title: A fluorometric assay for peptidyl alpha-amidation activity using high-performance liquid chromatography.
abstract: A rapid and sensitive method for the determination of peptidyl alpha-amidation activity has been developed and is based on reverse-phase high-performance liquid chromatographic separation and fluorometric detection. A dansylated tripeptide, N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay and the amount of alpha-amidation activity is determined by quantitating the extent of its conversion to product, N-dansyl-Tyr-Val-NH2. Both product and substrate can be detected in a single assay in quantities as low as 5 fmol by isocratic elution using C-18 reverse-phase columns. The method yields highly reproducible results and requires less than 3 min per sample for separation and quantitation. The assay procedure is applicable to the screening of a large number of samples under different pH conditions and is readily adaptable for use in a variety of studies. For example, the procedure is ideal for detecting alpha-amidation activity in various tissues, monitoring activity at the different stages during purification of a particular alpha-amidation enzyme, determining kinetic parameters of the purified enzyme, and identifying both competitive and noncompetitive inhibitors.
authors: Department of Protein Chemistry, Unigene Laboratories, Inc., Fairfield, New Jersey 07006.
authors: B N
authors: BN
authors: Jones
authors: P P
authors: PP
authors: Tamburini
authors: A P
authors: AP
authors: Consalvo
authors: S D
authors: SD
authors: Young
authors: S J
authors: SJ
authors: Lovato
authors: J P
authors: JP
authors: Gilligan
authors: A Y
authors: AY
authors: Jeng
authors: L P
authors: LP
authors: Wennogle
mesh_terms: D000577:Amides / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds / Q000032:analysis; D005470:Fluorometry / Q000379:methods*; D010455:Peptides / Q000032:analysis*
chemical_list: D000577:Amides; D003619:Dansyl Compounds; D010455:Peptides