A Photometric Method for the Determination of Proline

The characteristic red color produced when proline interacts with nin-hydrin in acidic solutions has recently been proposed for use in the quan-titative 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 pres-ent method, which is a modification of the procedure of Chinard (l), the interfering basic amino acids lysine, hydroxylysine, and ornithine are re-moved by shaking the solutions with Permutit. Under these circum-stances, 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 ab-sorption spectra of the acid (1) and neutral ninhydrin condensation prod-ucts (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). EXPERIMENTAL Proline Method-The following procedure was developed for the de-termination of proline. The solutions (1 to 5 X lO+ M proline) were shaken with approximately one-tenth their weight of Permutit ’ for 5 min-utes. 5 ml. of this solution, 5 ml. of glacial acetic acid, and 5 ml. of nin-hydrin reagent2 are heated in a water bath for 1 hour in test-tubes with plastic screw caps. The solutions are cooled to room temperature and extracted with 5 ml. of benzene by shaking them vigorously for 5 minutes.