The synthesis and utilization of 2,4-dinitrophenyl-labeled irreversible peptidyl diazomethyl ketone inhibitors.
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.
Hawthorne
S J
SJ
Medical Biology Centre, The Queen's University of Belfast, 97 Lisburn Road, Belfast, BT9 7BL, United Kingdom.
Pagano
M
M
Harriott
P
P
Halton
D W
DW
Walker
B
B
D019297:2,4-Dinitrophenol / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002852:Chromatography, Ion Exchange; D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry; D005210:Fasciola hepatica / Q000201:enzymology; D006801:Humans; D007659:Ketones / Q000138:chemical synthesis* / Q000737:chemistry; D007700:Kinetics; D013050:Spectrometry, Fluorescence
D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors; D007659:Ketones; D002403:Cathepsins; D019297:2,4-Dinitrophenol