Fluorometric rate assay of alpha-amylase using an intramolecularly-quenched fluorescent substrate (FG5P)

The complete hydrolysis of a fluorogenic derivative of rho-nitrophenyl alpha-maltopentaoside, FG5P, by human salivary alpha-amylase, resulted in a 5-fold increase in fluorescence. This is due to disruption of the intramolecular quenching of the fluorescence of the 2-pyridylamino residue by the rho-nitrophenyl residue by separation of the two residues. This change of fluorescence accompanying the cleavage of the glucosidic bond was exploited to develop a fluorometric rate assay of alpha-amylase in human serum.