Clinical Chemistry, Vol 15, 15-23, Copyright © 1969 by the American Association for Clinical Chemistry

A Shortened Method for the Fluorometric Determination of Phenylalanine

¹ National Communicable Disease Center, U. S. Public Health Service, Bureau of Disease Prevention and Environment Control, U. S. Department of Health, Education, and Welfare, Atlanta, Ga. 30333.

As a result of a study of the classic ninhydrin reaction with amino acids (which results in the production of Ruhemann’s purple), and the fluorescence reaction occurring between ninhydrin and phenylalanine in the presence of a dipeptide, the initial incubation period in the fluorescence reaction was shortened from 2 hr. to 16 min. This improvement in the fluorometric method for the determination of phenylalanine was accomplished by (1) decreasing the initial incubation pH from 5.8 to 5.0, and (2) increasing the incubation temperature from 60° to 85°. A study of the effect of pH on the fluorescence level in both the acid and alkaline reactions demonstrated the presence of fluorescence pH plateaus. These plateaus indicate that the fluorescence level was relatively unaffected by a change in pH within specified ranges. A study of the specificity led to the discovery that the inclusion of a peptide control was necessary to correct for one type of nonspecific fluorescence. To make this correction, it was necessary to add the reading of the serum blank to that of the reagent blank, and to subtract this total from the sample reading.

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