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Thioamide-Based Fluorescent Protease Sensors

[Image: see text] 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 ca...

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Autores principales: Goldberg, Jacob M., Chen, Xing, Meinhardt, Nataline, Greenbaum, Doron C., Petersson, E. James
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985465/
https://www.ncbi.nlm.nih.gov/pubmed/24472041
http://dx.doi.org/10.1021/ja412297x
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author Goldberg, Jacob M.
Chen, Xing
Meinhardt, Nataline
Greenbaum, Doron C.
Petersson, E. James
author_facet Goldberg, Jacob M.
Chen, Xing
Meinhardt, Nataline
Greenbaum, Doron C.
Petersson, E. James
author_sort Goldberg, Jacob M.
collection PubMed
description [Image: see text] 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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spelling pubmed-39854652015-01-28 Thioamide-Based Fluorescent Protease Sensors Goldberg, Jacob M. Chen, Xing Meinhardt, Nataline Greenbaum, Doron C. Petersson, E. James J Am Chem Soc [Image: see text] 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. American Chemical Society 2014-01-28 2014-02-05 /pmc/articles/PMC3985465/ /pubmed/24472041 http://dx.doi.org/10.1021/ja412297x Text en Copyright © 2014 American Chemical Society
spellingShingle Goldberg, Jacob M.
Chen, Xing
Meinhardt, Nataline
Greenbaum, Doron C.
Petersson, E. James
Thioamide-Based Fluorescent Protease Sensors
title Thioamide-Based Fluorescent Protease Sensors
title_full Thioamide-Based Fluorescent Protease Sensors
title_fullStr Thioamide-Based Fluorescent Protease Sensors
title_full_unstemmed Thioamide-Based Fluorescent Protease Sensors
title_short Thioamide-Based Fluorescent Protease Sensors
title_sort thioamide-based fluorescent protease sensors
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985465/
https://www.ncbi.nlm.nih.gov/pubmed/24472041
http://dx.doi.org/10.1021/ja412297x
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