[13] Fluorescence Resonance Energy Transfer

of the extent of binding. This effect (known informally as "Chip dip") is illustrated in Figs. 6 and 7 which concern the ethidium bromide/tRNA system. 41 In this case the free ethidium bromide has a lifetime of 1.86 nsec and a rotational relaxation time of 0.54 nsec. On binding the PhetRNA Phe the lifetime increases to 26 nsec and the rotational relaxation increases to 136 nsec. A single rotating species cannot give rise to a negative value for the differential phase delay (assuming excitation at a wavelength which gives a positive P0), and the negative excursion for this function shown in Fig. 6 is due to the presence of free ethidium bromide in equilibrium with bound material. Figure 7 shows the calculated effects for increasing amounts of bound material. We should note that, given the difference in quantum yield between the free and bound probe, the fractional intensities utilized in Fig. 7 actually represent small percentages of bound probe on a molar basis. In fact, considering the accuracy of the differential phase measurement (better than 0.1 °) one can detect, in this system, on the order of 0.1% bound probe. This phenomenon also occurs in time-domain measurements. Specifically, if one monitors the anisotropy decay of a system which displays multiple lifetimes associated with multiple rotational diffusion rates then one may observe a decline at short times of the anisotropy followed by a rise at latter times and subsequent decrease. This "dip and rise" effect has been observed by Millar and co-workers 22 in studies on protein-DNA interactions, specifically in the case of the interaction of a fluorescent DNA duplex with the Klenow fragment of DNA polymerase.