Colorimetric determination of citrulline residues in proteins

A method is described for the direct colorimetric determination of citrulline residues in proteins based on the reaction with diacetylmonoxime in the presence of lower concentrations of sulfuric acid. The reduced sensitivity due to the lower acid concentration was overcome by the addition of ferric chloride which also contributed to the color stabilization. Insoluble proteins or proteins resulting in turbidity can be analyzed following partial hydrolysis of those with enzyme or acid. The molar absorption coefficient (epsilon) for citrulline at 464 nm was 2.8 x 10(4). The method enabled us to determine low levels of protein-bound citrulline which are beyond the limitations of conventional methods using an amino acid analyzer. We determined the citrulline contents in the cornified cells of the epidermis of newborn rats, as well as soybean trypsin inhibitor.