New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain.

A series of new substrates for determining the catalytic activity of cysteine proteinases is described. The rate of hydrolysis by papain was monitored by a fluorescence continuous assay based on internal resonance energy transfer using 5-[(2-aminoethyl)amino]naphtalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) as fluorescent donor and quenching acceptor, respectively, in peptides with the general structure: DABCYL-Lys-Phe-Gly-Xxx-Ala-Ala-EDANS. The substrates were used to evaluate the effect of amino acid structure in the S1' position on the kinetic parameters for papain catalyzed hydrolysis.