α-胰凝乳蛋白酶在胍溶液中的变性、失活、复性、复活

DENATURATION, INACTIVATION, RENATURATION AND REACTIVATION OF α-CHYMOYRTPSIN IN GUANIDINE SOLUTIONS

前已报导,在脲或胍的作用下,肌酸激酶失活速度远快于酶分子整体构象变化的速度.本文报导利用在变性剂存在下研究底物反应的方法对分子较小,由单亚基组成,并有五个二硫键使分子结构更加稳定的胰凝乳蛋白酶,在盐酸胍作用下的变性,失活以及相应的复性,复活进行动力学的比较.结果表明失活仍快于构象变化速度,复活慢于构象的恢复速度.实验结果还表明已经充分复活的酶和未经变性的酶在溶液中的构象存在着某些差别.

In earlier studies it was shown that in guanidium chloride or urea solutions, rapid inactivation of creatine kinase occurs before any significant comformational changes, Such studies relating the change in activity with comformational change of the enzyme are of particular interest in the case of small monomeric enzymes stabilized by multiple disulfide bonds.In this work we present a comparison of the rates of comformational changes with those of inactivation of chymotrypsin in guanidinium chloride solution. In the first part of this study the inactivation of chymotrypsin in gua-nidine solution was followed by substrate reaction in the presence of denaturant in a stopped flow apparatus. Results show that the rate of inactivation is significantly faster than that of the unfolding of the enzyme molecule, and that the rate of reactivation of guanidine denatured chymotrypsin is slower than that of refolding of the enzyme molecule. Finally comparison of the spectra of native, fully active chymotrypsin and of the reactivated enzyme shows that there are differences in the comformations of these molecules.