The synthesis and utilization of 2,4-dinitrophenyl-labeled irreversible peptidyl diazomethyl ketone inhibitors

S.J. Hawthorne, M. Pagano, P. Harriott, D.W. Halton, B. Walker

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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 byFasciola 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 novelF. hepaticacathepsin L-like protease, when assayed fluorimetrically, displaying second-order rate constants (ki/Ki) of 7.93 × 102, 2.16 × 102, and 3.73 × 102M−1s−1, respectively
LanguageEnglish
Pages131-138
Number of pages8
JournalAnalytical Biochemistry
Volume261
Issue number2
DOIs
Publication statusPublished - 1998

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Cathepsin L
Cathepsin B
Peptide Hydrolases
Cysteine Proteases
Rate constants
Peptides
peptidyl diazomethyl ketones
Proteins
diazomethyl ketones

Cite this

Hawthorne, S.J. ; Pagano, M. ; Harriott, P. ; Halton, D.W. ; Walker, B. / The synthesis and utilization of 2,4-dinitrophenyl-labeled irreversible peptidyl diazomethyl ketone inhibitors. 1998 ; Vol. 261, No. 2. pp. 131-138.
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abstract = "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 byFasciola 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 novelF. hepaticacathepsin L-like protease, when assayed fluorimetrically, displaying second-order rate constants (ki/Ki) of 7.93 × 102, 2.16 × 102, and 3.73 × 102M−1s−1, respectively",
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The synthesis and utilization of 2,4-dinitrophenyl-labeled irreversible peptidyl diazomethyl ketone inhibitors. / Hawthorne, S.J.; Pagano, M.; Harriott, P.; Halton, D.W.; Walker, B.

Vol. 261, No. 2, 1998, p. 131-138.

Research output: Contribution to journalArticle

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T1 - The synthesis and utilization of 2,4-dinitrophenyl-labeled irreversible peptidyl diazomethyl ketone inhibitors

AU - Hawthorne, S.J.

AU - Pagano, M.

AU - Harriott, P.

AU - Halton, D.W.

AU - Walker, B.

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N2 - 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 byFasciola 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 novelF. hepaticacathepsin L-like protease, when assayed fluorimetrically, displaying second-order rate constants (ki/Ki) of 7.93 × 102, 2.16 × 102, and 3.73 × 102M−1s−1, respectively

AB - 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 byFasciola 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 novelF. hepaticacathepsin L-like protease, when assayed fluorimetrically, displaying second-order rate constants (ki/Ki) of 7.93 × 102, 2.16 × 102, and 3.73 × 102M−1s−1, respectively

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