Donor quenching

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. 

Absorbance and donor quenching activities by compounds are also frequently cited limitations of the technique (Comley, 2006). An additional disadvantage of the TR-FRET readout is the complexity of the biochemical system with several assay components and their multiple equilibria. These interactions may be disturbed by the compounds actually tested for their potency against the protease and thus lead to false positive results. 

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 1. Number of nai thalene donors quenched per trap n as a function of trap concentration for (O) 1>M1A-2A 9MA, (A) 2VN-PVK, and ( ) 2NMMA-9A WA in 2MeTHF at 77K ( 4 x 10- M in naphthalene). (Reprinted from Ref. 2. Copyright 1985 American Chemical Society.)...

Tian and co-workers have prepared and investigated the properties of flexible dendrons and dendrimers that have NI or PDI cores with carbazole (CZ) or oxadiazole (OXZ) peripheral units (Scheme 38) [103,104]. Excitation of the peripheral chromophores result in emission of the NI of PDI cores. Interestingly, in the case of the NI core dendrons, excitation of the peripheral oxadiazole residues of 75c results in a 3.9 times enhancement of core liuni-nescence, while excitation of the peripheral carbazole residues of 76c results in only a 20% emission intensity, suggesting a second pathway for donor quenching in the CZ systems. This pathway is probably photo-induced electron transfer (PET) from the CZ units to the NI core [104], The OXZ units, however, have a relatively higher electron affinity and no PET can take place. 

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. 

Richtol, H. H., and A. Belorit Simultaneous Donor Quenching and Acceptor Sensitization in Phosphorescence Studies of Triplet Energy Transfer The Biacetyl-Benzil System. J. Chem. Phys. 45, 35 (1966). 

Energy transfer efficiencies are most commonly evaluated by measuring donor quenching by the acceptor according to... 

Donor quenching is the most common method used for detecting energy transfer. Quenching of the donor fluorescence is due to acceptors at different distances as well as orientations, and due to the motions of donor or acceptor. Excitation is set at the wavelength of donor absorption and the emission of the donor is monitored. The emission wavelength of donor is selected such that uo coutrihution from the acceptor fluorescence is observed. 

The use of RET to measure protein association and distance is shown in Figure 1.23 for two monom s which associate to form a dimer. Suppose one monomer contains a tryptophan (trp) residue, and the other a dansyl group. The Forster distance is determined by the spectral overlap of the trp donor emission with the dansyl acceptor absorption. Upon association, RET will occur, which decreases the intensity of the donor emission (Figure 1.23). The extent of donor quenching can be used to calculate the donor-to-acceptor distance in the dimer (Eq. [1.12]). It is also important to notice that RET provides a method to measure protein association because it occurs whenever the donor and acceptor are within the Forster distance. 

The most common application of RET is to measure the distances between two sites on a macromolecule. Typically, a protein is covalently labeled with a donor and an accq>tor (Figure 13.1). The donor is (tften a tryptophan residue, but extrinsic donors are also used. If there is a sin e donor and acceptor, and if the D-A distance does not chan during the excited-state lifetime, then the D-A distance can be determined from the efficiency of energy transfer. The transfer efficiency can be detemuned by steady-state nr asurements of the extent of donor quenching due to the acceptor. 

For a high degree of RET donor quenching (FiWPb 1), a small percentage of unlabeled acceptor can result in a large change in the calculated transfer efficiency (Problem 13.9). 

One may readEfy visualize how energy qu icfaing data could be used to detennine ndiedier the < lributi is of donor and accepter are random. Using the calculmed vahie ofRo> one compares the measured extent of donor quench-... 

F re 21.27. Release of donor quenching during polymeraM chain loactioa. Rwn Ref. 56. 

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