Localization of the metal-induced conformational transition of bovine prothrombin.
The calcium-stabilized antigenic determinants on bovine prothrombin were localized to the NH2-terminal 1-42 residues using conformation-specific antibodies. Polyclonal antibodies to the bovine prothrombin-Ca(II) complex were raised in rabbits, and purified antibody subpopulations were isolated by sequential immunoabsorption and affinity chromatography. Anti-prothrombin-Ca(II) antibodies, characterized by their absolute specificity for the prothrombin-metal complex (Tai, M. M., Furie, B. C., and Furie, B. (1980) J. Biol. Chem. 255, 2790-2795), bound to prothrombin, fragment 1, reduced and carboxymethylated fragment 1, and CNBr fragment (1-72) in solution. However, these antibodies do not bind significantly to the gamma-carboxyglutamic acid-rich fragment (1-39), CNBr fragment (73-156), or prethrombin 1. To obviate the complex analysis of possible reasons for the lack of antibody binding to small peptides in solution, conformation-specific antibodies directed against defined regions of the whole prothrombin molecule were isolated. The influence of calcium ions on the binding of these site-specific antibody subpopulations to 125I-labeled prothrombin fragment 1 was evaluated. Anti-(1-39)N, anti-(1-42)N, anti-(1-72)N, and anti-(reduced and carboxymethylated fragment 1)N showed enhanced binding to prothrombin fragment 1 in the presence of Ca(II), indicating the presence of calcium-stabilized antigenic determinants within each of these regions on fragment 1. In contrast, calcium ions had no effect on the interaction of anti-des-(1-42)prothrombin, anti-prethrombin 1, anti-(43-72)N, and anti-(73-156)N antibodies with prothrombin fragment 1. These results indicate that the metal-induced conformational transition, monitored immunochemically, is localized to the NH2-terminal, gamma-carboxyglutamic acid-rich region of prothrombin between residues 1-42.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000936:Antigen-Antibody Complex; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D007106:Immune Sera; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism*
D000596:Amino Acids; D000936:Antigen-Antibody Complex; D007106:Immune Sera; D010446:Peptide Fragments; D011516:Prothrombin; D002118:Calcium