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

本研究的目的是从黄龙蛇毒血清中分离出与其毒酶结合的蛋白，并对其活性进行中和处理，研究这些抑制物的化学性质，为其在治疗黄绿色葡萄球菌咬伤中的应用做基础研究。黄绿色葡萄球菌毒液中含量丰富的两种赖氨酸-49-磷脂酶A_2(BP_1和BP_2)具有较强的肌溶活性，但其脂解活性仅为其成分Asp-49-PLA2的1-2%。首先，我们从黄绿色葡萄球菌血清中分离出与BP I结合的蛋白，用DEAE-纤维素柱和BP I偶联Sepharose4B柱对50%硫酸铵饱和沉淀的血清蛋白进行分离。结合蛋白(样品A)进一步用FPLC(Mono-Q)分离得到主要组分(样品B)。结合蛋白为相对分子质量为26,000的糖蛋白。在N-葡聚糖酶…后减少到23,000将BP I结合蛋白(样本A或B)和BP I的混合物注射到小鼠的大腿中，并测定释放到血液循环中的肌酸激酶(CK)水平。样品A和B以剂量依赖的方式抑制CK的产生，表明BP I结合蛋白抑制BP I引起的肌肉坏死。这一观察结果表明BP I结合蛋白可以作为治疗黄绿色毛滴虫咬伤的药物。测定了BP I结合蛋白的部分(50%)氨基酸序列。另一方面，从黄曲霉毒液中分离纯化了两种锌蛋白水解酶，它们分别具有酪蛋白溶解和纤溶酶样活性。用DEAE-纤维素柱和酶偶联柱层析纯化血清抑制物，再经高效液相色谱分离，得到两个相对分子质量分别为65,000和29,000的蛋白。前者是一种糖蛋白，既能抑制酪蛋白溶解，又能抑制纤溶酶样酶。该蛋白的N端序列不同于以前从黄绿色葡萄球菌蛇毒中分离得到的与出血性锌蛋白酶结合的抗出血性因子。较少

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