Iwasaki A, Shieh T C, Shimohigashi Y, Waki M, Kihara H, Ohno M
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
J Biochem. 1990 Nov;108(5):822-8. doi: 10.1093/oxfordjournals.jbchem.a123287.
A coagulant enzyme, named okinaxobin I, has been purified to homogeneity from the venom of Trimeresurus okinavensis (Himehabu) by chromatographies on Sephadex G-100 and CM-Toyopearl 650M columns. The enzyme was a monomer with a molecular weight of 37,000 and its isoelectric point was 5.4. The enzyme acted on fibrinogen to form fibrin clots with a specific activity of 77 NIH units/mg. Fibrinopeptide B was released at a rate much faster than fibrinopeptide A. The enzyme exhibited 2 to 3 times higher activity toward tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide than bovine thrombin. The esterase activity was strongly inhibited by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like thrombin. The N-terminal sequence was highly homologous to those of coagulant enzymes from T. flavoviridis and Bothrops atrox, moojeni venoms which preferentially release fibrinopeptide A. In order to remove most, if not all, of the bonded carbohydrates, the enzyme was treated with anhydrous hydrogen fluoride (HF), thereby reducing the molecular weight to 30,000. The protein contained approximately 260 amino acid residues when computation was based on this value. The HF-treated enzyme retained about 50% of the clotting and esterolytic (TAME) activities and preferentially released fibrinopeptide B from fibrinogen. The carbohydrate moiety is not crucial for enzyme activity but might be necessary for eliciting full activity.
一种名为okinaxobin I的凝血酶已通过Sephadex G - 100和CM - Toyopearl 650M柱色谱从冲绳竹叶青（Himehabu）的毒液中纯化至同质。该酶是一种分子量为37,000的单体，其等电点为5.4。该酶作用于纤维蛋白原形成纤维蛋白凝块，比活性为77 NIH单位/毫克。纤维蛋白肽B的释放速度比纤维蛋白肽A快得多。该酶对甲苯磺酰-L-精氨酸甲酯和苯甲酰-L-精氨酸对硝基苯胺的活性比对牛凝血酶高2至3倍。酯酶活性受到二异丙基氟磷酸酯和苯甲基磺酰氟的强烈抑制，而甲苯磺酰-L-赖氨酸氯甲基酮的抑制作用较小，表明该酶是一种类似凝血酶的丝氨酸蛋白酶。其N端序列与黄绿竹叶青和矛头蝮（莫杰尼）毒液中的凝血酶高度同源，后者优先释放纤维蛋白肽A。为了去除大部分（如果不是全部）结合的碳水化合物，该酶用无水氟化氢（HF）处理，从而使分子量降至30,000。根据该值计算，该蛋白质约含260个氨基酸残基。经HF处理的酶保留了约50%的凝血和酯解（TAME）活性，并优先从纤维蛋白原中释放纤维蛋白肽B。碳水化合物部分对酶活性不是至关重要，但可能是发挥全部活性所必需的。
