Nose T, Shimohigashi Y, Hattori S, Kihara H, Ohno M
Department of Chemistry, Faculty of Science, Kyushu University, Japan.
Toxicon. 1994 Dec;32(12):1509-20. doi: 10.1016/0041-0101(94)90309-3.
A coagulant enzyme, okinaxobin I, which was purified from Trimeresurus okinavenis (himehabu snake) venom, released specifically fibrinopeptide B from fibrinogen to form fibrin clots. In the present study, its isozyme denoted as okinaxobin II has been purified to homogeneity from the same venom by chromatographies on Sephadex G-100, CM-Toyopearl 650M, and FPLC Mono-Q columns. Differently from okinaxobin I, okinaxobin II specifically cleaved fibrinopeptides A and B from fibrinogen similarly as found for alpha-thrombin. The enzyme acted on fibrinogen with specific activity of 42 NIH units/mg at optimum pH of 8.0. Okinaxobin II was a monomeric glycoprotein with a mol. wt of 37,500 on SDS-PAGE, which was reduced to 29,500 after treatment with N-glycanase. Okinaxobin II was much more basic (pI = 8.1) than okinaxobin I (pI = 5.4). The N-terminal sequence was highly similar to those of okinaxobin I and some other snake venom coagulant enzymes such as flavoxobin (Trimeresurus flavoviridis), batroxobin (Bothrops atrox and Bothrops moojeni), and catroxobin (Crotalus atrox). Okinaxobin II hydrolyzed tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide. The esterase activity was strongly inhibited by diisopropylfluorophosphate and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like alpha-thrombin. In terms of amino acid composition, okinaxobin II was similar to okinaxobin I and dissimilar to alpha-thrombin.
从冲绳烙铁头蛇（himehabu snake）毒液中纯化得到的一种凝血酶——okinaxobin I，能特异性地从纤维蛋白原中释放出纤维蛋白肽B，从而形成纤维蛋白凝块。在本研究中，通过Sephadex G - 100、CM - Toyopearl 650M和FPLC Mono - Q柱层析，从同一种毒液中纯化得到了其同工酶，命名为okinaxobin II，并使其达到了均一性。与okinaxobin I不同，okinaxobin II能像α - 凝血酶一样，特异性地从纤维蛋白原中裂解出纤维蛋白肽A和B。该酶作用于纤维蛋白原时，在最适pH 8.0条件下的比活性为42 NIH单位/毫克。在SDS - PAGE上，okinaxobin II是一种分子量为37,500的单体糖蛋白，用N - 聚糖酶处理后分子量降至29,500。okinaxobin II比okinaxobin I碱性强得多（pI = 8.1，而okinaxobin I的pI = 5.4）。其N端序列与okinaxobin I以及其他一些蛇毒凝血酶如黄绿烙铁头蛇毒素（Trimeresurus flavoviridis）、矛头蝮蛇毒素（Bothrops atrox和Bothrops moojeni）和响尾蛇毒素（Crotalus atrox）的N端序列高度相似。okinaxobin II能水解甲苯磺酰 - L - 精氨酸甲酯和苯甲酰 - L - 精氨酸对硝基苯胺。酯酶活性受到二异丙基氟磷酸的强烈抑制，受到甲苯磺酰 - L - 赖氨酸氯甲基酮的抑制程度较小，这表明该酶是一种像α - 凝血酶一样的丝氨酸蛋白酶。就氨基酸组成而言，okinaxobin II与okinaxobin I相似，与α - 凝血酶不同。
