INVESTIGATION OF THE 35-62 CONSERVED HELIX AND DISULFIDE LOOP OF BOVINE PROTHROMBIN USING AN ANTIPEPTIDE ANTIBODY

A 28-residue peptide corresponding to the 35-62 region of bovine proth rombin fragment 1 (BF1) was synthesized by solid-phase methods. In BF1 this region consists of three conserved aromatic residues within an c c-helical region followed by a disulfide loop. This synthetic peptide was used to produce murine monoclonal antibodies (MAbs) that would rec ognize and bind native BF1. Antibody AH.Ab.E3, an IgG1 antibody that w as isolated and cloned, recognized and bound to both the synthetic pep tide and the BF1 molecule. Residues 55-59 (REKLN) were shown to be cri tical for antibody binding. This MAb was subsequently used to study th e 48-62 disulfide loop region of BF1. MAb AH.Ab.E3, which has been sho wn to bind the BF1 calcium-dependent conformation (BF1:Ca), does not a ppear to perturb the binding interaction between BF1:Ca and phospholip id (PL) vesicles as studied by light scattering methods. (C) Munksgaar d 1996.