The Stern-Volmer theory, in which the quantum yield ratio (Io/I) depends linearly on the quencher concentration, will typically be inapplicable to fluorescence quenching in membranes. Numerical analysis shows that diffusion-controlled quenching results in a nonlinear concentration dependence for diffusion coefficients less than or of the order of 10(-6) cm2 s-1 and probe fluorescence lifetimes in the region of 10-100 ns. Lateral diffusion coefficients in membranes are typically overestimated an order of magnitude or more by the Stern-Volmer theory. An alternative empirical method is presented, which represents nonlinear concentration curves by a single parameter linear approximation determined by a least-squares analysis. The fitting parameter, P, depends on the interaction distance, the membrane thickness, the maximum extent of quenching and, in the case of biexponential probe fluorescence decay, the fluorescence kinetic parameters. P is presented in tabular form for a useful range of these parameters. The method is used to estimate diffusion coefficients for plastoquinone and plastoquinol from pyrene fluorescence quenching in soya bean phosphatidylcholine liposomes. It is found that the diffusion coefficients are nearly equal and in the region of 1.3-3.5 X 10(-7) cm2 s-1 for interaction radii of 1.5-0.5 nm, respectively.

PYRENE FLUORESCENCE QUENCHING BY PLASTOQUINONE WAS USED TO ESTIMATE THE RATE OF PLASTOQUINONE LATERAL DIFFUSION IN SOYBEAN PHOSPHATIDYLCHOLINE PROTEOLIPOSOMES CONTAINING THE FOLLOWING INTEGRAL MEMBRANE PROTEINS: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc(1), and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 . 10(-7) cm(2) s(-1) in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc(1), and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration.

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