Purification of a murine IgM monoclonal antibody.

An IgM monoclonal antibody, S11-23.4, raised against the 47-62 amino acid sequence in bovine prothrombin fragment 1 (F-1, the amino-terminal 156 residues of prothrombin), was purified from tissue culture supernatants and ascites using different purification schemes to determine the best method. There are many different purification schemes for the purification of IgG antibodies, which are generally easier to purify than IgM antibodies.

Studies on the peptide corresponding to residues 34-47 of bovine factor X.

Calcium binding studies of a 14-residue peptide corresponding to the 37-46 sequence of bovine factor X were performed using calcium ion selective electrode titrations and equilibrium dialysis. The presence of gamma-carboxyglutamic acid residues at positions 36 and 40 coupled with the assumption that the peptide would bind calcium ions also prompted an investigation of possibly secondary conformational changes in the peptide by use of circular dichroism spectroscopy. Equilibrium dialysis revealed a single relatively weak calcium binding site (log Ka = 2.

Refinement of the NMR solution structure of the gamma-carboxyglutamic acid domain of coagulation factor IX using molecular dynamics simulation with initial Ca2+ positions determined by a genetic algorithm.

A genetic algorithm (GA) successfully identified the calcium positions in the crystal structure of bovine prothrombin fragment 1 bound with calcium ions (bf1/Ca). The same protocol was then used to determine the calcium positions in a closely related fragment, the Gla domain of coagulation factor IX, the structure of which had previously been determined by NMR spectroscopy in the presence of calcium ions. The most frequently occurring low-energy structure found by GA was used as the starting structure for a molecular dynamics refinement.

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.