Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
y-Carboxyglutamic acid
Methods of Analysis

8.12.2.1. Thin-Layer Chromatography. On aluminum plates coated with a 0.1 mm Cellulose layer (Merck) using an n-butanol — formic acid — Water (75:15:10) system, Gla yields a purple spot upon ninhydrin staining with an R_f of 0.24. A colorimetric METHOD FOR DETERMINING Gla using acetaldehyde and nitroprusside has been described [260].

8.12.2.2. Amino acid analyzer.

Cation-exchange Column. The elution time of Gla from the column strongly depends on the pH of the first buffer. On a Beckman 121 M analyzer at a nitrate buffer pH of 2.68, the elution times were 13.0, 17.4, and 30.5 min for Cya, taurine, and Gla, respectively. Other Amino Acids eluted later. The calculated ninhydrin response factor for Gla is 39% of that for Glu [145]. At pH 3.25, Gla is poorly resolved from pyroglutamic acid. To identify them, the eluate fraction is collected and divided into two parallel samples: the first for alkaline (2.5 M NaOH, 105 °C, 24 h) and the second for acidic (6 M HCl, similar conditions) Hydrolysis. Pyroglutamic acid is completely hydrolyzed to glutamic acid in both cases. The Gla concentration is determined from the difference between the amounts of glutamic acid formed upon acidic and alkaline hydrolysis. [¹⁴C]Cysteic acid is used as an internal standard for quantification. The total yield of Gla as Glu is 95–98% [179].

Anion-exchange column. On an anion-exchange column, Gla elutes after all other amino acids. For instance, using a column (125X9.0 mm, Bio-Rad) packed with Aminex A-27 or A-28 resin, equilibrated with 0.3 M sodium acetate buffer (pH 4.6), and eluted with the same buffer (flow rate 70 mL/h, 30 °C), Gla eluted at 55 minutes [361]. Similar results were obtained in [182].

8.12.2.3. HPLC [206].

Pre-column derivatization. Mix 50 µL of protein hydrolysate or urine with 50 µL of OPA reagent (Section 8.16.3.2) and vortex vigorously. After 2 min, add 100 µL of 0.1 M KH₂PO₄ in 66% acetonitrile. Inject 2–20 µL of the prepared solution onto the column.

HPLC. A short column (50X4.6 mm) packed with Nucleosil 5SB (particle size 5 µm; Macherey-Nagel) is used. This is a silica-based strong anion exchanger with N(CH₃)₃ functional groups. Isocratic elution is performed at 47 °C (flow rate 2 mL/min) using 0.2 M sodium citrate buffer (pH 5.28) — acetonitrile (1:1) as the Mobile phase. Repeated analyses can be carried out under the same conditions without column regeneration. Detection is performed at an excitation wavelength of 240 nm and an emission wavelength of 418 nm using a cutoff filter. Most amino acids co-elute within two minutes, followed by Glu, Asp, Cya, S-carboxymethylcysteine, and finally Gla (at 7 minutes). The peak area-to-Gla amount ratio is linear in the range of 0.3–100 pmol.

Note. Recently, the OPA derivative of Gla was determined by HPLC using β-carboxyaspartic acid as an internal standard [143].