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Exopeptidase-assay; Self-quenching substrates; Single-molecule imaging
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.
