A fluorometric assay for peptidyl α-amidation activity using high-performance liquid chromatography

✍ Scribed by Barry N. Jones; Paul P. Tamburini; Angelo P. Consalvo; Stanley D. Young; Susan J. Lovato; James P. Gilligan; Arco Y. Jeng; Lawrence P. Wennogle

• Publisher: Elsevier Science
• Year: 1988
• Tongue: English
• Weight: 663 KB
• Volume: 168
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(88)90318-1

✦ Synopsis

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.