Heterogeneity in human prothrombin: analysis of cause.

Two fractions of human prothrombin can be isolated from single donor plasma by the technique of heparin-agarose chromatography in (sodium) citrate buffer, pH 7.5, as previously reported for pooled plasma. The two fractions, designated H-II1 and H-II2, are found in a ratio of approximately 4:1. Both forms comigrate in sodium dodecyl sulfate gel electrophoresis; however, under nondenaturing electrophoretic conditions, each fraction migrates as a discrete entity with a different mobility. The larger fraction (H-II1) has a faster mobility towards the anode. Isoelectric focusing in urea of H-II1 reveals that it has two components, a minor component with a pl of 5.25 (H-II1a) and a major component with a pl of 5.40 (H-II1b). H-II2 has a pl of 5.6 H-II1 and H-II2 possess the same amino terminal residue (alanine, 0.87-0.92 mole/mole) and the same number of gamma -carboxyglutamic acid residues (9.8-10.5). Their amino acid composition is indistinguishable. However, the two fractions of prothrombin differ in their content of neutral sugar and of sialic acid residues. Removal of sialic acid with neuraminidase abolishes the electrophoretic heterogeneity. Thus, the charge heterogneity of the three variants of prothrombin found in normal human plasma appears to result exclusively from differences in the number of sialic acid residues attached to the protein moiety of the molecule.