Analysis of gamma-carboxyglutamic acid by reverse phase HPLC of its phenylthiocarbamyl derivative

A method is presented for analysis of gamma-carboxyglutamic acid based on its derivatization with phenylisothiocyanate and reverse phase HPLC analysis of the resulting phenylthiocarbamyl derivative. Proteins were hydrolyzed with sodium hydroxide and the hydrolysates were desalted on Dowex 50 eluted with ammonium hydroxide. The resulting amino acid mixtures were derivatized with phenylisothiocyanate and the phenylthiocarbamyl derivatives were separated under isocratic conditions on either C18 or C8 reverse phase columns using 0.14 M Tris, 0.05% triethylamine, titrated to pH 7.5 with glacial acetic acid, plus 2% acetonitrile, and detected by absorbance at 254 nm. The method is linear over the range from 10 to 1000 pmol of gamma-carboxyglutamic acid and the limit of detection is near 2 pmol. The utility of the method was verified for analysis of purified prothrombin yielding a value of 10.3 mol of gamma-carboxyglutamic acid per mole in agreement with sequence data. No gamma-carboxyglutamate was detectable for acid-hydrolyzed samples of prothrombin, nor in acid- or base-hydrolyzed samples of bovine serum albumin. Application of this method failed to corroborate the reported presence of gamma-carboxyglutamate in a putative mitochondrial gamma-carboxyglutamate-containing calcium-binding protein. The method was also tested for determination of beta-carboxyaspartate, beta-hydroxyaspartate, phosphoserine, phosphothreonine, and phosphotyrosine in an attempt to identify an unknown material which appeared in preparations of the mitochondrial protein.