Investigation of the 35-62 conserved helix and disulfide loop of bovine prothrombin using an anti-peptide antibody

A 28-residue peptide corresponding to the 35-62 region of bovine prothrombin fragment 1 (BF1) was synthesized by solid-phase methods. In BF1 this region consists of three conserved aromatic residues within an alpha-helical region followed by a disulfide loop. This synthetic peptide was used to produce murine monoclonal antibodies (MAbs) that would recognize and bind native BF1. Antibody AH.Ab.E3, an IgG1 antibody that was isolated and cloned, recognized and bound to both the synthetic peptide and the BF1 molecule. Residues 55-59 (REKLN) were shown to be critical for antibody binding. This MAb was subsequently used to study the 48-62 disulfide loop region of BF1. MAb AH.Ab.E3, which has been shown to bind the BF1 calcium-dependent conformation (BF1:Ca), does not appear to perturb the binding interaction between BF1:Ca and phospholipid (PL) vesicles as studied by light scattering methods.