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  • Title: A fluorometric assay for peptidyl alpha-amidation activity using high-performance liquid chromatography.
    Author: Jones BN, Tamburini PP, Consalvo AP, Young SD, Lovato SJ, Gilligan JP, Jeng AY, Wennogle LP.
    Journal: Anal Biochem; 1988 Feb 01; 168(2):272-9. PubMed ID: 3364727.
    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.
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