Purification and functional characterization of bothrojaractivase, a prothrombin-activating metalloproteinase isolated from Bothrops jararaca snake venom
Purification and functional characterization of bothrojaractivase, a prothrombin-activating metalloproteinase isolated from Bothrops jararaca snake venom
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DOI：
10.1016/j.toxicon.2007.09.005
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发表时间：
2008-03-15
期刊：
影响因子：
2.8
通讯作者：
Guimaraes, Jorge A.
中科院分区：
文献类型：
--
作者：
Berger, Markus;Pinto, Antonio F. M.;Guimaraes, Jorge A.
咬伤部位出血和/或全身性出血是贾拉拉卡波斯罗普蛇中毒时经常观察到的症状。在这项研究中，我们纯化并鉴定了一种可能与这些临床表现有关的从刺槐中提取的凝血酶原激活剂。该酶经凝胶过滤和离子交换色谱分离得到，命名为bothrojaractivase。它有一个单肽链，通过质谱测量分子量为22,829 Da。Bothrojaractivase从凝血酶原产生活性凝血酶，独立于辅助因子。凝血酶原激活产物的SDS-PAGE分析表明，bothrojaractivase将凝血酶原转化为减数凝血酶，产生的片段与A组凝血酶原激活剂产生的片段相似。此外，bothrojaractivase还能降解纤维蛋白原和纤维蛋白。螯合剂完全抑制酶活性，而丝氨酸和半胱氨酸蛋白酶抑制剂没有作用。4个肽段的氨基酸序列表明，bothrojar激活酶与P-I类蛇毒金属蛋白酶具有较高的相似性。因此，我们的研究结果表明，bothrojaractivase是一种新的金属蛋白酶，它作用于凝血级联的不同蛋白质因子，特别是在凝血酶原激活生成凝血酶中发挥关键和最相关的功能作用，其作用模式与a组激活剂相似。(C) 2007 Elsevier Ltd.版权所有。
Bleeding at the site of bite and/or systemic hemorrhage are symptoms frequently observed in envenomation by Bothrops jararaca snakes. In this study, we purified and characterized a prothrombin activator from B. jararaca that is probably involved in these clinical manifestations. The enzyme was isolated by a combination of gel filtration and ion exchange chromatographies and named bothrojaractivase. It has a single polypeptide chain with a molecular weight of 22,829 Da as measured by mass spectroscopy. Bothrojaractivase generates active thrombin from prothrombin, independently of cofactors. SDS-PAGE analysis of the prothrombin activation products shows that bothrojaractivase converts prothrombin into meizothrombin producing similar fragments to those generated by group A prothrombin's activators. In addition, bothrojaractivase degraded fibrinogen and fibrin. Chelating agents completely inhibited the enzymatic activity, whereas inhibitors of serine and cysteine proteinases had no effect. Amino acid sequence of four peptides demonstrated high similarity of bothrojaractivase with P-I class of snake venom metalloproteinases. Thus, our results indicate that bothrojaractivase is a new metalloproteinase that acts on different protein factors of the clotting cascade especially displaying a key and most relevant functional action in the generation of thrombin through prothrombin activation in a similar mode of action as that of group A activators. (C) 2007 Elsevier Ltd. All rights reserved.