Govers-Riemslag J W, Knapen M J, Tans G, Zwaal R F, Rosing J
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Biochim Biophys Acta. 1987 Dec 18;916(3):388-401. doi: 10.1016/0167-4838(87)90185-3.
A prothrombin activator from the venom of Bothrops neuwiedi was purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on a Zn2+-chelate column. The overall purification was about 200-fold, which indicates that the prothrombin activator comprises about 0.5% of the crude venom. The venom activator is a single-chain protein with an apparent molecular weight of 60 kDa. It readily activated bovine prothrombin with a Km of 38 microM and a Vmax of 120 mumol prothrombin activated per min per mg of venom activator. Venom-catalyzed prothrombin activation was not accelerated by the so-called accessory components of the prothrombinase complex, phospholipids plus Ca2+ and Factor Va. Gel-electrophoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg-323-Ile-324 bond of bovine prothrombin, since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze commercially available p-nitroanilide substrates and its prothrombin-converting activity was not inhibited by benzamidine, phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethyl ketone and soy-bean trypsin inhibitor. However, chelating agents such as EDTA, EGTA and o-phenanthroline rapidly destroyed the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could be partially restored by the addition of an excess CaCl2. These results indicate that the venom activator remarkably differs from Factor Xa and that the enzyme is not a serine proteinase, but likely belongs to the metalloproteinases. The structural and functional properties of the venom prothrombin activator from B. neuwiedi are similar to those reported for the venom activator from Echis carinatus.
从巴西矛头蝮蛇毒中纯化出一种凝血酶原激活剂，采用葡聚糖凝胶G - 100凝胶过滤、DEAE - 葡聚糖凝胶离子交换色谱和锌螯合柱亲和色谱法。总体纯化倍数约为200倍，这表明凝血酶原激活剂约占粗毒的0.5%。蛇毒激活剂是一种单链蛋白，表观分子量为60 kDa。它能轻易激活牛凝血酶原，Km为38 μM，Vmax为每分钟每毫克蛇毒激活剂激活120 μmol凝血酶原。凝血酶原酶复合物的所谓辅助成分磷脂加Ca²⁺和因子Va并不能加速蛇毒催化的凝血酶原激活。凝血酶原激活的凝胶电泳分析表明，蛇毒激活剂仅切割牛凝血酶原的Arg - 323 - Ile - 324键，因为凝血酶原激活的唯一产物是中凝血酶。该激活剂不水解市售的对硝基苯胺底物，其凝血酶原转化活性不受苯甲脒、苯甲基磺酰氟、丹磺酰 - Glu - Gly - Arg - 氯甲基酮和大豆胰蛋白酶抑制剂的抑制。然而，螯合剂如EDTA、EGTA和邻菲罗啉能迅速破坏蛇毒激活剂的酶活性。加入过量CaCl₂可部分恢复螯合剂处理后的蛇毒激活剂的活性。这些结果表明，蛇毒激活剂与因子Xa显著不同，该酶不是丝氨酸蛋白酶，而可能属于金属蛋白酶。巴西矛头蝮蛇毒凝血酶原激活剂的结构和功能特性与锯鳞蝰蛇毒激活剂所报道的相似。
