The Photometric Microdetermination of Tryptophan in Biologic Materials

Abstract

Abstract A modification of the Fischl (6) glyoxylic acid method for tryptophan was developed which permits the reaction to occur quantitatively in aqueous solution by substitution of 70% HCIO4 (w/v) for concentrated H2S04. Spectral analysis of the chromogen formed showed a strong adsorption band at 365 mµ for pure tryptophan which was 17 times more sensitive than that at 560 mµ. Factors were studied which influence the absorbance of the tryptophan chromogen at 365 mµ and the most favorable conditions for its formation. A new method for hydrolyzing proteins and peptides was evolved using 3.5% HCIO4 (w/v) which results in complete rupture of the peptide bond while retaining intact the indole nucleus. Application of the modified glyoxylic acid method to perchloric acid hydrolyzates of various proteins and peptides of known tryptophan content gave experimental values in close agreement with those calculated from the structural formulae. The over-all reproducibility of the procedure for separate hydrolyzates, run in duplicate, of the same protein or peptide sample was about ±3.0%. Comparison of the tryptophan values obtained for 19 different "purified" peptides and proteins checked rather well with some of the results reported in the literature for the same materials using other procedures, except for trypsin and ovomucoid.