Fret Substrates

August 26, 2021

Fret Substrates offered by Bachem

Fluorescence Resonance Energy Transfer (FRET) is the non-radiative transfer of energy from an excited fluorophore (or donor) to a suitable quencher (or acceptor) molecule. FRET is used in a variety of applications including the measurement of protease activity with substrates, in which the fluorophore is separated from the quencher by a short peptide sequence containing the enzyme cleavage site. Proteolysis of the peptide results in fluorescence as the fluorophore and quencher are separated. In this brochure we present a range of highly sensitive FRET protease substrates for a variety of enzymes.


Fluorophores are substances which, like chromophores, absorb light in the UV or visible range. In contrast to chromophores they re-emit part of the light as radiation. This process is called fluorescence and is illustrated by the Jablonski energy level diagram (Fig 1). Absorption of light (hνA) causes an electron to be promoted from its electronic ground state (designated as S0) to an excited state (usually S1).

Every energy state has several vibrational energy levels 0, 1, 2 etc. During the lifetime of the excited state, i.e. the time elapsed between excitation of the molecule and emission of the photon (usually between 1-10 ns), part of the energy is lost by internal vibration. As a result, the wavelength of the emitted light (hνF) is always longer than that of the exciting light.

This phenomenon is called the Stokes shift and allows the detection of emission against a background of light derived from excitation. Usually, the fluorescence excitation spectrum of a fluorophore in a diluted solution is identical to its absorption spectrum and under the same conditions, the fluorescence emission spectrum is independent of the excitation wavelength.

In a diluted solution, fluorescence intensity is linearly proportional to several parameters as deduced from Lambert-Beer’s law. These are the molar absorption coefficient, the path length, the intensity of the incident light, and the quantum yield which is the ratio of the number of emitted to the total number of absorbed photons. Fluorescence detection is dependent on the sensitivity of the instrument and is therefore measured in arbitrary units.

Higher concentrations of the fluorophore (> 0.1 absorption units) lead to deviations from the linearity due to loss of excitation intensity across the cuvette path length as the excitation light is absorbed by the fluorophore. This phenomenon is known as the inner filter effect. Other effects which influence fluorescence measurements are related to intrinsic or background fluorescence originating from sample preparations and buffer contaminants, respectively. To minimize fluorescence derived from contaminants, it is recommended to use materials of maximum purity.

Fluorescence spectra may also be dependent on the solvent. With some fluorophores, such as 2-acetylanthracene or tryptophan, a spectral shift to longer wavelengths (bathochromic shift or red shift) is observed in more polar solvents. The fluorescence spectra of fluorophores containing acidic or basic substituents (e.g. AMC) can depend on the pH of the solution.

Fig.1. Energy Level Diagram

Fluorescence Quenching

Any process which decreases the fluorescence intensity of a given substance can be referred to as quenching. Several types of quenching processes can be distinguished. Collisional or dynamic quenching can be considered as a reduction in fluorescence intensity due to a collision of the quencher with the fluorophore in the excited state. Upon contact the fluorophore returns to the ground state without light emission. One of the best known collisional quenchers which quenches almost all known fluorophores is molecular oxygen. It is therefore often required to remove dissolved oxygen to obtain reliable measurements.
In static quenching, a non-fluorescent complex is formed between the quencher and the fluorophore. In contrast to both of these quenching processes, FRET does not require contact of the quencher with the fluorophore. The energy transfer occurs without the appearance of a photon.

Fluorescence Resonance Energy Transfer (FRET)

Fluorescence resonance energy transfer (FRET) is the transfer of the excited state energy of a donor to an acceptor without the emission of light (Fig 2). The energy transfer can be considered as an energy exchange of an oscillating dipole to a dipole with similar resonance frequency. FRET can only take place when the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor.

The donor and acceptor have to be within a distance of 1-10 nm. The energy transfer efficiency depends on the extent of the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the relative orientation of the donor and acceptor transition dipoles, and the distance r between donor and acceptor. The energy transfer efficiency decreases exponentially by r6. The distance at which the efficiency of energy transfer is reduced by 50 % is a characteristic value for a given donor acceptor pair and is called the Förster distance R0.

Fig. 2. Fluorescence Resonance Energy Transfer (FRET)

Abz (2-Aminobenzoyl or Anthraniloyl) Substrates

Abz (F) substrates are generally used in combination with a number of quenchers (Q) such as Dnp (2,4-dinitrophenyl), EDDnp (N-(2,4 dinitrophenyl)ethylenediamine), 4-nitro-phenylalanine, or 3-nitro-tyrosine.

Substrate cleavage can be detected at 420 nm using an excitation wavelength of 320 nm.

Example: 4043877 Abz-Phe-Arg-Lys(Dnp)-Pro-OH

N-Me-Abz (N-Methyl-anthraniloyl) Substrates

N-Me-Abz substrates are generally used with Dnp as quencher (Q). The fluorescent group (F) is either linked to the N-terminal amino group or the ε-amino group of a lysine residue. Substrate cleavage can be detected at 440-450 nm using an excitation wavelength of 340- 360 nm.

Example: N-Me-Abz-Lys-Pro-Leu-Gly-Leu- Dap(Dnp)-Ala-Arg-NH2

Dansyl (5-(Dimethylamino)naphthalene-
1-sulfonyl) Substrates

In a few substrates the fluorescent dansyl group (F) serves as donor with 4-nitro-phenylalanine
as acceptor. Substrate cleavage can be assayed at 562 nm using excitation at 342 nm. More commonly the dansyl group is used as a quencher for tryptophan

Example: 4050412 Dansyl-D-Ala-Gly-4-nitro-Phe-Gly-OH

DMACA (7-Dimethylaminocoumarin-
4-acetyl) Substrates

DMACA (F) can be detected fluorometrically at 465 nm using an excitation wavelength of 350 nm. It can be quenched by NBD (7-Nitro-benzo[2,1,3]oxadiazol-4-yl) (Q).

Example: 4028275 NBD-ε-aminocaproyl- Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp- Lys(DMACA)-NH2

EDANS (5-[(2-Aminoethyl)amino]naphthalene-
1-sulfonic acid) Substrates

In these substrates, the fluorescence of the EDANS group (F) is generally quenched by the DABCYL (4-(4 dimethylaminophenylazo) benzoyl) group (Q). The DABCYL group is usually conjugated to the N-terminus and the EDANS group attached to the C-terminus of the peptide substrate. Substrate cleavage can be detected at 490 nm using an excitation wavelength of 340 nm.

Example: DABCYL-Tyr-Val-Ala Asp-Ala-Pro- Val-EDANS

FITC (Fluorescein isothiocyanate) Substrates

Only few FITC substrates have been described. The FITC label (F) can be quenched with Dnp (Q). Substrate cleavage can be detected at 520 nm using an excitation wavelength of 490 nm.

Example: 4027937 FITC-Tyr-Val-Ala-Asp-Ala-Pro-Lys(Dnp)-OH (contains FITC isomer I)

Lucifer Yellow (6-Amino-2,3-dihydro-1,3-dioxo-2-hydrazinocarbonylamino 1Hbenz[d,e]isoquinoline-5,8-disulfonic acid) Substrates

Lucifer Yellow (F) can be detected at 520 nm using excitation at 430 nm. It is efficiently quenched by Dabsyl (4-(4-Dimethylaminophenylazo)-benzenesulfonyl) (Q).

Example: H-Lys(Dabsyl)-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Gln-Lucifer Yellow

Mca ((7-Methoxycoumarin-4-yl)acetyl) Substrates

In this kind of substrates Mca (F) is bound to an amino group (usually the N-terminal amino group) of a peptide sequence and quenched by Dnp (Q). The cleaved peptide fragment with the attached Mca group can be detected fluorometrically at 392 nm using an excitation wavelength of 325 nm.

Example: Mca-Leu-Glu-Val-Asp-Gly-Trp-Lys(Dnp)-NH₂

Trp (Tryptophan) Substrates

Tryptophan (F) is a fluorescent amino acid which has been used in a variety of substrates with Dnp as a quencher (Q). Substrate cleavage can be detected at 360 nm using an excitation wavelength of 280 nm.

Example: 4030541 Dnp-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser-OH

Table 1. Fluorophores

FluorophoreExcitation Wavelength*Emission Wavelength*References
(2-Aminobenzoyl or Anthraniloyl)
320 nm420 nmCezari, M.H. et al. (2002); Bourgeois, L. et al.
(1997); Parameswaran, K.N. et al. (1997)
340-360 nm440-450 nmBickett, D.M. et al. (1993)
342 nm562 nmFlorentin, D. et al. (1984)
350 nm465 nmBickett, D.M. et al. (1994)
(5-[(2-Aminoethyl)amino]naphthalene-1-sulfonic acid)
340 nm490 nmMatayoshi, E.D. et al. (1990)
(Fluorescein isothiocyanate)
490 nm520 nmChersi, A. et al. (1990)
Lucifer Yellow
(6-Amino-2,3-dihydro-1,3-dioxo-2-hydrazinocarbonylamino- 1H-benz[d,e]isoquinoline-5,8-disulfonic acid)
430 nm520 nmGrüninger-Leitch, F. et al. (2002)
325 nm392 nmKondo, T. et al. (1997)
280 nm360 nmCezari, M.H. et al. (2002)

* the values listed are as reported in the cited literature.

Table 2. Donor/Acceptor Pairs

Donor (Fluorophore) Acceptor (Quencher) References
(2-Aminobenzoyl or Anthraniloyl)
Cezari, M.H. et al. (2002)
(2-Aminobenzoyl or Anthraniloyl)
Andrau, D. et al. (2003)
(2-Aminobenzoyl or Anthraniloyl)
Toth, M.V. and G.R. Marshall (1990)
(2-Aminobenzoyl or Anthraniloyl)
Breddam, K. and M. Meldal (1992)
(2-Aminobenzoyl or Anthraniloyl)
Stöckel, A. et al. (1997)
Bickett, D.M. et al. (1993)
Florentin, D. et al. (1984)
Matayoshi, E.D. et al. (1990)
Bickett, D.M. et al. (1994)
(Fluorescein isothiocyanate)
Korting, H.J. et al. (1977)
Lucifer Yellow
Grüninger-Leitch, F. et al. (2002)
Kondo, T. et al. (1997)
Cezari, M.H. et al. (2002)
Persson, A. and E.B. Wilson (1977)

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H.J. Korting et al.
Fluorometric determination of the quality of FITC conjugates.
Virologie 28, 41-43 (1977)

A. Persson and E.B. Wilson
A fluorogenic substrate for angiotensin- converting enzyme.
Anal. Biochem. 83, 296-303 (1977)

D. Florentin et al.
A highly sensitive fluorometric assay for “enkephalinase”, a neutral metalloendopeptidase that releases tyrosine-glycine-glycine from enkephalins.
Anal. Biochem. 141, 62-69 (1984)

A. Chersi et al.
Preparation and utilization of fluorescent synthetic peptides.
Biochim. Biophys. Acta 1034, 333-336 (1990)

E.D. Matayoshi et al.
Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer.
Science 247, 954-958 (1990)

M.V. Toth and G.R. Marshall
A simple, continuous fluorometric assay for HIV protease.
Int. J. Pept. Protein Res. 36, 544-550 (1990)

K. Breddam and M. Meldal
Substrate preferences of glutamicacid-specific endopeptidases assessed by synthetic peptide substrates based on intramolecular fluorescence quenching.
Eur. J. Biochem. 206, 103-107 (1992)

D.M. Bickett et al.
A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9).
Anal. Biochem. 212, 58-64 (1993)

D.M. Bickett et al.
A high throughput fluorogenic substrate for stromelysin (MMP-3).
Ann. N.Y. Acad. Sci. 732, 351-355 (1994)

L. Bourgeois et al.
Serpin-derived peptide substrates for investigating the substrate specificity of human tissue kallikreins hK1 and hK2.
J. Biol. Chem. 272, 29590-29595 (1997)

T. Kondo et al.
Activation of distinct caspase-like proteases by Fas and reaper in Drosophila cells.
Proc. Natl. Acad. Sci. U.S.A. 94, 11951-11956 (1997)

K.N. Parameswaran et al.
Hydrolysis of gamma:epsilon isopeptides by cytosolic transglutaminases and by coagulation factor XIIIa.
J. Biol. Chem. 272, 10311-10317 (1997)

A. Stöckel et al.
Specific inhibitors of aminopeptidase P. Peptides and pseudopeptides of 2-hydroxy-3-amino acids.
Adv. Exp. Med. Biol. 421, 31-35 (1997)

M.H. Cezari et al.
Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides.
Biochem. J. 368, 365-369 (2002)

F. Grüninger-Leitch et al.
Substrate and inhibitor profile of BACE (beta-secretase) and comparison with other mammalian aspartic proteases.
J. Biol. Chem. 277, 4687-4693 (2002)

D. Andrau et al.
BACE1- and BACE2-expressing human cells: characterization of beta-amyloid precursor protein-derived catabolites, design of a novel fluorimetric assay, and identification of new in vitro inhibitors.
J. Biol. Chem. 278, 25859-25866 (2003)

For further details, please see the following literature references

J. Bergmeyer and M. Grassl, eds.
Methods of Enzymatic Analysis, 3rd Edition, Vol. I, Fundamentals
Verlag Chemie GmbH, Weinheim (1983)

J. Bergmeyer and M. Grassl, eds.
Methods of Enzymatic Analysis, 3rd Edition, Vol. II, Samples, Reagents, Assessment of Results
Verlag Chemie GmbH, Weinheim (1983)

J.R. Lakowicz
Principles of Fluorescence Spectroscopy, 3rd Edition
Springer, New York (2006)

A.K. Carmona et al.
The use of Fluorescence Resonance Energy Transfer (FRET) peptides for measurement of clinically important proteolytic enzymes.
Anais da Academia Brasileira de Ciências (Annals of the Brazilian Academy of Sciences) 81, 381-392(2009)