A fluorescence-based assay for exopeptidases using self-quenching peptide probes with single-molecule sensitivity

Abstract

In recent years, the interest in assaying exopeptidases has become increasingly important because they are significantly involved in many diseases like cancer. To date, no generally applicable fluorescence-based detection method has been developed because commercially available doubly-labeled substrates are not always digested by exopeptidases. In this article we present a new method for the sensitive detection of exopeptidases based on fluorescently-labeled substrates containing only one fluorophore that is efficiently quenched by an adjacent tryptophan residue via photoinduced electron transfer. Because of their well-known properties we chose carboxypeptidase A (CPA) as a model system. The self-quenching probes were used in homogeneous solution as well as on cross-linked PEG-coated surfaces in combination with single-molecule imaging techniques. However, even with standard fluorescence spectrometers we achieved sensitivity below the picomolar range.

Keywords:

• Exopeptidase-assay
• Self-quenching substrates
• Single-molecule imaging

Acknowledgements

The authors would like to thank Prof. J. Wolfrum and Prof. M. Sauer for their support and stimulating discussions. Furthermore, Prof. Dr K.H. Drexhage (Universität-Gesamthochschule Siegen) is acknowledged for providing the oxazine derivative MR121. This work was supported by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Grants 311864 and 13N8349).