Bovine thrombin and activated factor X. Separation and purification.

Parke-Davis bovine thrombin was resolved into three distinct protein peaks on a Sephadex G-200 column. A stepwise chromatographic technique on diethylaminoethyl (DEAE) cellulose was developed for the complete separation and partial purification of thrombin and activated Factor X. Thrombin was quantitatively eluted in the first protein peak with 0.1 M NaCl, pH 7.0. A second peak with residual thrombin activity, containing more than 65% of the applied protein, was eluted with 0.14 M NaCl in 0.05 M sodium citrate, pH 5.8. When this fraction was rechromatographed, to remove the residual thrombin, Factors II, VII, IX, and nonactivated X were detected in this peak. Activated Factor X was eluted from the column with 0.2 M sodium citrate, pH 5.8. This activated Factor X fraction did not clot a standard fibrinogen solution during 24 hours either at 22’ or 37”, with or without added calcium, nor after removal of the citrate by dialysis against 0.14 M NaCI. The DEAE-cellulose-chromatographed thrombin was further purified by Sephadex G-200 chromatography. More than 68% of the resulting thrombin peak had a constant specific activity of approximately 7000 National Institutes of Health units per mg of tyrosine. At high dilutions, when the thrombin activity in the commercial bovine thrombin preparation was negligible, the activated Factor X activity was still detectable in appreciable quantity. The purified thrombin fraction was free of plasminogen and plasmin.