Fluorogenic Peptide Substrates for Assay of Aspartyl Proteinases

Via a combination of chemical and enzymatic synthesis, new hexapeptide substrates convenient for use in activity assessment of several aspartyl proteinases—porcine pepsin, human pepsin, gastricsin, and cathepsin D—were prepared. These peptide derivatives,o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala-p-nitroanilide andN-(o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala)-N′-2,4-dinitrophenyl ethylenediamine, contain a fluorescento-aminobenzoyl moiety as well asp-nitroaniline orN-2,4-dinitrophenyl ethylenediamine—the groups that cause fluorescence quenching. Aspartyl proteinases hydrolyze the Phe-Phe peptide bond in the substrates, which diminishes quenching due to separation of the fluorescent and quenching moieties and leads to an increase in the fluorescence intensity ofo-aminobenzoyl residue. Abz-Ala-Ala-Phe-Phe-Ala-Ala-Ded, being fairly well hydrolyzed by HIV proteinase, might be used for assay of this enzyme.