title: Highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer.
abstract: Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in  Escherichia coli  lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.
authors: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany.
authors: Thomas
authors: T
authors: Zauner
authors: Renate
authors: R
authors: Berger-Hoffmann
authors: Katrin
authors: K
authors: Müller
authors: Ralf
authors: R
authors: Hoffmann
authors: Thole
authors: T
authors: Zuchner
mesh_terms: D004765:Enteropeptidase / Q000032:analysis* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D014357:Trypsin / Q000032:analysis* / Q000378:metabolism
chemical_list: D014357:Trypsin; D004765:Enteropeptidase