Purification and Some Properties of Autoprothrombin C

Abstract

Methods were developed for the isolation of autoprothrombin C, which is the second enzyme obtained from purified bovine prothrombin. In the presence of lipids and standardized conditions 0.35 μg of the purified autoprothrombin C were sufficient for clotting recalcified plasma in 15 seconds. Some physicochemical properties are as follows: S20, w is 2.27S, the diffusion constant 8.4 × 10−7 cm2/second, and the partial specific volume 0.695. The molecular weight from physicochemical measurements was 21,500. All amino acids were found, but only 1 molecule of methionine. On the basis of amino acid composition the molecular weight was found to be 27,000, giving an average of 24,200 for our two determinations. Prothrombin contains sufficient of each amino acid residue to supply autoprothrombin C and thrombin. Autoprothrombin II, however, has only sufficient amino acids for either autoprothrombin C or thrombin, but not both. The purified autoprothrombin C contained 7% carbohydrate (orcinol) and 3.8% hexosamine. It was stable at pH 7.2 for more than a week at room temperature, and longer in subzero glycerol solution. Autoprothrombin C was used to obtain a single precipitin band in agar diffusion plates with antibody to prothrombin. The band also identified with a single plasma antibody.

Etude Immunologique de la Prothrombine et de la Thrombine Humaines

“…Seegers et coll. (17) ont observe en immunodiffusion avec du materiel bovin, une ligne de precipitation entre une preparation d' autoprothrombine C et un antiserum anti-prothrombine prepare selon la methode de Barnhart et coll. (3) .…”

The preparation of activated Factor X and its action on prothrombin

“…The activation of the Factor X by the venom involves cleavages of both the heavy and the light chain of the zymogen; this is in contrast to the relatively minor changes caused by the activation of Factor X by the intrinsic system (Fujikawa et al, 1971). Other forms of Factor Xa have been reported in which more than 50 % of the zymogen is removed (Seegers et al, 1963). All these forms of Factor Xa, although differing widely in their molecular weights, appear to have similar biological specificity.…”

Activation of Purified Prothrombin in Ammonium Sulfate Solutions: Purification of Autoprothrombin C

“…Purified bovine prothrombin (12) and purified thrombin (13) were obtained as described previously. For separation of autoprothrombin C, DEAE cellulose (0.94 meq/g) was obtained from Schleicher and Schnell Co., Keene, N. H., and used for chromatography according to the procedure of Seegers, Cole, Harmison and Marciniak (2). The assay for prothrombin (14) , thrombin (15) and autoprothrombin C ( 16) was performed as described.…”