High performance liquid chromatographic quantitation of carboxypeptidase activity secreted by human hep G2 cells

A new high performance liquid chromatographic method has been developed for the determination of carboxypeptidase N activity which quantitates the furylacryloyl-alanine released by enzymatic cleavage of furylacryloyl-alanyl-lysine or furylacryloyl-alanyl-arginine. A short isocratic gradient elutes the substrate and product in less than 7 min and multiple analyses are facilitated by an automatic sample injector. The microassay readily detects and quantitates carboxypeptidase N activity secreted into culture medium. It was determined that approximately 1 X 10(6) Hep G2 cells at early confluence secreted 1 ng of carboxypeptidase N in 24 h. The microassay will also detect as little as 51 pg of purified carboxypeptidase N or 8 pg of carboxypeptidase B.