The relationship between protein-protein and protein-lipid interactions and the immunological properties of bovine prothrombin and several of its fragments.

An investigation of the antigenicity of bovine prothrombin and several of its fragments: fragment 1 (NHz-terminal 156 amino acid residues of bovine prothrombin released by thrombin digestion) and NHz-terminal residues 1-39, employed a rabbit antibovine prothrombin fragment 1 antibody which had previously been fractionated by affinity chromatography and which shows an absolute specificity for the metal ioninduced conformation of prothrombin and fragment 1. This study indicates that, in the presence of calcium ions, 1-39 self-association, the formation of (1-39)/fragment 1 complexes, and prothrombin/phospholipid binding all mask the antigenic site. We hypothesize that in the presence of calcium ions a conformational change in the previously metal ion-free protein generates a hydrophobic region which is co-extensive with the antigenic region or whose conformational integrity is closely linked to the antigenic site and that this hydrophobic region includes at least the NHAerminal 39 residues of prothrombin. We further hypothesize that this hydrophobic region is involved in the protein/ protein and protein/lipid interactions noted above. Implicit in the interpretation of the protein-binding data has been the development of thermodynamic equations consistent with appropriate conceptual model systems. Appplication of these models has allowed the estimation of several association constants: 1) ios M-’ for (1-39)/fragment 1 complex formation; 2) 10” M’ for (1-39)/(1-39) dimer formation; and 3) 10’ M” for (1-39)/antibody complex formation. While the absolute values of these association constants may vary when this work is repeated using homospecific antibodies, the ability of the models to predict the observed relative changes in antigenicity is the important factor in generating the hypotheses presented. The derivation of the equations is independent of the number of antibody populations present and therefore applicable to any similar system. Further application of the model predicts that the antigenicity of the 1-39 dimer, the fragment l/(l-39) complex, and the prothrombin/