A COLORIMETRIC ASSAY FOR MEASURING PEPTIDYLGLYCINE ALPHA-AMIDATING MONOOXYGENASE USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

In many peptide hormones and neuropeptides, the carboxy-terminal α-amide structure is essential in eliciting biological activity. In the present study, a rapid and sensitive assay method for the determination of peptidylglycine α-amidating monooxygenase (PAM) 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-NH2 (Dabsyl-Gly-Phe-NH2), enzymatically formed from the substrate 4-dimethylaminoazobenzene-4′-sulfonyl-Gly-l-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This method is sensitive enough to measure Dabsyl-Gly-Phe-NH2 at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 5 min per sample for separation and quantitation. The concentrations of copper and ascorbic acid required for maximal enzyme activity were 1 μm and 2 mm, respectively. The pH optimum for PAM activity was 5.0 to 5.5. The Km and Vmax values were respectively 3.5 μm and 100 pmol/μg/h with the use of enzyme extract obtained from bovine pituitary. By using this method, PAM activity could be readily detected in a single rat saliva. The sensitivity of this assay method will also aid in the effort to examine the regulation of in vivo PAM activity. © 1991.