Fluorescence donor quenching

Bimolecular reactions with paramagnetic species, heavy atoms, some molecules, compounds, or quantum dots refer to the first group (1). The second group (2) includes electron transfer reactions, exciplex and excimer formations, and proton transfer. To the last group (3), we ascribe the reactions, in which quenching of fluorescence occurs due to radiative and nonradiative transfer of excitation energy from the fluorescent donor to another particle - energy acceptor. 

Additionally, since the acceptor is excited as a result of FRET, those acceptors that are fluorescent will emit photons (proportional to their quantum efficiency) also when FRET occurs. This is called sensitized emission and can also be a good measure of FRET (see Fig. 1). To quantitate FRET efficiency in practice, several approaches have been evolved so far. In flow cytometric FRET (7), we can obtain cell-averaged statistics for large cell populations, while the subcellular details can be investigated with various microscopic approaches. Jares-Erijman and Jovin have classified 22 different approaches that can be used to quantify energy transfer (8). Most of them are based on donor quenching and/or acceptor sensitization, and a few on measuring emission anisotropy of either the donor or the acceptor. Some of these methods can be combined to extend the information content of the measurement, for example two-sided FRET (9) involves both acceptor depletion (10) and... 

Indirect evidence for the formation of a complex intermediate in diffusional energy transfer processes may be provided by the measurement of kt from both donor quenching and acceptor sensitization at low temperatures where kQC kf = 1/T° in this case exciplex relaxation should reduce the quencher sensitization efficiency but leave the donor fluorescence quenching constant unchanged. 

In the substrate, the fluorescence of the donor is quenched by the quenching label in close proximity. Upon cleavage of the substrate by the protease, this proximity is lost and a strong increase in fluorescence can be recorded. Table 2.3 summarizes a selection of fluorescence donor and acceptor pairs frequently used for protease activity assays based on a FRET quench readout. In principle, the FRET quench effect can be achieved by labeling the substrate with two different dyes with overlapping spectra (hetero-double labeling). 

Examples of Fluorescence Donor and Acceptor Pairs Frequently Used for Protease Assays Based on FRET Quench Readout Principle... 

Abz was combined with a broad variety of non-fluorescent acceptors such as p-nitrobenzyl for leucine aminopeptidase (Carmel et al., 1977), pNA for trypsin (Bratanova and Petkov, 1987), 4-ni-trophenylalanine [Phe(NC>2)] for HIV protease (Toth and Marshall, 1990), and V-(2,4-di n itrophenyl) ethylenediamine (EDDnp) for thermolysin and trypsin (Nishino et al., 1992). Lecaille et al. (2003) described a FRET quench assay based on a specific substrate for cathepsin K labeled with Abz and EDDnp. This substrate is not cleaved by the other Cl cysteine cathepsins and serine proteases in contrast to methoxycoumarin (Mca)-based substrates described earlier (Aibe et al., 1996 Xia et al., 1999) and merely covered the non-primed site of the scissile bond. The 5-[(2-aminoethyl)amino] naphthalene-l-sulfonic acid (EDANS) compound is a second example of a fluorescence donor historically used for many FRET quench-based protease assays, e.g., in combination with tryptophan as a quencher in an ECE activity assay (Von Geldren et al., 1991). The FRET-1 example in Table 2.2 shows the typical dynamic range that can be achieved with an EDANS/DABCYL-based assay. 

In general, a FRET quench readout is simple. A broad range of available fluorescence donors and acceptors allows cost-efficient operations in an industrialized HTS and automated compound profiling environment. On the other hand, the readout can suffer from inner filter effects due to high absorption coefficients of the dyes and fluorescence artifacts by the tested compounds, resulting in enhanced false positive and false negative rates. Moreover, the readout is limited to substrates in which short distances between donor and acceptor dye can be realized without disturbing the interaction of enzyme and substrate. The flexibility of the peptide conformation makes the prediction of the effective distance between the dyes and consequently the prediction of the FRET effect difficult. The distance between donor and acceptor cannot be easily approximated by the mean hydrodynamic radii of the dyes. 

FRET-based readouts are the most common methods applied for endopeptidase activity assays. In general, the FRET readout is simple, numerous assay examples are published, many fluorescently labeled substrates are commercially available, and labeling procedures are well established. Furthermore, broad range of fluorescence donors and acceptors is available. FRET quench substrates allow fast and cost-efficient operations in an industrialized HTS environment. However, in many cases, the assays suffer from fluorescence artifacts from the tested compounds. This can lead to falsepositive and false-negative results because the most frequently used FRET quench dyes are excited in the ultraviolet range. 

Fig. 28 Photoinduced electron transfer studies carried out on 6-mer (not shown) and 7-mer hairpin DNA duplexes capped by a stilbene acceptor chromophore.31 167 168 The duplexes contain A-T base pairs and a single G-C base pair, whose position in the duplex is varied, (a) The photoinduced ET process is illustrated for 3G C, in which the G-C base pair is third removed from the stilbene (St) group. The stilbene fluorescence is quenched by electron transfer from the G donor, (b) Damping factors for charge separation, /3(CS), and subsequent charge recombination, /3(CR), for n G C, in which the G base is connected to the T-bearing strand, (c) Damping factors, /3(CS) and /3(CR), for n C G, in which the G base is connected to the A-bearing strand.

Relatively little has been done with C70. However, Verhoeven et al. showed that electron donors quench its fluorescence, and there is evidence for formation... 

Figure 7 compares the enhancement obtained with four donors under the same conditions. Also shown is the effect of these donors on cyanine dye aggregate fluorescence in the absence of acceptor in the monolayer assembly. Several of the donors quench this fluorescence, presumably because they can photoreduce the excited dye [reaction (4)]. Qualitatively the extent of radical yield enhancement or supersensitization correlates with the extent of cyanine fluorescence quenching by the donors. This raises the possibility that reactions (4) and (5) are involved in the supersensitization. However, even the donor which causes virtually no fluorescence quenching still results in some supersensitization. Thus, some other mechanism such as reaction (3) is probably also involved. 

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