Reversed-phase high-performance liquid chromatography used to monitor enzymatic cleavage of pyrrolidone carboxylic acid from regulatory peptides.

Sequence determination of peptides blocked by amino-terminal pyrrolidone carboxylic acid (PCA) has in the past been hampered by the lack of a reliable and efficient method for removing this residue. We report here a rapid, efficient enzymatic method for removal of PCA. Reversed-phase high-performance liquid chromatography is used to monitor the reaction and to separate unblocked peptide from the reaction product. The method allows direct sequence analysis of newly purified PCA blocked peptides, eliminating the need for complicated enzymatic digestions and chromatography to determine the amino-terminal residue. Only a few micrograms of peptide are required instead of the several milligrams needed in the past.