Undercarboxylation of recombinant prothrombin revealed by analysis of gamma-carboxyglutamic acid using capillary electrophoresis and laser-induced fluorescence

The gamma-carboxyglutamic acid (Gla) content of several variants of human p rothrombin has been measured by using capillary electrophoresis and laser-i nduced fluorescence (CE-LIF). Both plasma-derived prothrombin and recombina nt prothrombin contain ten residues of Gla per molecule of protein. In cont rast, a variant of human prothrombin (containing the second kringle domain of bovine prothrombin) was separated into two populations that differed in their Gla content. Direct measurement of the Gla content showed an associat ion with the presence or absence of the calcium-dependent conformational ch ange that is required for prothombinase function. Thus, the CE-LIF assay is useful in determining the carboxylation status of recombinant proteins. (C ) 1999 Federation of European Biochemical Societies.