Quenched fluorescent peptides
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 wavelength | Emission wavelength |
---|---|---|---|
Abz 2-Aminobenzoyl |
Dnp 2,4-dinitrophenyl |
320 nm | 420 nm |
EDANS 5-[(2-Aminoethyl)amino] naphthalene-1-sulfonic acid |
Dabcyl 4-([4′-dimethylamino)phenyl] azo)benzoyl |
340 nm | 490 nm |
Mca 7-Methoxycoumarin-4-yl)acetyl |
Dnp 2,4-dinitrophenyl |
325 nm | 392 nm |
Trp Tryptophan |
Dnp 2,4-dinitrophenyl |
280 nm | 360 nm |
FAM Carboxyfluorescein |
Dabcyl 4-([4′-dimethylamino)phenyl] azo)benzoyl |
492 nm | 517 nm |