Thioamide-based fluorescent protease sensors.
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.
Department of Chemistry, University of Pennsylvania , 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States.
Jacob M
JM
Goldberg
Xing
X
Chen
Nataline
N
Meinhardt
Doron C
DC
Greenbaum
E James
EJ
Petersson
D000818:Animals; D002478:Cells, Cultured; D057075:Enzyme Assays; D005347:Fibroblasts / Q000201:enzymology; D005456:Fluorescent Dyes / Q000737:chemistry*; D051379:Mice; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013854:Thioamides / Q000737:chemistry*
D005456:Fluorescent Dyes; D010455:Peptides; D013854:Thioamides; D010447:Peptide Hydrolases