Biophysics Oxygen permeability of phosphatidylcholine - cholesterol membranes ( pulse ESR / permeability coefficient / oxygen transport ) WITOLD

Oxygen transport in phosphatidylcholinecholesterol membranes has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed at various distances from the membrane surface using long-pulse saturation recovery ESR techniques. The collision rate was estimated for tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameter across the membrane were obtained as a function of cholesterol mol fraction and temperature in L-a-dimyristoylphosphatidylcholine ([Myr2]PtdCho) and L-a-dioleoylphosphatidylcholine ([Ole2]PtdCho) membranes. Membrane oxygen permeability coefficients were estimated from oxygen transport parameter profiles. At %300C, the oxygen permeability coefficients in the presence and absence of 50 mol % cholesterol are 22.7 and 125.2 cm/s, respectively, for [Myr2]PtdCho membranes, and 54.7 and 114.2 cm/s, respectively, for [Ole2]PtdCho membranes (compared with 60-80 cm/s for water layers with the same thicknesses as the membranes). The major results in the liquid-crystalline phase are as follows: (i) In the absence of cholesterol, membranes are not barriers to oxygen transport. (ii) Addition of 50 mol % cholesterol decreases oxygen permeability by a factor of =5 and =z2.5 in [Myr2]PtdCho and [Ole2]PtdCho membranes, respectively. The resistance to oxygen transport is located in and near the polar headgroup regions in the membrane. (iii) Cholesterol increases oxygen transport in the central regions of [Ole2]PtdCho membranes. We ask two fundamental questions about oxygen transport in lipid bilayers: (i) Can a biological membrane be a permeability barrier for molecular oxygen, and (ii) if so, what is the location of the major permeability resistance? We are additionally concerned with development of methodology to study molecular structure and diffusion in artificial and cellular membranes using molecular oxygen as a small nonelectrolyte probe. Our method is based on measurement of the bimolecular collision rate between oxygen and spin labels. Subczynski and Hyde (1) found that the average oxygen concentration in the hydrocarbon region of L-a-dimyristoylphosphatidylcholine [Myr2]PtdCho membranes in the liquid-crystalline phase is higher than in water, increases with temperature, and increases abruptly by 75% and 50% at pretransition and main phase-transition temperatures. Subczynski and Hyde (2) and Kusumi et al. (3) observed oxygen transport in membranes using the electron spin-lattice relaxation time (T1) as a basic "clock." The rationale of the spin-label T1 method is that the molecular probe can be placed at a known location in the membrane to observe local events and that the time scale of T1 (1-20 ,u s) is in the correct range to study many molecular processes (4). Kusumi et al. (3) defined an oxygen transport parameter W(x) = TT'(air, x) Tf'(N2, x). [1] Since W(x) is proportional to the collision rate ofoxygen with the spin-label nitroxide group, it is a function ofboth the local concentration [C(x)] and the local translational diffusion constant [D(x)] of oxygen at a "depth" x in the airequilibrated membrane, W(x) = AD(x)C(x), [2] whereA = 8irpr0. Here r. is the interaction distance between oxygen and the nitroxide radical spin labels (=4.5 A) and p is the probability that an observable event occurs when a collision occurs (2). They concluded that the oxygen transport parameter is a useful monitor of membrane fluidity that reports on translational diffusion of small molecules. In the present research, profiles of W(x) across various membranes have been investigated with emphasis on effects of cholesterol and alkyl chain unsaturation (5, 6). Using the theory of Diamond and Katz (7), we have assessed oxygen permeability across the membrane on the basis of W(x) profiles, assuming that oxygen diffusion is isotropic. We have previously shown that oxygen diffusion is almost isotropic in the liquid-crystalline phase of non-cholesterol-containing membranes (3). Diffusion of oxygen below the pretransition temperature was found to be more rapid in the transverse than in the lateral direction. Between the preand maintransition temperature, our data with respect to the anisotropy of oxygen diffusion are complex and no detailed interpretation was advanced (3). Diamond and Katz (7) derived an expression for the permeability coefficient PM of a nonelectrolyte in terms of resistances r' and r" in the polar headgroup regions (8), the solute (local) partition coefficient K(x), and the solute (local) diffusion constant D(x), taking the plane of the membrane as perpendicular to the x axis: PM = [r[ K(x)D() +r [3] The integration is taken across the hydrophobic portion ofthe membrane. Since a spin probe is placed in the polar regions as well as in the hydrophobic region in the present study, r' and r" can also be estimated within the integral. The coefficient K(x) is related to the local concentration, C(x), and the Abbreviations: [Myr2]PtdCho, L-a-dimyristoylphosphatidylcholine; [Ole2]PtdCho,L-a-oleoylphosphatidylcholine;5-SASL,5-doxylstearic acid spin label; 16-SASL, 16-doxylstearic acid spin label; T-PC, tempocholine phosphatidic acid ester. tTo whom reprint requests should be addressed at: National Biomedical Electron Spin Resonance Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. 4474 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 86 (1989) 4475 oxygen concentration in the aqueous phase equilibrated with air, Cw(air), by the equation K(x) = C(x)/Cw(air). Eq. 3 becomes PM A XC(a)1 h dx PM=A x Cw(air) [oW(x)J [4] where h is the entire thickness of the lipid bilayer. Eq. 4 allows us to evaluate permeability coefficients in terms of experimental observables W(x) and values of Cw(air) taken from published tables. This method is based on the profile of the local oxygen transport parameter across the membrane and does not require formation of an oxygen gradient. Several attempts have previously been made to obtain the oxygen permeability of erythrocyte membranes by creating an oxygen gradient by rapid mixing, but they were not successful because the presence of a thick (-2 ,um) unmixed water layer on the cell surface prevented immediate contact of oxygenated solution with the erythrocyte membrane (911). MATERIALS AND METHODS [Myr2]PtdCho and L-a-dioleoylphosphatidylcholine ([Ole2]PtdCho) were obtained from Sigma, cholesterol (crystallized) was from Boehringer Mannheim, 5-doxylstearic acid spin label (5-SASL) and 16-doxylstearic acid spin label (16-SASL) were from Molecular Probes, and 1-15N-1-oxyl 4-oxo-2,2,6,6tetramethylpiperidine-d16 (d-Tempone) was from Merck. Tempocholine phosphatidic acid ester (T-PC) was a generous gift from S. Ohnishi (Kyoto University, Kyoto, Japan). The buffer was 0.1 M borate at pH 9.5. To ensure that all probe carboxyl groups were ionized in PtdCho membranes, a rather high pH was chosen (3, 5, 12, 13). The structure of PtdCho membranes is not altered at this pH (3, 13). The membranes used in this work were multilamellar dispersions of lipids containing 1 mol% of spin label and were prepared as described (3, 5). The lipid dispersion (10 mM) was centrifuged briefly and the loose pellet [20%o (wt/wt) lipid] was used for ESR measurement. Dilution of the pellet did not induce any detectable changes in the membrane structure (5). The sample was placed in a capillary (i.d., 0.5 mm) made of gas-permeable methylpentene polymer. The concentration of oxygen in the sample was controlled by equilibrating the sample with the same gas that was used for temperature control-namely, a controlled mixture of nitrogen and dry air adjusted with flowmeters (Matheson Gas Products; model 7631H-604) (3). Spin-lattice relaxation times were measured at X-band using the long-pulse saturationrecovery technique (3). The method has been reviewed by Hyde (14). The apparatus used here was described by Yin et al. (4). RESULTS AND DISCUSSION Saturation-Recovery Measurement of the Oxygen Transport Parameter. All measurements of T1 were made on the central '4N hyperfine line (MI = 0) between 0WC and 45°C. Typical saturation-recovery curves are shown in Fig. 1. T11 values for T-PC, 5-SASL, and 16-SASL in [Myr2]PtdChocholesterol membranes in the presence and absence of oxygen are plotted against reciprocal temperature in Fig. 2 (Upper). The error in the estimate of T1 is within 5%. In the absence of oxygen, addition of cholesterol increases T1 for 5-SASL but decreases it for T-PC and 16-SASL. The following points can be made for samples equilibrated with air: (i) In the absence of cholesterol, abrupt changes of T1 at the main phase-transition temperature were observed for all spin labels. T1 is shorter by a factor of 5-10 compared I I I I I I Il A A IsI A A I