Waley S G
Biochem J. 1973 Sep;135(1):165-72. doi: 10.1042/bj1350165.
The refolding and reactivation of the glycolytic enzyme triose phosphate isomerase (EC 5.3.1.1) has been studied. The enzyme, which is a dimer, is disaggregated and unfolded in solutions of guanidinium chloride. Unfolding, followed by changes in E(233), took place quite rapidly in 3m-guanidinium chloride (i.e. with a half-life of about 1 min). Refolding also took place rapidly when the solution was diluted about tenfold; two first-order processes could be resolved. Regain of enzymic activity was followed by diluting the solution of the denatured enzyme in guanidinium chloride into assay mixture. The half-life (i.e. the time when the activity was half the final activity) depended markedly on the concentration of protein at low concentrations (about 100ng/ml), but at higher concentrations the half-life became independent of concentration. Thus at low concentrations dimerization was a rate-determining step and this is taken to indicate that the monomers showed little or no activity under these conditions. The rate of regain of enzymic activity was the same as the rate of the slower process of refolding, which was detected spectroscopically. The native enzyme was resistant to proteolysis; high concentrations of subtilisin prevented regain of activity, but at lower concentrations refolding competed with proteolysis.
已对糖酵解酶磷酸丙糖异构酶（EC 5.3.1.1）的重折叠和再激活进行了研究。该酶为二聚体，在氯化胍溶液中会解聚并展开。在3M氯化胍中，展开过程以及随后E(233)的变化相当迅速（即半衰期约为1分钟）。当溶液稀释约10倍时，重折叠也迅速发生；可以分辨出两个一级过程。通过将变性酶在氯化胍中的溶液稀释到测定混合物中来跟踪酶活性的恢复。半衰期（即活性为最终活性一半时的时间）在低浓度（约100纳克/毫升）时明显取决于蛋白质浓度，但在较高浓度时半衰期与浓度无关。因此，在低浓度下二聚化是限速步骤，这表明在这些条件下单体几乎没有或没有活性。酶活性恢复的速率与通过光谱检测到的较慢重折叠过程的速率相同。天然酶对蛋白水解具有抗性；高浓度的枯草杆菌蛋白酶会阻止活性恢复，但在较低浓度下重折叠与蛋白水解相互竞争。
