Bovine Thrombin: PURIFICATION AND CERTAIN PROPERTIES

Abstract

Abstract An examination is made of the fractionation of a crude thrombin preparation obtained by bioactivation, namely, Parke Davis thrombin, topical. Columns of carboxylic resin, cellulose phosphate, diethylaminoethyl cellulose, and Sephadex G-100 were studied. The starting material and all products obtained by using only carboxylic resin or cellulose phosphate are shown to be impure and to contain a nonenzymatic inhibitor. A simple procedure is given for isolating a stable thrombin of high, constant specific activity. The procedure involves removal from solution, first, of inhibitor and some nonthrombin protein by DEAE-cellulose at pH 7, Γ/2 = 0.1 (phosphate), and 23°. A cellulose phosphate column is next used under the same conditions to adsorb thrombin and pass some nonthrombin protein. After additional inactive protein is washed off at pH 7 and Γ/2 = 0.15, thrombin is eluted at Γ/2 = 1.0 to give stock thrombin. The procedure at this point gives a minimum yield of 91.1 ± 8.4% of the original activity. Gel filtration of stock thrombin on Sephadex G-100 separates an average additional 24% of the absorbance of stock thrombin as an inactive protein and gives a single thrombin peak. By comparison with known proteins the peak position corresponds to a molecular weight of 36,000 ± 1,600 (95% confidence limits). The last 0.6 of the peak has constant specific activity which corresponds to 1,160 ± 130 NIH units per absorbance unit or 2,260 ± 250 NIH units per mg. At ionic strength 1.0, pH 7, and 4°, stock thrombin preparations lose activity to the maximum extent of 0.1% per week. At Γ/2 = 0.1, pH 7, and 23°, diluted stock thrombin (20-sec clotting time) loses 1.6% of its activity per day. This is shown not to be due to an inhibitor, an extraneous enzyme, or the presence of 5 x 10-4 m KCN, which had to be added to prevent bacterial growth. The mechanism appears to be a zero order denaturation. Attempts to concentrate stock thrombin by ammonium sulfate precipitation or osmotic solvent removal have frequently led to the production of inactive aggregates. Acetone precipitation from 0.15 m calcium chloride at pH 7 has been successful in giving concentrations up to 10 absorbance units per ml.