Methods for alkaline hydrolysis of proteins and chromatographic resolution and quantitation of the calcium-binding amino acid y-carboxyglutamate (Gla) are presented. Gla is extremely acid labile and totally converted to glutamic acid during acid hydrolysis. Under the recommended hydrolysis conditions of 2 M KOH at 110°C for 22 hr, the recovery of Gla is quantitative. The ninhydrin color yield for Gla on our particular amino acid analyzer is only 3% of that for glutamic acid; this factor is readily obtained from the ratio of peak areas after acid conversion of Gla to glutamic acid. y-Carboxyglutamic acid (Gla) has recently been identified as a constituent of vitamin K-dependent blood coagulation proteins [factors IX, X, prothrombin (l-9), and, presumably, factor VII], acidic proteins indigenous to mineralized tissue (lo-12), a serum protein of unknown function (9,13), and one or more proteins formed in the kidney (14). Gla biosynthesis occurs post-translationally and involves vitamin K-dependent enzymatic carboxylation of specific glutamic acid residues in the polypeptide chain (15,16).
The interest in Gla as a newly discovered amino acid which confers on proteins both Ca2+-binding properties and the propensity for Ca2+mediated phospholipid interaction (17-21) has prompted development of methods for its quantitation.
Gla, as a malonic acid derivative, is readily decarboxylated to glutamic acid under the acidic conditions normally used for protein hydrolysis (5,lO). Enzymatic digestion and separation of specific peptides from prothrombin preceded the rigorous structural characterization of Gla by mass spectrometric techniques (l-4). Reductive modification of Gla with [3H]diborane allows isolation of a stable, labeled derivative from acid hydrolysates (7) but this elegant qualitative method is unsuitable for routine quantitation. Alkaline hydrolysis provides quantitative release of Gla from peptide linkage without significant destruction (10,22). The method reported here allows routine quantitative analysis of Gla in proteins using standard automatic amino acid analyzers. 212