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

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Spatz,  Joachim P.
Cellular Biophysics, Max Planck Institute for Medical Research, Max Planck Society;
Biophysical Chemistry, Institute of Physical Chemistry, University of Heidelberg, 69120 Heidelberg, Germany;

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Citation

Marmé, N., Weston, K. D., Staudt, T., Spatz, J. P., & Knemeyer, J.-P. (2005). A fluorescence-based assay for exopeptidases using self-quenching peptide probes with single-molecule sensitivity. International Journal of Environmental Analytical Chemistry, 85(9-11), 741-751. doi:10.1080/03067310500147720.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0010-27AB-D
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.