Vitamin E - Phospholipid Membrane Interactions: Esr and Stesr Study

The effect of a-tocopherol on a phospholipid model membrane has been investigated by Electron Spin Resonance and Saturation Transfer ESR by using stearic acid and perdeuterodi-t-butyl nitroxide spin probes. It is observed that in the gel phase it induces a decrease of order and increase in fluidity; while in the liquid crystalline phase an indication of a slight increase in ordering and a clear decrease in fluidity are registered. INTRODUCTION Vitamin E, whose main constituent is a-Tocopherol (aT), is an indispensable lipid component of biological membranes. It is a chain-breaking antioxidant preventing peroxidation of the highly unsaturated fatty acids in membrane lipids [1]. It has also been suggested that it might stabilize biological membranes by restricting the molecular mobility of their components [2]. In the present work we report the effects of aT, in the gel and liquid crystalline phases, both on the rate of the stearic acid spin label motion and on the phospholipid chain order by using conventional ESR and STESR spectroscopy. These results, together with the dynamical information obtained by using PDDTBN, are critically analysed in order to obtain a clearer insight on the effect of aT on lipid membranes. MATERIALS AND METHODS D-a-tocopherol and DipalmitoylLaphosphatidylcholine (DPPC) were obtained from Sigma Chem. Co. and used without any further purification. 5-keto and 16-keto stearic acid spin labels (5 SASL and 16 SASL, respectively) were purchased from SYVA, Palo Alto. C-depleted PDDTBN was kindly provided by Prof.W.Plachy of San Francisco State University. Stearic acids labelled DPPC liposomes were prepared according to the procedure reported in ref [3], but in the present case, stearic acid spin probes, dissolved in ethanol, were mixed with aT and DPPC at the beginning of the sample preparation (probe to lipid molar ratio was 1:100). Dry films were hydrated with borate buffer at pH 9.4. This pH has been chosen to ensure that all spin probe carboxyl groups are ionized in the phosphatidylcholine membranes [4].Samples were contained in lmm i.d. 100 /xl glass capillaries within standart 4 mm diameter quartz tubes. The ESR apparatus and settings were reported in ref.[3]. For randomly oriented liposome samples, where the stearic spin labels undergo rapid anisotropic motion, the order parameter S was estimated as reported in ref. [5]. For samples in which the spin label motion was slower (rc > 10~s), some sort of information about the relative order was obtained by taking into account the 2A|| splitting [6]. For the samples showing very slow motion (rc ~ 10~ — 10~ s) we used second harmonic absorption out-of-phase detection at high power (STESR). Effective correlation times were obtained by using the reference curves given in ref. [7]. RESULTS AND DISCUSSION Fig l.a and b shows the ESR spectra of 5 SASL and 16 SASL .respectively in 20 mol % aT containing DPPC liposomes at different temperatures. 5 SASL inserted into the bilayer is able to monitor the region near to the polar interface of the membrane and 16 SASL inserted into the bilayer is able to monitor the deep interior of the 278 Bulletin of Magnetic Resonance Fig.l. Conventional first harmonic in phase absorbtion ESR spectra of: 5 SASL (a) and 16 SASL (b) in DPPC liposomes containing 20 mol % aT at different temperatures. bilayer far away from the polar interface of the membrane. When aT is added the ESR spectra obtained are similar to those of pure DPPC liposomes except that the sudden change in lineheights, qualitatively monitoring the main phase transition, is not now observed. Fig.2 shows the variation of Amax for 5 SASL and 16 SASL as a function of temperature for DPPC liposomes with and without addition of aT. S order parameter values were also calculated. It is found that for 41° C, by using 5SASL, S=0.52 for pure DPPC liposomes and S=0.55 for 20 mol % aT containing DPPC liposomes. At 39° C, by using 16 SASL, S is found to be 0.31 for pure DPPC and 0.10 for 20 mol % aT containing DPPC liposomes. Examination of Fig.2 and order parameter values reveals that, below the phase transition temperature, addition of aT slightly decreases order in the region near the phospholipid polar heads; while above the phase transition an almost negligible increase is registered. Concerning the interior of the bilayer, deeply inside the acyl chains, 16 SASL monitors a significant decrease of the phospholipid chain order in the presence of aT below the main phase transition. The effect of aT on ordering of the biomembrane is usually mixed up throughout the related literature [8]., Our results agree with those of Wassail et al.[9]. Fig. 3 shows the second harmonic 90° out of phase ESR spectra (V3), recorded at different temperatures, for DPPC multilamellar dispersions labelled with 5 SASL in the absence (Fig. 10 20 30 40 50 TCC) Fig.2. Temperature dependence of the maximum hyperfine 2A|| splitting of DPPC liposomes with and without aT labelled with 5 SASL and 16 SASL. 0 mol % aT, 5 SASL (• ); 20 mol % aT, 5 SASL ( A ); 0 mol % aT, 16 SASL (o); 20 mol % aT, 16 SASL (•). Vol. 11, No. 3/4 279 Fig.3. STESR spectra recorded at different temperature values in the second harmonic, 90° out-ofphase, absorbtion mode of DPPC liposomes without (a) and with (b) aT labelled with 5 SASL.