High-performance liquid chromatographic-colorimetric assay for glycine carboxypeptidase activity

A rapid and sensitive assay method for the determination of glycine carboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe, enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a TSK gel ODS-80TM reversed-phase column by isocratic elution. This method is sensitive enough to measure 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 7.5 min per sample for separation and quantitation. The pH optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The Km and Vmax values were respectively 21.1 micromol and 3.73 pmol/microg/h with the use of enzyme extract obtained from bovine pituitary. Glycine carboxypeptidase activity was strongly inhibited by Ag+, Cu2+ and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in testis. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.