AI Article Synopsis
- Thioamide quenchers can be effectively combined with small fluorescent markers to create "turn-on" substrates for studying proteases.
- This method has been successfully applied to analyze various types of proteases, enabling real-time monitoring of their activity even in complex samples.
- Three key applications highlighted include: studying papain enzyme kinetics, targeting specific cleavage sites in peptides, and evaluating the specificity of a calpain inhibitor in cell samples.
Article Abstract
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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Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985465 | PMC |
| http://dx.doi.org/10.1021/ja412297x | DOI Listing |
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