Colorimetric determination of carboxypeptidase A activity in serum

This simple, reproducible colorimetric method for determining the activity of carboxypeptidase A (EC 3.4.17.1) is based on measuring the absorbance at 505 nm of a quinoneimine dye produced from the action of this enzyme on the new substrate p-hydroxybenzoyl-glycyl-L-phenylalanine. The enzyme acts on the substrate to produce p-hydroxybenzoyl-glycine and L-phenylalanine. The former is then hydrolyzed by hippuricase (EC 3.5.1.14) to produce p-hydroxybenzoic acid and glycine. Finally, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by sodium periodate forms a quinoneimine dye. The Km for the reaction with this substrate is 3.6 mmol/L; the optimum pH is 7.8. Our within-run and between-run CVs are 4.3% and 6.6%, respectively. The activity of carboxypeptidase A in serum correlates well with that of lipase (r = 0.96) and immunoreactive elastase-1 (r = 0.76).