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Characterization of bothrojaracin interaction with human prothrombin.
R Q Monteiro1, P E Bock, M L Bianconi
1Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Brazil.
Protein Science : a Publication of the Protein Society
|August 22, 2001
Summary
Bothrojaracin (BJC), a snake venom protein, binds specifically to human prothrombin's exosite I. This interaction, characterized quantitatively, reveals BJC as a potent probe for prothrombin's anion-binding site.
Area of Science:
- Biochemistry
- Protein-protein interactions
- Snake venom toxins
Background:
- Bothrojaracin (BJC) is a snake venom protein known to inhibit thrombin by binding to exosites I and II.
- The interaction of BJC with prothrombin, a precursor to thrombin, has not been quantitatively characterized.
- Understanding BJC's binding to prothrombin is crucial for elucidating its mechanism of action and potential applications.
Purpose of the Study:
- To quantitatively characterize the interaction between Bothrojaracin (BJC) and human prothrombin.
- To identify the specific binding site of BJC on human prothrombin.
- To compare the binding affinity of BJC with other known ligands for prothrombin's exosites.
Main Methods:
- Gel filtration chromatography to assess complex formation.
- Isothermal titration calorimetry (ITC) to determine binding thermodynamics and affinity.
- Competitive binding assays using fluorescently labeled ligands (BJC and hirudin) to identify binding sites.
Main Results:
- BJC forms a calcium-independent, 1:1 complex with prothrombin, with a dissociation constant (Kd) of 76 ± 32 nM.
- BJC binds to prothrombin's exosite I, as evidenced by competitive displacement assays with hirudin.
- BJC exhibits significantly higher affinity for prothrombin's exosite I compared to hirudin(54-65).
- Both BJC and hirudin show increased affinity for exosite I upon prothrombin activation to thrombin.
Conclusions:
- Bothrojaracin (BJC) is a potent and specific ligand for the partially exposed anion-binding exosite (proexosite I) of human prothrombin.
- BJC serves as a valuable molecular probe for studying prothrombin and thrombin structure-function relationships.
- The findings provide new insights into the molecular recognition mechanisms governing interactions with prothrombin and its activation pathway.