Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates

Sensitive fluorogenic substrates for trypsin, chymotrypsin, and elastase were prepared. These substrates are amides of an acyl amino acid or peptide with 7-amino-4-methylcoumarin (AMC). The substrates with their respective K cat K m ratios given in parentheses are: for chymotrypsin, glutaryl-Phe-AMC (78) and Ala-Ala-Phe-AMC · TFA (1660); for trypsin, benzoyl- dl-Arg-AMC (800) and Carbobenzoxy (Cbz)- l-Arg-AMC (5300); for elastase, N-acetyl-Ala-Ala-Pro-Ala-AMC (15,000). The detection limits obtained by using the best substrates and short incubation times are: chymotrypsin, 25 ng; trypsin, 5 ng; and elastase, 2 ng.