Chang M C, Huang T F
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei.
Toxicon. 1995 Aug;33(8):1087-98. doi: 10.1016/0041-0101(95)00035-k.
A thrombin-like enzyme, grambin, was purified to homogeneity by gel filtration, affinity and ion-exchange chromatography from the venom of Trimeresurus gramineus. Its mol. wt was estimated to be 45,400 by SDS-PAGE under reduced conditions. The mass of neutral sugars in grambin is estimated to be 20.7% of total mass. Grambin's NH2-terminal ten amino acid residues show a high homology to other venom thrombin-like enzymes. It clots human fibrinogen with a specific activity of 220-250 NIH thrombin-equivalent units/mg protein. It preferentially releases fibrinopeptide A accompanied by a slow release of trace amounts of fibrinopeptide B as monitored by HPLC following enzyme treatment of fibrinogen. EDTA, aprotinin, hirudin and heparin did not affect the fibrinogen-clotting activity of grambin in purified human fibrinogen solution. Diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and leupeptin inhibited the clotting activity of grambin whereas iodoacetamide did not affect its activity, indicating that grambin is a serine protease rather than a cysteine protease. In addition, it caused defibrinogenation and showed a marked antiplatelet effect when administered intravenously to mice. It also significantly prolonged the time lapse of platelet-rich thrombus formation in the irradiated mesenteric venules of fluorescein sodium-treated mice. Therefore, grambin may be used as a therapeutic agent not only in treatment of venous thrombosis but also in prevention of arterial thrombosis.
从竹叶青蛇毒中通过凝胶过滤、亲和层析和离子交换层析纯化得到一种类凝血酶——绿链蛇毒素，并达到了均一性。在还原条件下，通过SDS-PAGE估计其分子量为45,400。绿链蛇毒素中中性糖的质量估计占总质量的20.7%。绿链蛇毒素的氨基末端十个氨基酸残基与其他蛇毒类凝血酶具有高度同源性。它能使人类纤维蛋白原凝结，比活性为220 - 250 NIH凝血酶当量单位/毫克蛋白质。在用纤维蛋白原进行酶处理后，通过HPLC监测发现，它优先释放纤维蛋白肽A，同时缓慢释放微量的纤维蛋白肽B。在纯化的人纤维蛋白原溶液中，EDTA、抑肽酶、水蛭素和肝素均不影响绿链蛇毒素的纤维蛋白原凝结活性。二异丙基氟磷酸、苯甲基磺酰氟和亮抑酶肽抑制绿链蛇毒素的凝结活性，而碘乙酰胺不影响其活性，这表明绿链蛇毒素是一种丝氨酸蛋白酶而非半胱氨酸蛋白酶。此外，静脉注射给小鼠时，它会导致去纤维蛋白原作用，并表现出显著的抗血小板作用。它还能显著延长荧光素钠处理小鼠的辐照肠系膜小静脉中富含血小板血栓形成的时间间隔。因此，绿链蛇毒素不仅可作为治疗静脉血栓形成的药物，还可用于预防动脉血栓形成。
