摘要
本文报道自尖吻蝮蛇中分离、纯化三个凝血酶样酶(Thrombin-like Enzymes,TLEs),TLE-A,TLE-Ⅵ及TLE-Ⅶ。双向免疫扩散分别出现单一沉淀线,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分别呈单一区带,并依此测定分子量分别为66、31及14KD。试管内TLES能使纤维蛋白原或血浆形成纤维强白微凝块。凝血酶样酶制剂(TLES)150μg/ml效价与牛凝血酶1.0U/ml接近。肝素不能对抗其凝血效应,以对甲苯磺酰精氨酸甲基酯(TAME)为底物,其精氨酸酯酰活力可达2000μmol/min·mg左右。动物在体实验中,犬(n=6)静脉注射100μg/kg后4h药效达高蜂,血浆纤维蛋白原含量明显降低(P<0.02),凝血酶时间明显延长,血浆副凝血试验强阳性,血小板聚集功能明显抑制,家兔(n=10)每日静脉注射300μg/kg连用7天,对血液凝固系统效应与犬相似。药后第8天处死家兔病理解剖无明显变化。小鼠一次静脉注射的LD_(50)及其95％可信限为14.5±2.2(12.3～16.6)mg/kg。
Three thrombin-like enzymes were separated and purified from Agkistrodon acutus veno by column chromatography on DEAE-Sephadex A-50, gcl filtration on Scphadex G-75, and rcchromatography on CM-Scphadex C-50 consccutivcly. They were homogenous on soditm dodccylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at pII 8.3. Each of thcm showed a precipitateted line by outchterlony double diffusion method. Their mole cufar weights were 66, 31 and 14 kD respectively as determined in SDS-PAGE. In vitro, 0.8% fibrinogen(in Tris-HC1 buffer) or blood plasma of rabbit was coagulated as microclot by TLEs. The arginine esterase activity of TLEs was about 2000 μmol TAME/mg. min. TLEs showed anti-plarma clot retraction induced by thrombin. In vivo, TLEs caused blood blasma fibrinogen level obvious decrease; fibrin degraded products increase, thrombin time prolong and platelet aggregation inhibition when iv into dogs. The same effects of TLEs were observed in rabbits. The acute toxicjty were determined on mice by intravenous injection, the LD_50 and 95％ confidential limit appeared to be 14.5±2.2(12.3～16.6)mg/kg.
关键词
尖吻蝮蛇毒
凝血酶样酶
生化药理
Agkistrodon acutus
thrombin-like enzymes
venom
fibrinogen
