After ammonium sulphate precipitation, Sephadex G-75 gel filtration, Lys-Sepharose 4B affinity chromatography and elution from electrophoresis, the fibrinolytic protease (TAFP) was isolated and purified from the extract of T. amaenus Walker gut. It appeared a single band corresponding to molecular weight of approximately 67kD on SDS-PAGE and an probably pI of 7.2 on IEF. On fibrin plate and plasminogen-free fibrin plate (heated at 85℃ for 30 minutes to eliminate plasminogen), TAFP showed same fibrinolytic activity. The result might indicate that TAFP is a fibrinolytic enzyme degrading fibrin, as well as a plasminogen activator degrading fibrin via activating plasminogen. The result of chromogenic substrates indicated that TAFP possesses trypsin-like activity specifically degrading argininyl amide bond or peptide bond, but has no chymotrypsin activity. TAFP was almost inhibited powerfully by antipain, PMSF, soybean trypsin inhibitor and soybean Bowman-Birk inhibitor. However, leupeptin, antitrypsin and TLCK was more powerful effective inhibitors of TAFP. Optimal reaction pH of TAFP was 7.5, and it was stable in 5.5 - 7.0 of pH range.%经过75%饱和度硫酸铵沉淀、Sephadex G-75凝胶过滤层析、Lys-Sepharose 4B亲和层析和电泳制备洗脱,从华广虻(Tabanus amaenus Walker)腹部组织匀浆液中分离纯化出分子量约为67KD的溶纤活性蛋白TAFP.经纤维蛋白平板测定表明,TAFP不仅具有纤溶酶作用,还具有激活纤溶酶原的作用;通过三肽生色底物测定发现,TAFP能分解纤溶酶原激活剂的生色底物-Chromozym UK及S-2288.还能水解胰蛋白酶专-底物Bz-Phe-Val-Arg-NA及CBZ-Gly-Pro-Arg-NA,表明TAFP具有类胰蛋白酶活性,专-水解精氨酸形成的酰胺键(或肽键).TAFP无胰凝乳蛋白酶活性.
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