On the preparation of bovine alpha-thrombin.

An improved method for the preparation of bovine alpha-thrombin is described. The procedure involves that activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2.200-2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strength and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of alpha-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to "aged" thrombin. In addition, the catalytic properties of alpha-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.