. The HPHIC packings with weak hydrophobicity was found to have good separation efficiency and to have almost continuous and many peaks denoting these intermediates, while that with strong hydrophobicity was found to have higher bioactivity recovery. The numbers of the intermediates of urea-unfolded alpha-amylase were much more than that of bovine pancreatic trypsin inhibitor (BPTI) separated by Weissman et al. The effect of temperature on the separations and the renaturations were investigated too. The result showed that the higher the column temperature, the more the refolded intermediates of unfolded alpha-amylase separated and the lower bioactivity recovery were obtained from HPHIC.