Electron-electron double resonance and saturation-recovery studies of nitroxide electron and nuclear spin-lattice relaxation times and Heisenberg exchange rates: lateral diffusion in dimyristoyl phosphatidylcholine.

Abstract

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...

Multifrequency Pulsed EPR and the Characterization of Molecular Dynamics

“…The T 1 values at the two maxima in the distributions obtained with UPEN were in good agreement with the two values obtained by fitting the recovery curves to the sum of two exponentials. These observations are consistent with the expectation of T 1N < T 1 at higher resonance frequencies (Hyde et al, 1984; Popp & Hyde, 1982). …”

“…For example, for nitroxide radicals in fluid solution the nitrogen nuclear spin relaxation T 1N depends on τ and ω (Hyde et al, 1984; Popp & Hyde, 1982; Robinson et al, 1994). In the fast tumbling limit T 1N is equal to the electron spin T 1 .…”

Relaxation Time Determinations by Progressive Saturation EPR: Effects of Molecular Motion and Zeeman Modulation for Spin Labels

“…These may be compared with values for the intrinsic T 1 -relaxation time that have been obtained from saturation recovery EPR measurements. In the fluid phase these are T 1e ϭ 2.25 s for 16-C atom labeled stearic acid (16-SASL) in dimyristoyl phosphatidylcholine (DMPC) at 27°C (28) and T 1e ϭ 1.6 s in DOPC at 54°C (29). In the gel phase, T 1e ϭ 8.5 s has been obtained by saturation recovery for 6-PCSL in DPPC at 1°C (30) and T 1e ϭ 8 s for 5-SASL in DMPC at 4°C (31).…”

Autobiography of James S. Hyde

“…The first of these, by C. A. Popp and J. S. Hyde [133], appeared in 1982. It provided the first saturation recovery data in a spin-labeled model membrane system (dimyristoylphosphatidylcholine [DMPC]).…”