Fluorescence donor-acceptor pair

In 1948 Forster proposed a theory for the resonance energy transfer. He postulated that the rate of transfer depends on the inverse sixth power of the distance between the donor and the acceptor. This predicted distance dependence was verified later by experimental studies of fluorescent donor-acceptor pairs separated by a known distance in defined systems. 

Let us consider tire case of a donor-acceptor pair where tire acceptor, after capturing excitation from tire donor, can emit a photon of fluorescence. If tire excitation light is linearly polarized, tire acceptor emission generally has a different polarization. Common quantitative expressions of tliis effect are tire anisotropy of fluorescence, r, or tire degree of polarization,... 

Proteases are enzymes that break peptide bonds in proteins. As such they lend themselves to a variety of homogeneous assay techniques. Most employ labeling both ends of the substrate with a different tag, and looking for the appearance (disappearance) of the signal generated in the intact substrate (product). As an example, for a fluorescence quench assay, the N-terminal of a peptide is labeled with DNP and the C-terminal with MCA. As such, the peptide is fluorescently silent since the fluorescence from DNP is quenched by absorption by the MCA. Another very popular donor/acceptor pair is EDANS 5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid and DABCYL 4-(4-dimethylaminophenylazo)benzoic acid) (a sulfonyl derivative (DABSYL) [27], Upon peptide cleavage, the two products diffuse, and due to a lack of proximity, the fluorescence increases. 

Sq635-b and Sq660 were also utilized as donor-acceptor pairs in combination with an HSA/anti-HSA system, in a fluorescence energy transfer (FRET)-based immunoassay [95, 96]. 

Schobel, U., Egelhaaf, H. J., Brecht, A., Oelkrug, D. and Gauglitz, G. (1999). New donor-acceptor pair for fluorescent immunoassays by energy transfer. Bioconjug. Chem. 15, 1107-14. 

CFP-YFP donor-acceptor pair, YFP is several times brighter than CFP [62]. Lastly, for studying dynamic protein associations in plants, the presence of chlorophyll pigments in leaf and stem cells is an additional limitation. These pigments directly absorb the fluorescence, which decreases blue fluorescence intensity for BFP and CFP donors that can be erroneously interpreted as reduced donor fluorescence quantum yield caused by FRET [18]. If sensitized emission or FSPIM is the only available method for quantifying FRET, then it is very important to restrict measurements to chlorophyll free areas within the cells. 

Excitation and emission spectra of molecules for donor-acceptor pairs can be found at one of the following Web sites Becton-Dickinson Fluorescence Spectrum Viewer (http //www. bdbiosciences.com/spectra), Invitrogen-Molecular Probes Fluorescence Spectra Viewer (http //www.probes.invitrogen. com/servlets/spectraviewer). 

Weller24 has estimated enthalpies of exciplex formation from the energy separation vg, — i>5 ax of the molecular 0"-0 and exciplex fluorescence maximum using the appropriate form of Eq. (27) with ER assumed to have the value found for pyrene despite the doubtful validity of this approximation the values listed for AHa in Table VI are sufficiently low to permit exciplex dissociation during its radiative lifetime and the total emission spectrum of these systems may be expected to vary with temperature in the manner described above for one-component systems. This has recently been confirmed by Knibbe, Rehm, and Weller30 who obtain the enthalpies and entropies of photoassociation of the donor-acceptor pairs listed in Table XI. From a detailed analysis of the fluorescence decay curves for the perylene-diethyl-aniline system in benzene, Ware and Richter34 find that... 

We note that all the films designed by e,f and g in Fig. 12 are transparent and exhibit no phase structure when examined by TEM, but differ somewhat in the degree of mixing when studied by the fluorescence technique. In fact, it was found that Ic/Ia for film e was sHghtly but definitely larger than those of the others. For the donor-acceptor pair used in this study, the characteristic distance is 2.7-2.9 nm [106], so that composition fluctuation on this scale in the films could be detected by routine TEM observations. The failure to see the phase structure in e, therefore, may be due to a great decrease in composition fluctuation by a strong intercomponent interaction. 

A simplified theory of FRET is sufficient to describe affinity sensors used in fluorescence transduction of glucose concentrations. A key quantity that describes the potential FRET interaction between a donor-acceptor pair is the Forster distance, Ro, the distance at which half the donor molecules are quenched by the acceptor molecules. Ro is proportional to several parameters of the fluorophores, in accordance with Ro = K6 Jx2n 4 cf>DJ l], where K is a constant. The variable k2 refers to the relative spatial orientation of the dipoles of D and A, taking on values from 0 to 4 for completely orthogonal dipoles and collinear and parallel transitional dipoles k2 = 4,... 

M Meldal, K Breddam. Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin. Anal Biochem 195 141-147, 1991. 

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