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Fluorescence Resonance Energy Transfer is a method that allows the detection of a ‘distance-dependent’ interaction between the excited states of two distinct, dye-linked, molecules, i.e. the ‘fluorophore’ and the ‘quencher’. Quenched fluorescent peptides (‘FRET peptides’) are widely used as suitable substrates in enzyme studies.
Their synthesis requires that the peptide is conjugated with both a fluorophore and a quencher dye. ‘Fluorescence/quencher’ pairs do require distinct overlap between the fluorescence emission spectrum of the fluorophore and the UV-absorbance spectrum of the quencher. So, when the fluorophore and quencher are conjugated to one and the same peptide, with a limited distance, the quencher efficiently blocks the emission of the fluorophore. However, when a peptide bond is cleaved, e.g. by enzymatic degradation, the distance between fluorophore and quencher is suddently increased significantly, and as a result of this the fluorophore is activated. The fluorescence signal can be detected continuously, allowing quantification of the enzyme activity.
Frequently used fluorophore/quencher combinations are:
|Fluorophore||Quencher||Excitation Wavelenghs||Emission Wavelenghs|
|320 nm||420 nm|
5-[(2-Aminoethyl) amino] naphthalene-1-sulfonic acid
|340 nm||490 nm|
|325 nm||392 nm|
|280 nm||360 nm|
|492 nm||517 nm|