A novel one-step purification of human α-thrombin after direct activation of crude prothrombin enriched from plasma

Abstract

Crude prothrombin enriched from human plasma was directly activated to generate alpha-thrombin without prior exhaustive purification of the proenzyme using a combination of several different types of chromatographic techniques, as in all previously described methodologies. Activated thrombin was separated from other components in a single step by taking advantage of its highly specific affinity to heparin immobilized on a matrix support of Sepharose CL-6B. On the basis of the data presented herein, we have demonstrated the ease with which at least 25 mg of a highly purified enzyme (greater than 97% homogeneous by laser densitometry) can be obtained per litre of plasma. Our product exhibits a specific activity of at least 4000 National Institutes of Health units/mg and is stable after being freeze-dried for the purpose of long-term storage.