Rapid fluorimetric assay for the detection of the peptidyl alpha-amidating enzyme intermediate using high-performance liquid chromatography.
Peptidylglycine alpha-amidating enzyme catalyzes the conversion of glycine-extended peptides to their corresponding amidated peptides via a stable alpha-hydroxyglycine intermediate. Using a new rapid fluorimetric reversed-phase high-performance liquid chromatographic assay, we have demonstrated that the substrate and product of the amidation reaction, as well as both stereoisomers of the alpha-hydroxyglycine intermediate, can be separated and detected in quantities as low as 1 pmol. The method is highly reproducible and requires less than 11 min for separation and quantification.
Consalvo
A P
AP
Department of Analytical Protein and Organic Chemistry, Unigene Laboratories, Inc., Fairfield, NJ 07004.
Young
S D
SD
Merkler
D J
DJ
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds; D006899:Mixed Function Oxygenases / Q000032:analysis*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D013050:Spectrometry, Fluorescence
D003619:Dansyl Compounds; D009097:Multienzyme Complexes; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase