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
Abz – Dnp

Abz – Dnp

EDANS – Dabcyl

EDANS – Dabcyl

Mca – Dnp

Mca – Dnp

Tryptophan – Dnp

Tryptophan – Dnp

FAM – Dabcyl

FAM – Dabcyl