A procedure is described for retaining bovine plasma Ac-globulin activity as one part of the protein from plasma for every 1000 parts removed. The yields averaged 15%. The procedure involves removal of prothrombin with barium carbonate, isoelectric fractionation, fractionation with ammonium sulphate, chromatography on Amberlite IRC-50, and a second fractionation with ammonium sulphate. The procedure requires 2 days; however, the first day completes up to chromatography and the concentrate at that time is quite useful for many purposes. It is more stable than the product obtained after chromatography and the yields are higher. In absence of salts Ac-globulin is quite insoluble at pH 5.0. The final product usually contained some impurity. With the analytical ultra-centrifuge the S20in 0.1 M potassium chloride solution was found to be 4.2 at a protein concentration of 12.4 mg/ml. The specific activity was 1500 U./mg dry weight. Bovine plasma contains 120 U./ml or about 9 mg/100 ml. Assuming the same specific activity for human plasma the concentration is most likely near 1 mg/100 ml. The best stability conditions found were: 50% glycerol, pH 7.0, and 0.1 M calcium chloride. Under those conditions at room temperature all activity was retained 6 to 7 hours, at refrigerator temperature 24 hours, and at −60 °C for 1 month. In rabbits, antibodies were readily produced. Oxidizing agents destroyed the activity, while reducing agents did not, nor did they tend to stabilize. SH blocking agents destroyed the activity. The loss of activity in the presence of 0.0025 M parachloromercuribenzoate was recovered with 0.04 M cysteine. The molecule deteriorated while attempts were made to obtain physical chemical data; consequently, the molecular weight was calculated from an amino acid analysis and found to be 98,800. The reliability of this value is problematical. Human plasma was analyzed and found to contain 13 U./ml Ac-globulin. After 4 days storage, at room temperature, the prolonged prothrombin time of that plasma was completely restored with 13 units of Ac-globulin, which is equivalent to 8 μg.
Outstanding Characteristics of Thrombokinase Isolated from Bovine Plasma
