Premium
Characterization of bothrojaracin interaction with human prothrombin
Author(s) -
Monteiro Robson Q.,
Bock Paul E.,
Bianconi M. Lucia,
Zingali Russolina B.
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.09001
Subject(s) - chemistry , dissociation constant , isothermal titration calorimetry , thrombin , stereochemistry , biochemistry , biology , platelet , receptor , immunology
Abstract 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 (SO 3 − ), displaced competitively [5F]BJC from prothrombin. Titration of the fluorescent hirudin 54–65 derivative, [5F]Hir 54–65 (SO 3 − ), with human prothrombin showed a dissociation constant of 7.0 ± 0.2 μM, indicating a ∼100‐fold lower binding affinity than that exhibited by BJC. Both ligands, however, displayed a similar, ∼100‐fold increase in affinity for exosite I when prothrombin was activated to thrombin. BJC efficiently displaced [5F]Hir 54–65 (SO 3 − ) 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 (SO 3 − ) 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 I) of human prothrombin.