DETERMINATION OF THE RATIOS OF THE AROMATIC AMINO-ACID-RESIDUES BY FIRST-DERIVATIVE OR 2ND-DERIVATIVE UV SPECTROMETRY FOR A SIMPLE CHARACTERIZATION OF PEPTIDES

A method for the simultaneous determination of the ratios of the three aromatic amino-acid residues in peptides was set up in acidic conditi ons. Binary and ternary mixtures of these amino acids were prepared, a nd first- and second-derivative spectra then calculated from their 0.1 nm resolution spectra between 240 and 320 nm. Certain spectral bands were chosen to differentiate tyrosine from tryptophan on the first-der ivative spectra, and phenylalanine from tyrosine and tryptophan on the second-derivative spectra. Variation of the amplitude of the chosen b ands was shown to be a linear function of the ratio of the aromatic am ino acids in the mixture. This technique was validated with peptides w hose sequence was known. The difference between theoretical and experi mentally determined ratios was lower than 10%. Since the results are o btained as ratios, neither the concentration nor the nature of the pep tide has to be known. The feasibility of application using a photodiod e array detector with high resolution in reversed-phase high-performan ce liquid chromatography is discussed. (C) Munksgaard 1995.