Energy transfer is enhanced by translational diffusion of the donor and acceptor [Steinberg, I. Z. & Katchalski, E. (1968) J. Chem. Phys. 48,2404-24101. The effect of diffusion on energy transfer depends on Dro/s2, in which D is the sum of the diffusion coefficients of the donor and acceptor, 7o is the lifetime of the donor in the absence of transfer, and s is the mean distance between donors and acceptors. In most previous studies, Dro/s2 << 1, corresponding to the static limit. We report here steady-state and kinetic fluorescence experiments showing that Dro/s2 >> 1, the rapid-diffusion limit, can be attained by usingTh3+ chelated to dipicolinate as a long-lived energy donor (To = 2.2 msec). The concentration of rhodamine B, the energy acceptor, result in 50% transfer was 0.67 pM, which is three orders of magnitude less than the concentration giving 50% transfer in the static limit. The dependence of the transfer efficiency on diffusion coefficients varying from 5 X 10-li to 1.5 X 10-4 cm2/sec, spanning the range from the static limit to the rapid-diffusion limit, is in excellent agreement with theory. It is evident that energy donors with millisecond or longer excited state lifetimes can be used to probe translational motions in membranes and other assemblies. Energy transfer in the rapid diffusion limit is sensitive to the distance of closest approach (a) of the donor and acceptor. For Elkana et al. (6). Haas et al. (7) have recently determined the diffusion coefficients of the ends of oligopeptide chains in solution by analyzing the effect of diffusion on energy transfer. In the present study, we consider the limiting case in which energy transfer is maximally enhanced by diffusion-i.e., a further increase in the diffusion coefficient has no further effect on the transfer efficiency. Our theoretical analysis of this rapid-diffusion limit shows that these experiments are sensitive to the distance of closest approach between donor and acceptor and that measurements are possible at much lower acceptor concentrations than in the absence of diffusion (the static limit). Calculations using the theory of Steinberg and Katchalski (5) show that, for small molecules in aqueous solution, the rapiddiffusion limit cannot be reached with conventional fluorescent donors having excited state lifetimes in the nanosecond range. This limit can be attained only if the fluorescent donor has a much longer lifetime, in which extensive diffusion can occur.In order to attain the rapid-diffusion limit and test the theory, we have used as a donor the fluorescent lanthanide ion Tb3+, which has a lifetime in the millisecond range (8, 9). Tb3+ was chelated to dipicolinic acid (DPA), resulting in a fluorescence enhancement of about 104 (10). This Tb-(DPA)3 chelate consists of three molecules of DPA liganded to Tb3+ and has a net charge of -3 above pH 5 (10). Experiments using Tb.(DPA)3 as the donor and either rhodamine B or nitrobenzodioxazole (NBD) diethanolamine as the acceptor verify the validity of the theory over the ent...
A new picosecond resonance Raman technique shows that resonance Raman lines characteristic of a distorted all-trans retinal appear within 30 picoseconds after photolysis of rhodopsin or isorhodopsin. This finding suggests that isomerization is nearly complete within picoseconds of the absorption of a photon.
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