Thioamide-Based Fluorescent Protease Sensors

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• 作者：Jacob M. Goldberg ; Xing Chen ; Nataline Meinhardt ; Doron C. Greenbaum ; E. James Petersson
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：February 5, 2014
• 年：2014
• 卷：136
• 期：5
• 页码：2086-2093
• 全文大小：343K
• ISSN：1520-5126

文摘

Thioamide quenchers can be paired with compact fluorophores to design 鈥渢urn-on鈥?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.