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取猪心组织用丙酮去脂,pH4.2、0.45mol/L醋酸钾缓冲液抽提组织型纤溶酶原激活剂(t-PA)。抽提液经硫酸铵分段盐析,然后用纤维蛋白-Sepharose柱吸附t-PA,2mol/L硫氰酸钾洗脱的t-PA溶液经Sephadex G-150凝胶过滤,得到纯的t-PA。其比活性为39038.5 IU/mg,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定其分子量为67000。提纯过程中应用了蛋白酶抑制剂(aprotinin)以及6-氨基己酸,以抑制单链t-PA转变为双链t-PA。t-PA活性测定以尿激酶(UK)为参考标准。
Pig heart tissue was removed with acetone, pH4.2, 0.45mol / L potassium acetate buffer extracted tissue-type plasminogen activator (t-PA). The extract was fractionated by ammonium sulfate and then t-PA was adsorbed on fibrin-Sepharose column. The t-PA solution eluted with 2 mol / L potassium thiocyanate was gel-filtered on Sephadex G-150 to obtain pure t -PA. The specific activity was 39038.5 IU / mg, and its molecular weight was 67000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In the purification process, aprotinin and 6-aminocaproic acid are used to inhibit the conversion of single-stranded t-PA to double-stranded t-PA. t-PA activity was measured using urokinase (UK) as a reference standard.