FRET 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 Wavelenghs

Emission Wavelenghs

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


EDANS – Dabcyl


Mca – Dnp


Tryptophan – Dnp




FAM Dabcyl