Amino Acid Analysis and Enzymatic Sequence Determination of Peptides by an Improved o-Phthaldialdehyde Precolumn Labeling Procedure

Abstract

Primary amino acids react with o-phthaldialdehyde in the presence of mercaptans to form intensely fluorescent derivatives. By the use of reverse-phase high-performance liquid chromatography, a mixture containing 26 of these derivatives was efficiently resolved with an analysis time of less than 35 minutes. The quantitation of the individual amino acids was reproducible with an average relative deviation of ± 1.4% and had a detection limit of approximately 50 femtomoles. Improvements in the stability and fluorescence response of lysine and hydroxylysine were obtained by the incorporation of sodium dodecyl sulfate in the derivatizing medium. Applications of the chromatography system involving the amino acid analysis of peptides after either acid or enzymatic hydrolysis are presented. Methods for the sequence analysis of picomole quantities of peptides by time course hydrolysis with exopeptidases were also developed, which employed the above separation procedure for the identification and quantification of the released amino acids.