Characterization of bothrojaracin interaction with human prothrombin
Characterization of bothrojaracin interaction with human prothrombin
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DOI：
10.1110/ps.09001
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发表时间：
2001-09-01
期刊：
影响因子：
8
通讯作者：
Zingali, RB
中科院分区：
文献类型：
--
作者：
Monteiro, RQ;Bock, PE;Zingali, RB
Bothrojaracin （BJC）是一种来自于Bothrops jararaca的27-kD蛇毒蛋白，被认为是一种有效的凝血酶抑制剂。BJC与外源位点I和II结合，解离常数为0.7 nM，影响但不阻断蛋白酶的催化位点。BJC还通过一种尚未被描述的相互作用结合凝血酶原。本研究首次定量表征了BJC与凝血酶原的相互作用，并鉴定了BJC与人凝血酶原的结合位点。凝胶过滤层析显示，荧光素标记的BJC （[5F]BJC）与凝血酶原之间形成了不依赖钙的1:1复合物，而与凝血酶原的激活片段1或2没有观察到相互作用。等温滴定量热法显示BJC与凝血酶原的结合是吸热的，解离常数为76 +/- 32 nM。外源i特异性配体水蛭素（54-65）(Hir（54-65) (SO3-)）竞争性地取代凝血酶原中的BJC [5F]。水蛭素（54-65）荧光衍生物[5F]Hir(54-65)（SO3-）与人凝血酶原的解离常数为7.0 +/- 0.2 nM，表明其结合亲和力比BJC低100倍。然而，当凝血酶原被激活为凝血酶时，这两种配体对外源位点I的亲和力都显示出类似的100倍的增加。BJC有效取代了与凝血酶或凝血酶原形成的络合物中的[5F]Hir(54-65)(SO3-)，解离常数分别为0.7 +/- 0.9 nM和11 +/- 80 nM，表明BJC和Hir(54-65)（SO3-）在这些分子上竞争相同的外源位点。结果表明，BJC是人凝血酶原部分暴露阴离子结合外源体（proexosite 1）的有效特异性探针。
Bothrojaracin (BJC) is a 27-kD snake venom protein from Bothrops jararaca that has been characterized as a potent thrombin inhibitor. BJC binds to exosites I and II, with a dissociation constant of 0.7 nM, and influences but does not block the proteinase catalytic site. BJC also binds prothrombin through an interaction that has not been characterized. In the present work we characterize the interaction of BJC with prothrombin quantitatively for the first time, and identify the BJC binding site on human prothrombin. Gel filtration chromatography demonstrated calcium-independent, 1:1 complex formation between fluorescein-labeled BJC ([5F]BJC) and prothrombin, whereas no interactions were observed with activation fragments 1 or 2 of prothrombin. Isothermal titration calorimetry showed that binding of BJC to prothrombin is endothermic, with a dissociation constant of 76 +/- 32 nM. The exosite I-specific ligand, hirudin(54-65) (Hir(54-65) (SO3-), displaced competitively [5F]BJC from prothrombin. Titration of the fluorescent hirudin(54-65) derivative, [5F]Hir(54-65)(SO3-), with human prothrombin showed a dissociation constant of 7.0 +/- 0.2 nM, indicating, a similar to 100-fold lower binding affinity than that exhibited by BJC. Both ligands, however, displayed a similar, similar to 100-fold increase in affinity for exosite I when prothrombin was activated to thrombin. BJC efficiently displaced [5F]Hir(54-65)(SO3-) from complexes formed with thrombin or prothrombin with dissociation constants of 0.7 +/- 0.9 nM and 11 +/- 80 nM respectively, indicating that BJC and Hir(54-65)(SO3-) compete for the same exosite on these molecules. The results indicate that BJC is a potent and specific probe of the partially exposed anion-binding exosite (proexosite 1) of human prothrombin.