The isolation and characterization of a specific antibody population directed against the prothrombin activation fragments F2 and F1 + 2.
We have raised antisera against human prothrombin activation fragment F2 in rabbits and have chromatographed the respective immunoglobulin G fractions on prothrombin-Sepharose, Pr1-Sepharose, and F2-Sepharose immunoadsorbents. The specific antibody population obtained was utilized to construct a double antibody radioimmunoassay capable of measuring as little as 0.8 ng/ml of this component. Our studies suggest that the immunoreactive site defined by this antibody population is most probably located within the negatively charged COOH-terminal region of F2. The immunologic expression of this area is unaffected by denaturation or reduction-alkylation of F2 as well as by attachment of polypeptide to the NH2-terminal of this component. However, the presence of covalently bound polypeptide at the COOH-terminal of F2 reduces its immunologic reactivity by 300- to 400-fold. Prothrombin, Pr1, and Pr*1, which contain the F2 region as part of their covalent structure, are at least 4000 to 7000 times less immunoreactive than F2 on a molar basis. Conversion of these components to thrombin as well as activation fragments generates the theoretically predicted level of immunoreactivity. Masking of the immunoreactive site within these zymogens is due to two phenomena. Firstly, covalent attachment of polypeptides on the COOH-terminal of the F2 segment significantly depresses the reactivity of this region. Secondly, a critical S--S bridge aids in the sequenstration of the immunoreactive site. This cross-link may facilitate interactions between the COOH-terminal of the F2 segment and other regions of the zymogen.
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D003429:Cross Reactions; D004789:Enzyme Activation; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D011516:Prothrombin / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D011863:Radioimmunoassay
D000906:Antibodies; D006023:Glycoproteins; D010446:Peptide Fragments; D011516:Prothrombin