Isolation and characterization of bovine factor XII (Hageman factor)

Abstract

Factor XII was purified approximately 14,000-fold from bovine plasma by ammonium sulfate fractionation followed by heparin-agarose, DEAE-Sephadex, CM [carboxy methyl]-cellulose, arginine-agarose and benzamidine-agarose column chromatography. By this method, about 15 mg of protein was purified from 15 l of plasma with an overall yield of 18%. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal analysis. Bovine factor XII is a glycoprotein with a MW of 74,000 as determined by sedimentation equilibrium centrifugation. It contains 13.5% carbohydrate including 3.4% hexose, 4.7% N-acetylhexosamine and 5.4% N-acetylneuraminic acid. Factor XII is a single polypeptide chain with an NH2-terminal sequence of Thr-Pro-Pro-Trp-Lys-Gly-Pro-?-Lys-His-. This sequence is homologous to the reactive-site regions of a number of protease inhibitors. The amino acid sequence of a carboxy-l-terminal fragment prepared by cyanogen bromide digestion was Leu-Cys-Ala-Gyl-Phe-Leu-Glu-Gly-Gly-Thr-Asp-Ala-Cys-Gln-Gly-Asp-SeR-Gly-Gly-Pro-Leu-Val-Cys-Glu-Asp-Glu. This sequence is homologous with the active site of several plasma serine proteases including thrombine, factor IXa, factor Xa and plasmin. Bovine factor XII is apparently a precursor to a serine enzyme with an inhibitor sequence and a catalytic site located in the same single polypeptide chain.