Protein Chemistry and Basic Research for Application of Inhibitors from Trimeresurus flavoviridis Serum against Its Venom Enzymes

本研究的目的是从竹叶青蛇血清中分离与其毒酶结合的蛋白质并测定其活性，研究其化学性质，为竹叶青蛇毒酶抑制剂在治疗竹叶青蛇咬伤中的应用做基础研究。黄绿胸蛙毒素中含有两种丰富的赖氨酸-49-磷脂酶A_2（BP Ⅰ和BP Ⅱ），它们的解脂活性仅为毒素组分Asp-49-PLA_2的1 - 2%。首先，我们打算从黄绿毛滴虫血清中分离与BP I结合的蛋白质。通过FPLC（Mono-Q）进一步分级分离结合的蛋白质（样品A），得到主要级分（样品B）。结合的蛋白质是MW 26，000的糖蛋白。在N-聚糖酶作用下， ...更多信息 将BPI结合蛋白（样品A或B）和BPI的混合物注射到小鼠大腿中，并定量释放到血液循环中的肌酸激酶（CK）水平。样品A和B以剂量依赖性方式抑制CK的产生，表明BP I结合蛋白抑制BP I引起的肌肉坏死。这种观察表明BP I结合蛋白可用作治疗黄绿毛滴虫叮咬的药物。测定了BPI结合蛋白的部分（50%）氨基酸序列。另一方面，两个锌蛋白酶具有酪蛋白溶解和纤溶酶样活性，分别从T. flavovoraciens毒液中纯化。经DEAE-cellulose柱层析和蛋白酶结合柱层析及HPLC纯化，得到分子量分别为65，000和29，000的蛋白质。前者是一种糖蛋白，可抑制酪蛋白溶解和纤溶酶样蛋白酶。该蛋白的N-末端序列与抗出血因子的N-末端序列不同，抗出血因子与先前从黄绿锥虫毒液中分离的出血性锌蛋白酶结合。少

The object of this study is to isolate proteins from Trimeresurus flavoviridis serum which are bound to its venom enzymes and meutralize their activities, and to investigate chemical properties of these inhibitors and to make basic study for application of these inhibitors to therapy of T.flavoviridis bite. Two lysine-49-phospholipases A_2(PLA_2)(BP I and BP II) which are involved abundantly in T.flavoviridis venom were found to have strong myolytic activity although they were lipolytically 1-2% as active as Asp-49-PLA_2, a component of the venom. At first, we intended to isolate protein from T.flavoviridis serum which is bound to BP I.Serum proteins precipitated at 50% saturation of ammonium sulfate were fractionated on DEAE-cellulose column and BP I-conjugated Sepharose 4B column. The bound proteins (sample A) were further fractionated by FPLC (Mono-Q) to give a major fraction (sample B). The bound protein was a glycoprotein of MW 26,000. which was reduced to 23,000 after N-glycanase … More digestion.Mixtures of BP I-binding protein (sample A or B) and BP I were injected into thigh of mice and creatine kinase (CK) levels released to blood circulaton were quantitated. Samples A and B depressed CK production in dose-dependent manner, indicating that BP I-binding protein represses muscle necrosis caused by BP I.Such observation suggests that BP I-binding protein could be utilized as a drug for therapy of T.flavoviridis bite. Partial(50%) amino acid sequence of BP I-binding protein has been determined. On the other hand, two zinc proteases which have caseinolytic and plasmin-like activity, respectively, were purified from T.flavovoridis venom. Purification of serum inhibitors against these proteases were made by DEAE-cellulose column and protease-conjugated column chromatographies followed by HPLC.Two proteins of MW 65,000 and 29,000, respectively, were obtained. The former, a glycoprotein, inhibited both caseinolytic and plasmin-like proteases. The N-terminal sequence of this protein was different from that of anti-hemorrhagic factor which is bound to hemorrhagic zinc protease which had previously been isolated from T.flavoviridis venom. Less