Biotinylated diazomethyl ketones have been much used to detect cysteine proteases on Western blots, but high background may occur due to the presence of endogenously biotinylated proteins. In order to overcome this problem, we have examined the potential utility of a peptidyl diazomethyl ketone labeled with a 2,4-dinitrophenyl (DNP) group as a potential inhibitor and disclosing agent of cathepsin B/L-like proteases. This DNP-labeled peptide, DNP-Ahx-Gly-Phe-Ala-CHN2, was used to detect a 30-kDa cathepsin L-like protease, produced by Fasciola hepatica, on Western blots with high sensitivity and relatively low background staining. The DNP-labeled diazomethyl ketone was also found to be a good inhibitor for bovine cathepsin B, human cathepsin L, and a novel F. hepatica cathepsin L-like protease, when assayed fluorimetrically, displaying second-order rate constants (ki/Ki) of 7.93 x 10(2), 2.16 x 10(2), and 3.73 x 10(2) M-1 s-1, respectively.
Copyright 1998 Academic Press.