[3H]diborane reduction of vitamin K-dependent calcium-binding proteins. Identification of a unique amino acid.

gamma-Carboxyglutamic acid has recently been identified as a component of the vitamin K-dependent region of bovine prothrombin (Nelsestuen, G. L., Zytkovicz, T. H., and Howard J. B. (1974) J. Biol Chem. 249, 6347-6350). The presence of this amino acid has been substantiated here by the reduction of vitamin K-dependent proteins with [3H]-DIBORANE. The reduction product of gamma-carboxyglutamic acid, 5,5'-[3H]dihydroxyleucine, was shown to be present in hydrolysates of reduced rat prothrombin, bovine prothrombin, and bovine factor X. The results are consistent with a minimum of 10 gamma-carboxyglutamic acid residues in the nonthrombin-generating region of bovine prothrombin but no such residues in the thrombin precursor portion of prothrombin. It is concluded that amino acid analyses of [3H]diborane-reduced proteins provides a sensitive, qualitative method for the identification of proteins which contain gamma-carboxyglutamic acid and are vitamin K-dependent.