A fluorometric assay for peptidyl alpha-amidation activity using high-performance liquid chromatography.
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.
Jones
B N
BN
Department of Protein Chemistry, Unigene Laboratories, Inc., Fairfield, New Jersey 07006.
Tamburini
P P
PP
Consalvo
A P
AP
Young
S D
SD
Lovato
S J
SJ
Gilligan
J P
JP
Jeng
A Y
AY
Wennogle
L P
LP
D000577:Amides / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds / Q000032:analysis; D005470:Fluorometry / Q000379:methods*; D010455:Peptides / Q000032:analysis*
D000577:Amides; D003619:Dansyl Compounds; D010455:Peptides