Wu H L, Shi G Y, Bender M L
Biochemistry Department, Medical College, National Cheng-Kung University, Taiwan, Republic of China.
Proc Natl Acad Sci U S A. 1987 Dec;84(23):8292-5. doi: 10.1073/pnas.84.23.8292.
A catalytically active, human microplasmin was produced by incubation of [Lys]plasmin in buffer at pH 11.0 for up to 12 hr. The microplasmin was purified by affinity chromatography that used lysine-Sepharose and soybean trypsin inhibitor-Sepharose columns. It is homogeneous and pure by electrophoretic analysis in NaDodSO4/polyacrylamide gels and by gel filtration on a Superose 12 column. The molecular weight of the microplasmin determined by NaDodSO4 gel electrophoresis is 29,000 and 26,500 under reducing condition, whereas the molecular weight of native plasmin is 76,500. Microplasmin consists mainly of the ligh (B) chain of native human plasmin and possesses one active site per protein molecule when titrated with p-nitrophenyl p'-guanidinobenzoate. Microplasmin hydrolyzes the peptide substrate NH2-D-Val-Leu-Lys-p-nitroanilide (S-2251) with a Km of 0.361 +/- 0.017 mM and a kcat of 40.3 +/- 3.3 s-1 at pH 7.4 and 37 degrees C, whereas native plasmin has a Km of 0.355 +/- 0.002 mM and a kcat of 27.9 +/- 0.3 s-1 under the same conditions.
通过在pH 11.0的缓冲液中孵育[赖氨酸]纤溶酶长达12小时，可产生具有催化活性的人微纤溶酶。微纤溶酶通过使用赖氨酸-琼脂糖和大豆胰蛋白酶抑制剂-琼脂糖柱的亲和色谱法进行纯化。通过在NaDodSO4/聚丙烯酰胺凝胶中的电泳分析以及在Superose 12柱上的凝胶过滤，它是均一且纯净的。在还原条件下，通过NaDodSO4凝胶电泳测定的微纤溶酶分子量为29,000和26,500，而天然纤溶酶的分子量为76,500。微纤溶酶主要由天然人纤溶酶的轻（B）链组成，在用对硝基苯基对'-胍基苯甲酸酯滴定法测定时，每个蛋白质分子具有一个活性位点。在pH 7.4和37℃条件下，微纤溶酶水解肽底物NH2-D-缬氨酸-亮氨酸-赖氨酸-对硝基苯胺（S-2251）的Km为0.361±0.017 mM，kcat为40.3±3.3 s-1，而在相同条件下天然纤溶酶的Km为0.355±0.002 mM，kcat为27.9±0.3 s-1。
