Sensitive enzyme assays based on the production of chemiluminescent leaving groups

Abstract A new type of synthetic peptide substrate for amidase assay has been devised. The substrates are luminogenic, with potential for extremely high sensitivity, and are here exemplified by Boc- and Z -Ala-Ala-Phe-isoluminol amide. The synthetic substrates were designed to release isoluminol when hydrolyzed by enzyme; isoluminol production was determined by measuring its chemiluminescence. Kinetic constants of the luminogenic substrates were measured with α-chymotrypsin; and levels of the enzyme as low as 50 ng were determined conveniently. A comparison of similar luminogenic, chromogenic, and fluorogenic substrates is presented.