Visual detection of peptidase activity using fluorogenic substrates in a microtiter plate assay.

A simple, inexpensive, and sensitive assay for peptidase activity has been devised. The assay was performed in a microtiter plate and was based on fluorogenic peptide substrates, many of which are commercially available. 7-Amino-4-methyl coumarin the fluorescent product liberated during an incubation period of between 1 and 16 h, was detected by inspection of the plate under ultraviolet light of wavelength 356 nm. A fluorometer was not required. Using alpha-chymotrypsin as a model enzyme, with succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-methyl-coumaryl-7-amide as substrate, it was shown that as little as 4 fmol of enzyme could be detected. The method was non-quantitative and was particularly suited to location of enzyme activity in fractions during a purification procedure. The validity of the assay was demonstrated by detection of activity of a known enzyme, alpha-chymotrypsin, after its purification by size-exclusion high-performance liquid chromatography. The method was used to locate two forms of aminopeptidase activity, in fractions from size-exclusion chromatography of an extract from reproductive tissue of Helix aspersa, using L-leucine 4-methyl-coumaryl-7-amide as substrate.