华广虻(Tabanusamaenus Walker)溶纤活性蛋白的纯化及生物活性分析

Purification and Bioactivity Assay of the Fibrinolytic Protein of Tabanus amaenus Walker

经过 ７５％ 饱和度硫酸铵沉淀、 Ｓｅｐｈａｄｅｘ Ｇ７５ 凝胶过滤层析、 Ｌｙｓ Ｓｅｐｈａｒｏｓｅ ４ Ｂ 亲和层析和电泳制备洗脱，从华广虻（ Ｔａｂａｎｕｓ ａｍ ａｅｎｕｓ Ｗ ａｌｋｅｒ）腹部组织匀浆液中分离纯化出分子量约为 ６７ｋ Ｄ 的溶纤活性蛋白 Ｔ Ａ Ｆ Ｐ经纤维蛋白平板测定表明， Ｔ Ａ Ｆ Ｐ 只具有纤溶酶作用，不具有激活纤溶酶原的作用；但 Ｔ Ａ Ｆ Ｐ 能分解纤溶酶原激活剂的生色底物—— Ｃｈｒｏｍ ｏｚｙｍ Ｕ Ｋ 及 Ｓ２２８８还能水解胰蛋白酶专一底物 Ｂｚ Ｐｈｅ Ｖａｌ Ａｒｇ Ｎ Ａ 及 Ｃ Ｂ Ｚ Ｇｌｙ Ｐｒｏ Ａｒｇ Ｎ Ａ，表明 Ｔ Ａ Ｆ Ｐ具有类胰蛋白酶活性，专一水解精氨酸形成的酰胺键（或肽键） Ｔ Ａ Ｆ Ｐ无胰凝乳蛋白酶活性

After ammonium sulphate precipitation,Sephadex G 75 gel filtration,Lys Sepharose 4B affinity chromatography and elution from electrophoresis,the fibrinolytic protein(TAFP)was separated from the homogenate of abdomen tissue of T.amaenus Walker A single band appeared corresponding to molecular weight of approximately 67 kD on SDS PAGE On fibrin plate and plasminogen free fibrin plate(heated at 85℃ for 30 minutes to eliminate plasminogen),TAFP showed the same fibrinolytic activity The result might indicate that TAFP was a fibrinolytic enzyme degrading fibrin,not a plasminogen activator degrading fibrin via activating plasminogen TAFP showed stronger activity against Chromozym UK and S 2288 (chromogenic substrates for plasminogen activator)as well as Bz Phe Val Arg NA and CBZ Gly Pro Arg NA(specific substrates of trypsin) Thus TAFP possessed trypsin like activity specifically degrading argininyl amide bond or peptide bond,but had no chymotrypsin activity