Rickli E E, Otavsky W I
Eur J Biochem. 1975 Nov 15;59(2):441-7. doi: 10.1111/j.1432-1033.1975.tb02472.x.
A method is described by which the heavy chain of human plasmin, obtained by partial reduction of urokinase-activated plasminogen with 2-mercaptoethanol, is adsorbed on lysine coupled to polyacrylamide. The heavy chain is recovered from the adsorbent by elution with 6-aminohexanoic acid (yield 60-65%). Sulfhydryl titrations of the heavy chain showed that the partial reduction involved primarily the cleavage of the sole interchain disulfide bridge of plasmin. Dodecylsulfate-polyacrylamide electrophoresis gave essentially a single band corresponding to a component of about 60000 molecular weight. The NH2-terminal amino acid was predominantly threonine. 6-Aminohexanoic acid at different concentrations caused significant variations of the sedimentation and diffusion constants of the heavy chain indicating inhibitor-induced conformational alterations of the protein. The present results suggest that in plasmin only the heavy chain is capable of interacting with 6-aminohexanoic acid, and it appears that it is primarily this chain which plays an important role in the inhibition of the enzyme by 6-aminohexanoic acid.
本文描述了一种方法，通过该方法，用2-巯基乙醇对尿激酶激活的纤溶酶原进行部分还原得到的人纤溶酶重链，可吸附于偶联到聚丙烯酰胺上的赖氨酸上。通过用6-氨基己酸洗脱从吸附剂中回收重链（产率60 - 65%）。对重链的巯基滴定表明，部分还原主要涉及纤溶酶唯一的链间二硫键的断裂。十二烷基硫酸钠-聚丙烯酰胺电泳基本上得到一条对应于约60000分子量组分的单一带。氨基末端氨基酸主要是苏氨酸。不同浓度的6-氨基己酸导致重链的沉降和扩散常数发生显著变化，表明抑制剂诱导了蛋白质的构象改变。目前的结果表明，在纤溶酶中只有重链能够与6-氨基己酸相互作用，并且似乎主要是这条链在6-氨基己酸对该酶的抑制中起重要作用。
