Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Arginine
Determination Methods

8.10.1.1. Arginine in intact protein [366].

Reagents

A. 0.1% a-naphthol in 50% ethanol (prepared fresh daily).

B. 10% KOH.

C. 5% urea.

D. Potassium hypobromite solution (0.64 mL Br₂ in 100 mL of 5% KOH; prepared fresh daily).

E. Stock arginine solution, 1.0 µmol/mL.

Procedure. Arginine may be free or protein-bound. Sequentially add 1 mL of reagents A, B, and C to 1 mL of the test solution containing 0.1–1.0 µmol of arginine. Mix thoroughly and rapidly add 2 mL of solution D with continuous shaking (crucial step!). Simultaneously, run a blank determination following the same sequence of operations using 1 mL of Water instead of the arginine solution. Let stand for 20 min at room Temperature, then measure the absorbance at 520 nm. Determine the concentration from a calibration curve in the range of 0.1–1.0 µmol/mL.

8.10.1.2. Determination with n-nitrophenylglyoxal [410].

Reagents

A. 0.1 M sodium pyrophosphate + 0.15 M sodium ascorbate, pH 9.0. Prepared fresh daily using nitrogen-free water.

B. n-Nitrophenylglyoxal (10% solution, w/v) in methanol. Prepared fresh daily.

C. Subtilisin (0.1% solution, w/v) in 0.01 M sodium phosphate, pH 7.9.

D. Bovine Trypsin (0.1% solution, w/v) in 1.0 mM HCl + 20 mM CaCl₂.

n-Nitrophenylglyoxal is prepared from n-nitroacetophenone According to the procedure described in [355]. A solution of 39 g (0.30 mol) of selenious acid in 24 mL of water, 150 mL of glacial acetic acid, and 49.8 g (0.30 mol) of n-nitroacetophenone is refluxed for 1 h. Cool and filter off the precipitated selenium. Upon distillation, water and acetic acid are removed first (at 15 mm Hg), followed by the reaction product at 118–128 °C (0.6 mm Hg). Upon standing, the yellow viscous liquid turns into a hard glassy mass with a melting point of 37–46 °C. Yield, 0.15 mol.

Procedure. To 0.20 mL of a protein sample solution (1–2 mg) containing 0.1–1 µmol of arginine in deionized water or 0.1 M sodium pyrophosphate (pH 9.0), add 40 µL each of reagents C and D. Incubate at 37 °C for 3 h. Cool and dilute to 3.0 mL with buffer A. Add 25 µL of reagent B with stirring and incubate at 30 °C for 30 min. Cool below 20 °C in an ice bath and measure the absorbance at 475 nm against a blank. Determine the arginine content from a calibration curve constructed using an arginine solution of known concentration (in the range of 0.03–0.33 mmol/L L-arginine in 3 mL of reagent A) + 25 µL of reagent B; for arginine, ε₄₇₅ = 0.715.