A photometric method for the determination of proline.

The characteristic red color produced when proline interacts with ninhydrin in acidic solutions has recently been proposed for use in the quantitative assay of this imino acid (1,Z). These methods could not, however, be applied directly to the estimation of proline in biological fluids, since other amino acids interfere to some extent and must be removed either by chromatography (1) or by destruction with nitrous acid (2). In the present method, which is a modification of the procedure of Chinard (l), the interfering basic amino acids lysine, hydroxylysine, and ornithine are removed by shaking the solutions with Permutit. Under these circumstances, the method becomes entirely specific for proline when applied to protein hydrolysates, urine, or plasma. The reaction product of proline in this acidic ninhydrin reaction has been assumed to be the compound isolated by Grassmann and von Arnim (3, 4) from a neutral reaction mixture (1, 2). The distinctly different absorption spectra of the acid (1) and neutral ninhydrin condensation products (5) required elucidation. The acid condensation product has been isolated and appears to have properties of a tautomer of the compound formed in the neutral condensation (3, 4).