USING CAPILLARY ELECTROPHORESIS IN THE OPTIMIZATION OF A CARBOXYPEPTIDASE-Y CATALYZED TRANSPEPTIDATION REACTION

A peptide amide, R-Arg-NH2, was produced by carboxypeptidase Y (CPDY)- catalyzed transpeptidation of a peptide, R-Ala-OH in presence of a lar ge excess of Arg-NH2. Baseline separation of R-Ala-OH and R-Arg-NH2 wa s achieved by free solution capillary electrophoresis (CE) analysis. W ith CE the reactions could be closely followed with an analysis freque ncy of 3-6 h-1. Due to a low consumption of sample per CE analysis (1- 5 nL introduced, 5-6 muL in the sample vial), the reactions were perfo rmed in 100 and 250 muL volumes. Consequently, the optimization experi ments consumed limited amounts of enzyme and substrate only. At optimi zed experimental conditions approximately 90% conversion of the starti ng peptide, R-Ala-OH, to R-Arg-NH2 was achieved.