{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,27]],"date-time":"2025-10-27T04:46:13Z","timestamp":1761540373361},"reference-count":44,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2008,12,31]],"date-time":"2008-12-31T00:00:00Z","timestamp":1230681600000},"content-version":"vor","delay-in-days":2678,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2001,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Bothrojaracin (BJC) is a 27\u2010kD snake venom protein from <jats:italic>Bothrops jararaca<\/jats:italic> 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\u2010independent, 1:1 complex formation between fluorescein\u2010labeled 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 \u00b1 32 nM. The exosite I\u2010specific ligand, hirudin<jats:sup>54\u201365<\/jats:sup> (Hir<jats:sup>54\u201365<\/jats:sup> (SO<jats:sub>3<\/jats:sub><jats:sup>\u2212<\/jats:sup>), displaced competitively [5F]BJC from prothrombin. Titration of the fluorescent hirudin<jats:sup>54\u201365<\/jats:sup> derivative, [5F]Hir<jats:sup>54\u201365<\/jats:sup>(SO<jats:sub>3<\/jats:sub><jats:sup>\u2212<\/jats:sup>), with human prothrombin showed a dissociation constant of 7.0 \u00b1 0.2 \u03bcM, indicating a \u223c100\u2010fold lower binding affinity than that exhibited by BJC. Both ligands, however, displayed a similar, \u223c100\u2010fold increase in affinity for exosite I when prothrombin was activated to thrombin. BJC efficiently displaced [5F]Hir<jats:sup>54\u201365<\/jats:sup>(SO<jats:sub>3<\/jats:sub><jats:sup>\u2212<\/jats:sup>) from complexes formed with thrombin or prothrombin with dissociation constants of 0.7 \u00b1 0.9 nM and 11 \u00b1 80 nM, respectively, indicating that BJC and Hir<jats:sup>54\u201365<\/jats:sup>(SO<jats:sub>3<\/jats:sub><jats:sup>\u2212<\/jats:sup>) compete for the same exosite on these molecules. The results indicate that BJC is a potent and specific probe of the partially exposed anion\u2010binding exosite (proexosite I) of human prothrombin.<\/jats:p>","DOI":"10.1110\/ps.09001","type":"journal-article","created":{"date-parts":[[2002,7,26]],"date-time":"2002-07-26T23:59:58Z","timestamp":1027727998000},"page":"1897-1904","source":"Crossref","is-referenced-by-count":26,"title":["Characterization of bothrojaracin interaction with human prothrombin"],"prefix":"10.1002","volume":"10","author":[{"given":"Robson Q.","family":"Monteiro","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul E.","family":"Bock","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M. 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