Colorimetric determination of arginine residues in proteins by p-nitrophenylglyoxal

A new method is presented for the colorimetric determination of arginine residues in proteins. Under mildly alkaline conditions, p-nitrophenylglyoxal reacted with arginine to produce a stable colored solution in the presence of 0.15 m sodium ascorbate. Complete color development was obtained after 30 min at pH 9.0 and 30°C. The color produced at 475 nm obeyed Beer's law in the range 0.03–0.33 mm arginine. This color reaction was used to determine the number of arginine residues in several proteins of known arginine content. Best results were obtained when the protein samples were digested with a mixture of trypsin and subtilisin prior to assaying. The arginine contents obtained by this method agreed well with either the published values or with the results of amino acid analysis.