PURIFICATION AND PROPERTIES OF BOVINE PROTHROMBIN

A rapid, simple, and reproducible method for the purification of bovine prothrombin, starting with a commercial preparation, is reported. By carrying out isoelectric precipitation, chromatography on DEAESephadex, and ultracentrifugation, a product in 18% yield, free of contaminants as evaluated by the sensitive criterion of disc gel electrophoresis, was obtained. Chemical and physiocochemical characterization studies were performed to determine the size and shape of the molecule. Prothrombin in phosphate buffer has a sedimentation equilibrium molecular weight of 74,000 ± 4100, a sedimentation coefficient of 4.80 S, intrinsic viscosity of 3.4 cc/g, a value for β of 2.07 × 106, possesses low helix content, and contains 2.3% carbohydrate as hexose. In the denaturing solvents 6 m guanidine hydrochloride and 6 m guanidine hydrochloride plus 0.1 m β-mercaptoethanol, prothrombin has essentially the same molecular weight as in buffer, indicating that the protein molecule consists of a single chain. Values for s and [η] in these solvents are also consistent with those for proteins known to be made up of one chain in these solvents. We conclude that prothrombin is a globular compact protein molecule of one chain, possessing little helix content. Preliminary activation, studies are also reported, and indicate that pure prothrombin does not activate autocatalytically. © 1969, American Chemical Society. All rights reserved.