Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 136, Issue 5, Pages 2086-2093Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja412297x
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Funding
- NIAID NIH HHS [AI09727301] Funding Source: Medline
- NIGMS NIH HHS [T32 GM007133, T32 GM07133] Funding Source: Medline
- NINDS NIH HHS [NS081033, R01 NS081033] Funding Source: Medline
- Direct For Mathematical & Physical Scien
- Division Of Chemistry [1337449] Funding Source: National Science Foundation
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Thioamide quenchers can be paired with compact fluorophores to design turn-on fluorescent protease substrates. We have used this method to study a variety of serine-, cysteine-, carboxyl-, and metallo-proteases, including trypsin, chymotrypsin, pepsin, thermolysin, papain, and calpain. Since thioamides quench some fluorophores red-shifted from those naturally occurring in proteins, this technique can be used for real time monitoring of protease activity in crude preparations of virtually any protease. We demonstrate the value of this method in three model applications: (1) characterization of papain enzyme kinetics using rapid-mixing experiments, (2) selective monitoring of cleavage at a single site in a peptide with multiple proteolytic sites, and (3) analysis of the specificity of an inhibitor of calpain in cell lysates.
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