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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2014
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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. |
format | Online Article Text |
id | pubmed-3985465 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT goldbergjacobm thioamidebasedfluorescentproteasesensors AT chenxing thioamidebasedfluorescentproteasesensors AT meinhardtnataline thioamidebasedfluorescentproteasesensors AT greenbaumdoronc thioamidebasedfluorescentproteasesensors AT peterssonejames thioamidebasedfluorescentproteasesensors |