The Preparation and Activation of [sialyl-3H]Prothrombin

Abstract

Abstract Bovine prothrombin with tritium incorporated into its sialic acid residues has been prepared by the method of Van Lenten and Ashwell ((1970) J. Biol. Chem. 246, 1889–1894). The resulting radiolabeled protein ([sialyl-3H]prothrombin) was electrophoretically indistinguishable from the untreated protein. The thrombin activity generated by factor Xa activation of [sialyl-3H]prothrombin was about 90 % of that obtained from nonlabeled prothrombin. The sole products of the reaction of thrombin with [sialyl-3H]prothrombin are intermediates 1 and 3. The products of the activation of [sialyl-3H]prothrombin with factor Xa are intermediates 1,2,3, and 4. Analysis of the carbohydrate distribution during activation suggests that about two-thirds of the sialic acid is present in intermediate 3, and about one-third of the sialic acid is attached to intermediate 1. Upon further activation, the carbohydrate which was attached to intermediate 1 is found in intermediate 2, and subsequently in α-thrombin. Intermediate 4 contains no sialic acid. Activation of [sialyl-3H]prothrombin in serum-thromboplastin produced intermediates identical in apparent molecular weight with those produced both upon activation in purified systems (Heldebrant, C. M., Butkowski, R. J., Bajaj, S. P., and Mann, K. G. (1973) J. Biol. Chem. 248, 7149–7163) and upon the activation of fluorescein-labeled prothrombin in serum-thromboplastin (Fass, D. N., and Mann, K. G. (1973) J. Biol. Chem. 248, 3280–3287). The sialic acid distribution during serum-thromboplastin activation was identical with that observed in purified systems. The radiolabeling procedure has also been successfully applied to the labeling of bovine intermediate 3, bovine factor X, and human prothrombin.