HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC COLORIMETRIC ASSAY FOR GLYCINE CARBOXYPEPTIDASE ACTIVITY

A rapid and sensitive assay method for the determination of glycine ca rboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4 '-sulfonyl-G-L-Phe, enzymatically formed from the substrate dimethylam inoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-perf ormance 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 concentrat ions as low as 1 pmol and yield highly reproducible results and requir es less than 7.5 min per sample for separation and quantitation. The p H optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The K- m and V-max values were respectively 21.1 mu mol and 3.73 pmol/mu g/h with the use of enzyme extract obtained from bovine pituitary: Glycine carboxypeptidase activity was strongly inhibited by Ag+, Cu2+ and p-c hloromercuriphenylsulfonic 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 exam ine the physiological role of this peptidase. (C) 1997 Elsevier Scienc e B.V.