Fluorescence-quenched peptide substrates have been used to quantitate various enzyme activities. Fluorescence resonance energy transfer (FRET) occurs when the excitation energy is transferred from an excited fluorescent donor to a quenching acceptor in a distance-dependent manner. Cleavage of a scissile peptide bond within a fluorescence quenched substrate leads to the separation of the intramolecular donor-acceptor pair, thus allowing the increase of the fluorescence. The fluorescence increase is proportional to the amount of peptide hydrolyzed.
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