Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

Abstract

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.

Fig. 1

Chemical structures of spin labels used in this work: n-PC, T-PC, 9-SASL, and ASL. Chemical structures of POPC and cholesterol molecules are indicated to illustrate approximate localization of these molecules and nitroxide moieties across the membrane.

Fig. 2

Panel of EPR spectra of 5-, 10-, and 16-PC in membranes made of lens lipids, POPC/Chol equimolar mixture, and POPC. Spectra were recorded at 35°C. Measured values for evaluating the order parameter are indicated. The positions of certain peaks were evaluated with a high level of accuracy by recording them at 10-times higher receiver gain and, when necessary, at higher modulation amplitude.

Fig. 3

Profiles of the molecular order parameter for membranes made of lens lipids, POPC/Chol equimolar mixture, and POPC (order parameter is plotted in a log scale as a function of nitroxide position (n) along the alkyl chain in spin labels) at 35°C. Points for pure POPC were taken from our previous work [30].

Fig. 4

Molecular order parameter of 5-, 10-, and 16-PC in membranes made of lens lipids (A) and POPC/Chol equimolar mixture (B) plotted as a function of temperature.

Fig. 5

EPR spectra of 14-PC in membranes made of lens lipids, POPC/Chol equimolar mixture, and POPC measured at −163°C. The measured 2A_z value (z component of hyperfine interaction tensor) is indicated.

Fig. 6

Hydrophobicity profiles (2A_z) across membranes made of lens lipids (A) and POPC/Chol equimolar mixture (B). Upward changes indicate increases in hydrophobicity. 2A_z for 16-PC in the aqueous phase was calculated from the isotropic hyperfine constant of the nitroxide spin-label as shown in [40]. Because T-PC contains a different nitroxide moiety than n-PC and n-SASL, its points are not connected with other points. However, the relative changes of the hydrophobicity in the polar headgroup region can be evaluated. Approximate localizations of nitroxide moieties of spin labels are indicated by arrows.

Fig. 7

Representative saturation-recovery signals and fitted curves of 5-PC in the membrane made of lens lipids at 35°C for the sample equilibrated with 100% nitrogen gas and with a gas mixture of 50% air and 50% nitrogen. The solid lines indicate the fit to single-exponential curves with recovery times of 1.6 μs (with 50% air) and 3.9 μs (with 0% air). The difference between the experimental data and the fitted curve is shown in the lower part of each recovery curve.

Fig. 8

T₁⁻¹ for 5-, 10-, and 16-PC in the membrane made of lens lipids at 25°C plotted as % air in the equilibrating gas mixture. Experimental points show a linear dependence up to 50% air, and extrapolation to 100% air is performed to indicate a way of calculating oxygen transport parameters. W_5-PC, W_10-PC, and W_16-PC are oxygen transport parameters measured for 5-, 10-, and 16-PC, respectively.

Fig. 9

Profiles of the oxygen transport parameter (oxygen diffusion-concentration product) across membranes made of lens lipids (A) and POPC/Chol equimolar mixture (B) obtained at 15 (♦), 25 (●), and 35°C (○). The symbol × indicates the oxygen transport parameter in the aqueous phase. It does not change significantly because temperature dependences of oxygen diffusion and concentration are opposite. Approximate localizations of nitroxide moieties of spin labels are indicated by arrows.

Fig. 10

The enlarged part of profiles of the oxygen transport parameter for the membrane made of lens lipids presented in Fig. 9A with added points for the cholesterol analogue spin label, ASL, at 15 (◊), 25 (□), and 35°C (■).