A colorimetric assay for measuring peptidylglycine α-amidating monooxygenase using high-performance liquid chromatography

Abstract

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-NH 2 (Dabsyl-Gly-Phe-NH 2), 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-NH 2 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 μ m and 2 m m, respectively. The pH optimum for PAM activity was 5.0 to 5.5. The K m and V max values were respectively 3.5 μ m 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.