Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
y-Carboxyglutamic Acid
Protein Hydrolysis

y-Carboxyglutamic acid (Gla) was first discovered in prothrombin and later identified in other Proteins and urine. Various Methods have been proposed for its quantification, including Amino acid analysis [134, 145, 146, 179, 182, 234, 361], GLC [247], colorimetric analysis (ranging from 0.5∙10:5х10-4 mol/L) [290], and HPLC [205, 206].

Gla undergoes complete decarboxylation under conditions of acid (1 M HCl, 100 °C, 16 h, vacuum) and alkaline Hydrolysis typically used for proteins [205]. Peptides containing Gla residues can be detected using the so-called "thermal diagonal" method. The peptide mixture is subjected to paper Electrophoresis at pH 6.5. The paper is dried by heating at 80 °C for 1 h, followed by a second-dimension electrophoresis at pH 6.5. Decarboxylated peptides thereby shift off the diagonal [235] (see also H. Morris, personal communication).

1. Hydrolyze the protein in 2.5 M KOH at 100 °C (16 h, vacuum or N2). Centrifuge. Cool the supernatant in an ice bath and acidify to pH 4–5 by the dropwise addition of first 60% and then 6% HClO4. Leave on ice for 30 min. Centrifuge to remove the KClO4 precipitate. Use 2–10 µL of the supernatant for analysis [205].

2. Hydrolyze the protein in 0.4–1.0 mL of 2.0 M KOH at 110 °C for 22 h. Add 0.2 mL of freshly prepared saturated KHCO3 solution. While stirring, neutralize with 70% HClO4 to pH 7 (using indicator paper). Centrifuge and analyze the clear supernatant. Gla is stable at pH 7 (—20 °C) [145].



Last update: 06/08/2026

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