Luminescence resonance energy transfer
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Fluorescence resonance energy transfer (FRET), in which a fluorescent donor molecule transfers energy via a nonradiative dipole-dipole interaction to an acceptor molecule (which is usually a fluorescent molecule), is a standard spectroscopic technique for measuring distances in the 10-70 Angstrom range. We have used a luminescent europium chelate as donor and an organic dye, CY-5, as acceptor. This luminescence resonance energy transfer (LRET) has several advantages over the more conventional FRET. The distance at which 50% of the energy is transferred (R[sub 0]) is large, 70 [angstrom]; the donor lifetime is single exponential and long (0.63 ms in H[sub 2]O; 2.5 ms in D[sub 2]O), making lifetime measurements facile and highly accurate; the orientation dependence (k[sup 2]) of energy transfer is minimized by the donor's multiple electronic transitions and long lifetime, limiting uncertainty in the measured distance due to orientation effects to [+-]12% in the worst case; the sensitized emission of the acceptor can be measured with little or no interfering background, yielding a >50-fold improvement in signal to background over standard donor-acceptor pairs and enabling distances several times R[sub 0] to be measured. 13 refs., 4 figs.