Vertical fluctuations of the terminal methyl groups of stearic acid acyl chains toward the surface of dimyristoylphosphatidylcholine (DMPC) bilayers have been investigated by using spin-label electron-electron double-resonance ( ELDOR ) methodology. Spin-label pairs consisting of two populations of stearic acid spin-labels were employed, each at 0.25 mol% concentration, where the nitroxides of the first population were 15N substituted and the nitroxides of the second contained 14N. Various combinations of labels with the nitroxide moieties located at carbons 5, 12, or 16 (C5, C12, C16) were used. ELDOR permits measurement of collision frequencies between the two constituents of the pair, for example, between 15N spin-labels at C5 and 14N labels at C16. Intramolecular contributions to the ELDOR effect including nitrogen nuclear relaxation are eliminated by the use of spin-label pairs. Above the main phase transition temperature, bimolecular collisions between C5 and C16 occur with about half the frequency of C16:C16 collisions. It is concluded that vertical fluctuations are very pronounced. A dependence of these fluctuations on temperature and pH has been observed. Lateral diffusion constants calculated from the bimolecular collision frequencies of C16:C16 pairs are 4.56 X 10(-8), 5.77 X 10(-8), and 8.09 X 10(-8) cm2/s at 27, 37, and 47 degrees C. These values are in good agreement with previous measurements of lipid diffusion in DMPC.

Lateral diffusion constants of the stearic acid nitroxide radical spin label 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolindinyl oxide in dispersions of dimyristoyl phosphatidylcholine have been measured. Electron-electron double resonance methods were used to determine the product of the bimolecular collision frequency and Tie, the electron spin-lattice relaxation time. Tie in turn was measured by the technique of saturation recovery. The theoretical model of Trauble and Sackmann was then used to relate the bimolecular collision frequencies to the diffusion constants. Results are in agreement with other methods. Lower spin-label concentrations than were used in previous electron paramagnetic resonance studies are needed (label-to-lipid ratio < 0.5 mol%). Analysis of the data also yields values of the nitrogen nuclear spin-lattice relaxation time of the nitroxide moiety. These values are indicative of membrane fluidity.Lateral diffusion is considered to be important for a broad range ofbiological functions (1, 2, 3), and a variety of techniques have been used for measuring lateral diffusion constants, D. A summary of results obtained from various methods is included in the discussion by Kuo and Wade (4). There is general agreement that D for lipid in membranes above the phase transition temperature falls within the range 10-9 to 10-7 cm2 S-1.Early EPR studies of lateral diffusion in membranes were carried out by Devaux and McConnell (5) and Sackmann and Trauble (6-8) and were based on Heisenberg exchange effects on nitroxide spin-label linewidths. The latter authors made simulations of linewidths to obtain Heisenberg exchange rates, WHex, from which diffusion constants were calculated. Both studies used high spin-label concentrations and may have been complicated by structural perturbations by the probe molecules. Sackmann and Trauble's results may also have been affected by inhomogeneous broadening from coupling to protons of the spin label and by electron-electron dipolar interactions between spin labels.We present here the use of electron-electron double resonance (ELDOR) (9) and saturation-recovery (10) techniques to study lateral diffusion in model membranes. ELDOR and saturation-recovery methods permit determination of the Heisenberg exchange rate, which is effectively the bimolecular collision frequency. When this frequency is determined by simple translational diffusion, the lateral diffusion constant can be calculated from the theory of Trauble and Sackmann (8). As will become apparent, much lower values of WHex can be measured than are possible from analysis of linewidths, thus permitting the use of lower spin-label concentrations.Our method for determining WHeX differs from EPR spin-label linewidth studies in the following ways. Both ELDOR and saturation-recovery require saturation ofthe spin system-i.e., the application of large incident microwave fields. They also require the use of microwave cavities that can support two microwave fields simultaneously, with one field used to sat...

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