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The phenolic group of #-tocopherol anchors in the lipid-water interface of fully saturated membranes Alessio Ausili, Alejandro Torrecillas, Ana M deGodos, Senena Corbalan-Garcia, and Juan C. Gomez-Fernandez Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04142 • Publication Date (Web): 15 Feb 2018 Downloaded from http://pubs.acs.org on February 25, 2018

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85x69mm (600 x 600 DPI)

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The phenolic group of α-tocopherol anchors in the lipid-water interface of fully saturated membranes

Alessio Ausili, Alejandro Torrecillas, Ana M. de Godos, Senena Corbalán-García, Juan C. Gómez-Fernández*

Departamento de Bioquímica y Biología Molecular “A”, Facultad de Veterinaria, Regional Campus of International Excellence Mare Nostrum, Universidad de Murcia, Apartado de Correos 4021, E-30080-Murcia, Spain.

*Corresponding author. E-Mail: [email protected] Telephone: +34868884766 Fax: + 34 968364147

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Abstract α-Tocopherol is considered to carry on a very important role as antioxidant of membranes and lipoproteins and other biological roles as membrane stabilizers and bioactive lipids. Given its essential role, it is very important to fully understand its location in the membrane. In this work, the vertical location of vitamin E in saturated membranes has been studied using biophysical techniques. Small- and wide-angle X-ray diffraction experiments show that α-tocopherol alters the water layer between bilayers in both DMPC and DPPC, indicating its proximity to this surface. The quenching of the intrinsic fluorescence of αtocopherol indicates a low quenching efficiency by acrylamide and a higher quenching by 5-doxyl-PC than by 9- and 16-doxyl-PC. These results suggest that in both DMPC and DPPC membranes the chromanol ring is not far away from the surface of the membrane but within the bilayer. 1H-NMR-NOESY MAS-NMR studies showed that α-tocopherol is localized similarly in DMPC and DPPC membranes, with the chromanol ring embedded in the upper part of the hydrophobic bilayer. Using ATR-FTIR spectroscopy it was observed that the tail chain of α-tocopherol lies nearly parallel to the acyl chains of DMPC and DPPC. Taking these results together it was concluded that in both DMPC and DPPC, the hydroxyl group of the chromanol ring will establish hydrogen-bonding with water at the membrane surface and the main axis of the α-tocopherol molecule will be perpendicular to the bilayer plane.


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Key words α -tocopherol; membrane location; saturated phosphatidylcholines; X-ray diffraction; NMR; fluorescence quenching; FT-IR.

Abbreviations: POPC, 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-snglycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine;; MLV, multilamellar vesicle; SAXD, small-angle X-ray diffraction; NMR, nuclear magnetic resonance.


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Introduction α-Tocopherol is a lipid soluble vitamin that it is considered to play a very important antioxidant role and its deficiency has been associated to different pathological conditions such as infertility or deterioration of tissues such as the nervous systems 1-3. Since αtocopherol is a hydrophobic molecule it is found in membranes and lipoproteins and it will protect from peroxidation the unsaturated fatty acyl chains of the phospholipids. In order to understand how this vitamin acts as an antioxidant agent it is important to discover its location and orientation in the membranes 4. It has been described that α-tocopherol might modulate the molecular mobility of membrane components 5 and it may form complexes with other molecules unsaturated fatty acids 6-7 or lysophosphatidylcholine and other similar phospholipids 8. It also buffers the permeability of phosphatidylcholine liposomes 9. It is interesting that vitamin E may stabilize liposomes during long-term storage decreasing the breakdown of phosphatidylcholine to lysophosphatidylcholine and decreasing the level of peroxidation of unsaturated fatty acyl chains 10.


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Fig. 1. Structure of α-tocopherol. Methyl group C24 (A), methyl groups C25, C26 and C28(B) and C4 are labelled.

Since α-tocopherol may have an so important role in biomembranes it is interesting to understand the molecular mechanism of action of α-tocopherol in its organization in the membrane and if it is well mixed with phospholipids or if it may form aggregates. It was suggested that it could have affinity for polyunsaturated phospholipids 11. It was described that when α-tocopherol was incorporated in membranes formed by heteroacid phosphatidylcholines, fluid immiscibility was detected by DSC, probably arising from αtocopherol-rich domains 12. It was also found that α-tocopherol will have preference for the most fluid phase when it is incorporated to a membrane where phase separation occurs and this suggests that, in this way, α-tocopherol, being located in the most fluid phase, will be very well situated to protect unsaturated phospholipids from peroxidation 13. Also important is to understand its vertical location in the membrane. However this is an issue still subject to discussions. Several proposals were put forward at this respect 14. The first of them (a) was that whereas hydrophobic tail is anchored into the hydrophobic 5 ACS Paragon Plus Environment

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palisade the chromanol ring will be floating near the bilayer-water interface 15-18. In a second model (b) the phenolic group of the chromanol ring is situated anchored in the lipidwater interface but with the chromanol ring within the lipidic bilayer interacting with the segments of the phospholipid acyl chains close to the beginning of these chains 13, 19-22. A third point of view (c) suggested that the chromanol resides deep in the membrane bilayer 23-26

. More recently it was suggested that the type of fatty acyl chains of the phospholipids

determine different locations of α-tocopherol in the membrane 27 and these authors interpreted their data as if in DPPC the chromanol group resides in among the choline groups of the phospholipids (model a), whereas in DOPC and PAPC membranes the 5methyl group is located near the glycerol backbone (model b) 27 and in the case of DMPC the location of the chromanol group is the center of the bilayer (c) 28. This same group proposed later on that α-tocopherol´s location in membranes is the same for DPPC, POPC and POPE with the C5-merhyl group above the glycerol/phosphate group (model a) but they proposed that it sits lower in POPS and egg sphingomyelin 29. In this last work the authors also suggested that the α-tocopherol´s tail mostly parallel to the surface of the membrane and far from the center of POPC bilayers. It was concluded from very recent results obtained by incorporating α-tocopherol to membranes with phosphatidylcholines with different levels of unsaturation, that the vertical location of α-tocopherol in phosphatidylcholine membranes is not altered as a function of the degree of unsaturation of the fatty acyl chains 30. In this paper we try to clarify if the location of α-tocopherol in saturated phosphatidylcholine such as DMPC and DPPC is or not similar to that found in unsaturated


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membranes. We have used for this objective small- and wide-angle X-ray diffraction, fluorescence quenching and 1H NOESY MAS-NMR. Our conclusion is that, in all cases, the most likely disposition of vitamin E will be determined by the hydroxyl group of the chromanol ring that will be anchored to water at the lipid-water interphase. We have also used ATR-FTIR to study the orientation of the hydrophobic tail of tocopherol and we conclude that both in DMPC and DPPC membranes is nearly parallel to the acyl chains of the phospholipids.


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Materials and Methods Materials 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine glycero-3-phosphocholine

(DPPC),

(POPC),

1,2-dipalmitoyl-sn-

1,2-dipalmitoyl(D62)-sn-glycero-3-phosphocholine

(DPPC-d62), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl(D54)sn-glycero-3-phosphocholine (DMPC-d54), 1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3phosphocholine phosphocholine

(5-doxyl-PC), (12-doxyl-PC),

1-palmitoyl-2-stearoyl-(12-doxyl)-sn-glycero-3and

1-palmitoyl-2-stearoyl-(16-doxyl)-sn-glycero-3-

phosphocholine (16-doxyl-PC) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). α-Tocopherol, acrylamide and butylated hydroxytoluene (BHT) were acquired from Sigma-Aldrich Química S.L. (Madrid, Spain). All other reagents and solvents were of the highest purity commercially available. Multilamellar vesicles preparation Samples of each pure phospholipid in the presence and in the absence (only for X-ray diffraction) of α-tocopherol were basically prepared with the same procedure and buffer for all the experiments to form multilamellar vesicles (MLVs). Typically, appropriate amounts of phospholipids and α-tocopherol (5:1 molar ratio in X-ray, in NMR and fluorescence experiments, and 3:1 molar ratio and ATR-FTIR experiments) dissolved in chloroform/methanol (1:1), were mixed, adding 0.5 mol% of BHT. For doxyl-PC quenching experiments, different amounts of 5-, 12- and 16-doxyl-PC dissolved in chloroform/methanol (1:1), was also added to obtain 4, 8, 12, 16 and 20 mol% doxylPC/phospholipid. Then, the organic solvent was evaporated under a stream of oxygen-free 8 ACS Paragon Plus Environment

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nitrogen and subsequently by high vacuum for at least 2 hours. The dried samples were hydrated in a buffer containing 10 mM Hepes at pH 7.4, and the MLVs were generated by vortexing vigorously at temperatures above the transition temperatures. X-ray diffraction X-ray samples were prepared as described above using 10 mg of phospholipids. MLVs were centrifuged at 13,000g and the pellets were placed in a steel holder with cellophane windows, which provide a good thermal contact with the Peltier heating unit. The concentration was 130 mM for DMPC samples and 150 mM for DPPC samples. Simultaneous small- (SAXS) and wide- (WAXS) angle X-ray diffraction measurements were performed at 20 °C for POPC samples, and below and above the transition temperature for DMPC (8 and 32 °C) and DPPC samples (25 and 50 °C) after 10 min for temperature equilibration, with exposure times of each measurement of 10 min. Experiments were carried out in a modified Kratky compact camera (MBraun–Graz– Optical Systems, Graz Austria), incorporating two coupled linear position sensitive detectors (PSD, MBraun, Garching, Germany) to monitor the s-ranges [s = 2sin θ/λ, 2θ = scattering angle, λ = 1.54 Å] between 0.0075–0.07. Nickel-filtered Cu Kα X-rays were generated by a Philips PW3830 X-ray Generator operating at 50 kV and 30 mA. The detector position was calibrated by using Ag-stearate (small-angle region, d-spacing at 48.8 Å) as reference materials. Background corrected SAXS data were analyzed using the program GAP (global analysis program) written by Georg Pabst and obtained from the author 31-32. This program allowed us to retrieve the membrane thickness, dB=2(zH + 2σH), from a full q-range analysis of the SAXS patterns 33. The parameters zH and σH are the position and width respectively of the Gaussian used to describe the electron-dense 9 ACS Paragon Plus Environment

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headgroup regions within the electron density model. The water layer was then given by dw=d - dB where d stands for the lamellar repeat distance. Fluorescence spectroscopy experiments Acrylamide and doxyl-PC quenching experiments were performed on a Fluoromax3 fluorescence spectrometer (Jobin Yvon, Longjumeau, France) equipped with a thermostated quartz cuvette with constant stirring at temperatures above the transition temperatures. α-Tocopherol intrinsic fluorescence emission was monitored at 329 nm (15nm slit width), exciting the sample at 297 nm (5-nm slit width). All the experiments were done in triplicate, and the means and standard deviations were calculated. Results were elaborated and quenching parameters calculated according to Stern-Volmer equation and, in the case of acrylamide quenching experiments, corrected for static quenching as described 34. Samples for acrylamide quenching experiments were prepared as described using 0.1 µmol of pure phospholipids. In these experiments, large unilamellar vesicles (LUVs) were used in order to optimize the acrylamide quenching effect. LUVs were generated by extruding 11 times MLVs through two stacked 100-nm polycarbonate filters (Millipore Inc., Bedford, USA). The emission of α-tocopherol was measured in the presence of increasing concentration of acrylamide from 0 to 1.894 M. The total concentration of lipids in the cuvette was 50 µM. Acrylamide quenching experiments were performed in triplicate for all the lipid samples. Sampling error was calculated as standard deviation from mean


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value. The lipid samples were prepared independently while the acrylamide used to quench the α-tocopherol fluorescence was the same for all the samples. For doxyl-PC quenching experiments, MLVs were prepared as described above adding different concentration of 5-, 12- and 16-doxyl-PC to the pure phospholipids for a total amount of 0.1 µmol. The effect of quenching was evaluated measuring the αtocopherol fluorescence in the presence of 0, 4, 8, 12, 16 and 20 mol% of each doxyl-PC. The total concentration of lipids in the cuvette was 50 µM. ATR-FTIR measurements For ATR-FTIR experiments, the vesicles were prepared by mixing a total amount of 4 µmol of DMPC-d54 or DPPC-d62 with the appropriate amount of α-tocopherol. MLVs, generated as described above, were placed directly onto the ATR germanium crystal surface and dried by means of a nitrogen stream. The crystal was formerly washed with alkaline detergent, rinsed with deionized water and then washed with methanol to render the surface hydrophilic. Finally, the crystal was placed in a liquid sample holder with connections to allow an air stream (wet in H2O) to hydrate the samples for 30 min before collecting the spectra. The spectra, with both parallel and perpendicular polarization of the incident beam, were recorded on a Bruker Vector 22 Fourier transform infrared spectrometer equipped with a liquid-nitrogen-cooled MCT detector and using a germanium ATR plate (Specac) (52 mm x 20 mm x 2 mm) with an aperture angle of 45° as internal reflection element at room temperature. A total of 128 scans were averaged for each spectrum with a nominal resolution of 4 cm-1. Before each spectrum acquisition, a background of ATR crystal was 11 ACS Paragon Plus Environment

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recorded as a single beam at the same conditions and parameters. During the experiments and at least 24h before the measurements, the instrument was continuously purged with dry air. Spectra were processed and areas calculated using the Opus-NT 2.0 software from Bruker. Orientation of CH2 and CD2 chains of α-tocopherol and phospholipids, was calculated as previously described 35-36. Briefly, the orientation of the acyl chains with respect to the ATR surface normal can be calculated by using spectra collected with parallel and perpendicular polarization of the incident IR beam. The CD2 (for the phospholipids) and CH2 (for α-tocopherol) characteristic bands of the IR spectra recorded are used to calculate the dichroic ratio (RATR) determined by the following equation:

=

where and are the integrated areas of the parallel and perpendicular polarized spectra, respectively. From RATR of CH2 and CD2, the corresponding order parameters can be derived by the following equation:

+ − 〈 〉 = − 2 − + 2

where 〈 〉 is the order parameter, RCH2 is the dichroic ratio of the integrated areas of α-tocopherol CH2 asymmetric stretching vibration band at 2920 cm-1, and also the 12 ACS Paragon Plus Environment

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phospholipid CD2 stretching vibration bands both at 2090 cm−1 (symmetric CD2 band) and !

2195 cm−1 (asymmetric CD2 band), and " and ! #

!$ !#

are the ratios of the radiation electric

field vectors, corresponding respectively to 0.853 and 1.147 37. From the previous equation derives the effective tilt of the acyl chain defined in terms of the Legendre polynomial:

〈 〉 =

1 &3 ()) − 1* 2

Where ()) is the angle that α-tocopherol CH2 and phospholipids CD2 chains form with the normal to the ATR plate normal. The effective angle between α-tocopherol and phospholipid acyl chains is the difference between the corresponding ()) . Each sample was analysed in triplicate and expressed as mean value ± standard deviation. 1

H NOESY MAS-NMR 25 mg of phospholipid (DMPC or DPPC) and the appropriate amount of α-

tocopherol (3:1 molar ratio) were used to prepare multilamellar vesicles in deuterated water, using the same procedure described above for X-ray diffraction measurements. The concentration of lipids were 220 mM for DMPC samples and 230 mM for DPPC samples. A Bruker Avance 600 spectrometer, operating at 600 MHz, equipped with an HRMAS probe and using ZrO2 BL4 rotors of 4 mm with Kel-f BL4 caps at 25 ºC. The spin rate was 8 kHz; 1024 data points were obtained from 16 scans; the spectral width was 20 ppm. The relaxation delay was 3.5 seconds and the mixing time was 300 ms. Two-dimensional 13 ACS Paragon Plus Environment

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NOESY experiments were acquired using 90º pulses of 5.5 µs. Data were processed using TopSpin 3.5 software, supplied by Bruker. The location probability was estimated from data obtained at a mixing time of 300 ms, according to a previous work 38 σij=(Aij(tm))/(Ajj(tm) x tm) where σij is the cross-relaxation rate, Aij is the cross-peak volume, Ajj is the diagonal peak volume and tm is the mixing time of the NOESY spectrum. It has been suggested 38 that a single mixing time MAS-NOESY experiment is sufficient for characterizing intermolecular interactions in membranes as concluded from previous extensive work 39. Molecular model The molecular models that are shown in Figure 11 are a representation of αtocopherol localization into a DPPC membrane to graphically recapitulate the results obtained in this study. The figure has been prepared by using PyMol (http://www.pymol.org) based on the DPPC lipid bilayer .pdb file created by Peter Tieleman 40 (http://www.ucalgary.ca/tieleman). The molecules of α-tocopherol were generated by means of the builder module of Pymol.


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Results

X-ray diffraction experiments show that α-tocopherol alters the water layer between bilayers in both DMPC and DPPC. Small-angle X-ray diffraction experiments were performed on samples containing mixtures of the saturated PCs studied here, with α-tocopherol at molar ratios 5:1 (phospholipid/tocopherol) at different temperatures. Fig. 2 depicts a membrane bilayer pattern in all the cases, with first, second and third order peaks correlated as 1:1/2:1/3 41, for the pure phospholipids POPC at 20 ºC (62.9/31.5/21.0 Å), DMPC 8 ºC (63.0/31.4/20.9 Å), DMPC 32 ºC (64.6/32.3 Å), DPPC 25 ºC (65.1/36.2/21.7 Å) and DPPC 50 ºC (66.2/33.0/ 22.1 Å). Very small changes were observed when comparing the first spacings of these saturated lipids between them and at temperatures below Tc (8 ºC-DMPC and 25 ºC-DPPC) and above Tc (32 ºC-DMPC and 50 ºC-DPPC). These d-spacings are composed of the bilayer thickness and the thickness of the water layer between bilayers 42.


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Fig. 2. X-ray diffraction profiles of pure POPC, DMPC and DPPC (A: small-angle and B: wide-angle); and POPC/α-tocopherol, DMPC/α-tocopherol and DPPC/α-tocopherol mixtures (C: small-angle and D: wide-angle). Molar ratio was 5:1 phospholipid/αtocopherol in all the cases. Temperatures are indicated on the diffractograms.


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The addition of α-tocopherol to these phospholipids produced an increase in the d-spacings in all the cases, the first order being now 66.3 Å (POPC), 72.8 Å (DMPC at 8 ºC), 65.1 Å (DMPC at 32 ºC), 74.2 Å (DPPC at 25 ºC) and 69.2 Å (DPPC at 50 ºC). Wide-angle X-ray diffractions were also obtained for these samples, to investigate the organization of the membranes. Diffractograms for samples above the phase transitions, as it was the case of mixtures of pure POPC, pure DMPC (32 ºC) and pure DPPC (50 ºC) and of mixtures of α-tocopherol with POPC, DMPC (32 ºC) and DPPC (50 ºC) (see Fig. 2) were wide and diffuse curves indicating fluid phases. However those diffractograms obtained from samples below the phase transition showed patterns that can be associated with gel phases; in the case of pure DPPC (25 ºC) a sharp reflection at 4.19 Å and a broad shoulder at 4.10 Å can be seen indicating a Lβ´ phase. The sample DPPC/α-tocopherol (25 ºC) showed a reflection different to that of pure DPPC at this temperature but also different from the pattern seen for POPC at the same temperature. This pattern consist on of a broad peak with a maximum centered at 4.20 Å and it may indicate that a reflection coming from the gel phase was superimposed to a broad peak, indicating the presence of a mixture of fluid (Lα) and gel (Lβ) phases. This interpretation is supported by previous studies using DSC showing that the transition phase for this DPPC/tocopherol sample was very broad and going down below 30 ºC 20, 43. The pure DMPC sample (8 ºC) gave place to a sharp reflection at 4.21 Å and a broad shoulder at 4.09 Å can be seen indicating a Lβ´ phase the DMPC/α-tocopherol sample (8 ºC) showed a very broad peak centered at 4.20 Å similar to that of DPPC/α-tocopherol at 25ºC, probably indicating a mixture of fluid (Lα) and gel (Lβ) phases; again this interpretation is supported by previous DSC studies showing a very broad transition for this tocopherol concentration in DMPC 43. 17 ACS Paragon Plus Environment

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To gain more information about the structure of the bilayers and the effect of incorporating α-tocopherol background corrected SAXD data were analyzed using the program GAP and the electronic density profiles were obtained (Fig. 3). From these profiles some interesting parameters were deduced as indicated in Fig. 3 and shown in Table 1. In the case of POPC (at 20 ºC) the increase in d-spacing appearing after the addition of α-tocopherol is explained by an increase in dw, i.e. in the water layer associated to the bilayer, indicating an alteration in the lipid-water interface, probably due to the


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Fig. 3. One dimensional electron density profiles calculated from SAXD profiles at 25 ºC of DMPC at 8 ºC and 32 ºC and of DPPC at 25 and 50 ºC in the presence and in the absence of α-tocopherol (5:1 molar ratio).

insertion of α-tocopherol that alters the hydration of the polar groups of the phospholipids. In the case of DMPC, after the phase transition there was a small increase in the dspacing (63.0 Å at 8 ºC and 64.6 Å at 32 ºC) and an increase in dw (12.0 Å at 8 ºC and 16.6 Å at 32 ºC) and this is due in part to a 3 Å decrease in dHH and dB (i.e. the decrease is reflecting a decrease in the hydrophobic thickness of the membrane). In the case of DPPC there was a small increase in d-spacing as a consequence of the transition (65.1 Å at 25 ºC and 66.2 Å at 50 ºC). It is noticeable that in this case there was an increase of 5.5 Å in dw, bigger than the increase observed for DMPC at a consequence of the phase transition. However the decrease observed for dHH and dB as a consequence of the transition were the same for both DMPC and DPPC. Small cChanges in the membrane dimension were observed as the consequence of the addition of α-tocopherol and the changes in d-spacings observed were mainly due to the increase in the thickness of the water layers between the concentric membranes (dw) as shown in Table 1. In the case of DMPC at 12 8 ºC the addition of α-tocopherol occasioned an increase of 9.8 Å in d-spacing and 9.8 in dw, and the modification in the water layer was the only therefore a significant change. At 32 ºC there was a small increase in d-spacing of only 0.5 Å and an increase in dw of 1.9 Å showing that the change in the water layer was the main effect of α-tocopherol insertion on the parameters of the membrane measured through this technique. Of similar magnitude were the effects observed for DPPC as the


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consequence of incorporating α-tocopherol, and at 25 ºC the increase was of 9.1 Å in both d-spacing and 10.2 in dw. Also at 50 ºC d-spacing was increased in 3.0 Å and dw in 3.4 Å. The largest increases observed in dw as a consequence of the addition of α-tocopherol were seen below the transition temperature for both DMPC and DPPC membranes. Small but significant changes were observed in σC (width of the Gaussian describing the hydrocarbon tails) both as a consequence of the phase transition and because of the insertion of α-tocopherol in the bilayers. In both DMPC and DPPC, changes of about 1 Å can be observed at temperatures below of the phase transition and even lower at temperatures above the phase transition, indicating changes in the hydrophobic part of the membranes, which can be easily discerned examining Fig. 3 and the decrease in electronic density (rho) taking place in these cases within the hydrophobic part of the membrane.


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Table 1. Fitting parameters, σC stands for standard variation (width) of the Gaussian describing the hydrocarbon tails; dHH, headgroup peak-peak distance; dB, total bilayer thickness (polar head plus hydrophobic layer) ;dw, water layer, obtained by using the GAP program, as described in Materials and Methods.

POPC POPC/Toc DMPC DMPC DMPC/Toc DMPC/Toc DPPC DPPC DPPC/Toc DPPC/Toc

T (°C) 20 20 8 32 8 32 25 50 25 50

d (Å) 62.9 66.3 65.0 62.1 66.7 65.1 65.1 66.2 74.2 69.2

σC (Å) 7.7 7.6 5.6 6.2 5.9 6.4 5.5 7.4 6.4 7.1

dHH (Å) 36.2 36.0 40 36 39 36 42 38 42 38

dB (Å) 48 48 52 48 51 48 54 50 54 50

dw (Å) 14.7 18.3 13.0 14.1 15.7 17.1 10.7 16.2 20.9 19.6

The intrinsic fluorescence quenching of α-tocopherol indicates a low quenching efficiency by acrylamide and a higher quenching by 5-doxyl-PC than by 9- and 16-doxyl-PC The quenching of a fluorophore embedded in a membrane informs about its location. In this case two approaches were used. Firstly acrylamide a quencher that does not penetrate in the membrane 44 was used and the quenching efficiency informs about the accessibility of α-tocopherol to the aqueous medium. Fig. 4 shows a plot of fluorescence intensity versus acrylamide concentration, in which three membranes are compared, DMPC, DPPC and POPC all of them containing 16.7 mol% of α-tocopherol. Static quenching can be seen as indicated by upwards plots 45. This static quenching of αtocopherol in membranes has been previously observed and described 30, 34, 46-48. When comparing the three membranes studied, it can be seen that the efficiency of quenching of α-tocopherol follows the order DPPC>DMPC>POPC and this is the accessibility order from the aqueous medium. Note that this order is similar to the alteration observed for dw


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induced by the presence of α-tocopherol that we have noted after the electronic profiles deduced from the X-ray diffraction profiles.

Figure 4. Stern-Volmer plots for the quenching by acrylamide of α-tocopherol fluorescence in large unilamellar vesicles. In the figure (F0/F-1) vs [Q] is plotted and the different KD values are reported, calculated as described in the Materials and Methods section. F0 stands for the fluorescence intensity in the absence of quencher and F for fluorescence intensity in the presence of quencher. Squares, circles and triangles up correspond to POPC, DMPC and DPPC, respectively. Measurements were carried out at 25 ºC (POPC), 30 ºC (DMPC) and 50 ºC (DPPC).

The intrinsic fluorescence of α-tocopherol was also quenched using 5-, 12- and 16-doxylPC with the aim of localizing the chromanol ring in the hydrophobic palisade. These quenchers have been widely used previously for the same purpose. It is assumed that their ability to better quench a fluorophore within the membrane will depend on their proximity to the different doxyl groups located at different depths in the bilayer and quenching will only take place when one of the doxyl groups is close enough to the chromanol ring 30, 49-52. 22 ACS Paragon Plus Environment

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Fig. 5 shows that maximum quenching efficient efficiency is always observed for 5-doxylPC followed for 12-doxyl-PC and 16-doxyl-PC. This implies that the chromanol ring of αtocopherol is relatively close to the lipid-water interface in POPC at 25 ºC, in DMPC at 30 ºC and in DPPC at 50 ºC (all these membranes are fluid).

Fig. 5. Stern-Volmer plots for the quenching by n-doxyl-PC of α-tocopherol fluorescence in large unilamellar vesicles. F0 stands for the fluorescence intensity in the absence of quencher and F for fluorescence intensity in the presence of quencher. The assays were done at 25 ºC in the case of POPC, 30 ºC in DMPC samples and 50 ºC in DPPC samples.

1

H-NMR and 1H NOESY MAS-NMR studies: α-tocopherol is localized similarly in

DMPC and DPPC membranes, perpendicular to the plane of the membrane and with the chromanol ring embedded in the upper part of the hydrophobic bilayer 1

H-NMR-MAS was used to locate the chromanol ring of α-tocopherol in the DMPC

and DPPC membranes. These measurements were only carried out at temperatures above


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the Tc onset transition temperature (30 ºC for DMPC and 50ºC for DPPC) due to low spectral resolution in the gel state. Fig. 6 depicts the 1H-MAS NMR 1D spectrum of the DMPC membrane at 30 ºC in the presence of α-tocopherol (at a 5:1 phospholipid to vitamin E molar ratio) in which the resonances are assigned to different protons of DMPC and vitamin E (an identical assignation was carried out in the DPPC membrane)

Fig. 6. 1H-MAS NMR spectra of DMPC/α-tocopherol (30 ºC) and DPPC/α-tocopherol (50 ºC). Molar ratio was 5:1 phospholipid/α-tocopherol in both cases. The identification of the resonances was done according to the nomenclature for the different carbons used in Fig. 1. Note that underlined letters are used for α-tocopherol in order to avoid confusion with POPC carbons.

A way of inferring the location of the chromanol group is to measure the shifts of the phospholipid signals in the presence of tocopherol in comparison with the pure phospholipids. These shifts are supposed to be due to the ring current of the aromatic


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chromanol group of the vitamin. Fig. 7 shows that all signals arising from the phospholipids are shifted upfield. Shifts can be expected to be bigger from closer proximity to the chromanol ring. It can be seen that in both DMPC and DPPC membranes the biggest shifts come from the signals from C2, C3 and G1, i.e. from the protons bound to the first part of the acyl chains and C1 from glycerol. From the fact that the shifts are upfield we may deduce that the phospholipid protons may be located above of the aromatic ring 53, which is compatible with parallel orientation of the long axis of the vitamin molecule with the main axis of the phospholipid molecule. Nevertheless the mobility of the molecules that form the membrane may explain why there is a notable shift of all the protons.


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Fig. 7. Chemical shifts observed for the different protons of DMPC and DPPC, induced by the presence of α-tocopherol. Data obtained by comparison of spectra shown in Fig. 6 with those of pure phospholipids (not shown).

In order to investigate the location of α-tocopherol in the membrane we used 1HNMR-MAS-2D-NOESY and correlations were established between protons of αtocopherol and those of the phospholipids deduced form the volumes of the observed crosspeaks. 26 ACS Paragon Plus Environment

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The 2D-NOESY spectrum of a DMPC/α-tocopherol sample is depicted in Fig. 8. Although not many resonances originated from tocopherol protons are clearly discernible from those of phospholipids, it was possible to identify three of them corresponding to protons located in the chromanol group that correspond to the three protons of the methyl group (C24) bound to carbon 2 of the chromanol group (labelled as A in Fig. 1), the protons bound to three methyls groups (C25, C26 and C28, labelled as B in Fig. 1) and to the two protons bound to C4 of the chromanol group (labelled as C in Fig. 1).

Fig. 8. 1H-MAS NMR NOESY spectrum of a DMPC/α-tocopherol. Molar ratio was 3:1 phospholipid/α-tocopherol. Temperature was 30 ºC. The spectrum was obtained at a mixing time of 300 ms. Carbons to which protons are bound are indicated. Underlined A, B and C are given to designate the protons bound to carbons of α-tocopherol, as shown in Fig. 1 and in Fig. 6.


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Crosspeaks related to tocopherol protons named as A, B and C in Fig. 1 are indicated in Fig. 8, these symbols being underlined. Crosspeaks volumes are proportional to the proximity between phospholipid and αtocopherol protons but to correctly measure these interactions it is necessary to analyze cross-relaxation rates that will inform about phospholipid-tocopherol contacts 54. The volumes of crosspeaks between protons of phospholipids and α-tocopherol were plotted, as shown in Fig. 9, versus the different phospholipid groups ordered after their location from the center of the bilayer to the membrane lipid-water interface. In the case of DMPC/α-tocopherol the highest cross-relaxation rates are seen for protons A, B and C with C3, C2 and CH3, indicating that the chromanol ring is predominantly located in the half of the acyl chain close to the polar interface. In the case of DPPC/α-tocopherol again a similar location can be deduced although protons B and C do not show proximity to CH3, i.e. to the methyl groups. The difference between DMPC and DPPC is that if the hydroxyl group of α-tocopherol is anchored to the lipid-water interface in both membranes, the chromanol ring will be closer to the terminal methyl groups of the acyl chains in the DMPC membrane than in the DPPC membrane due to the shorter chains of the former phospholipid. It should be expected that protons A should be closer to the methyl groups to the phospholipids if α-tocopherol is anchored in the lipidwater interface and with its main axis perpendicular to the plane of the membrane, as it is actually seen for the DPPC membrane (see Fig. 1 and Fig. 9).


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Fig. 9. Cross relaxation rates obtained from the analysis of the 1H-NMR NOESY spectrum of DMPC/α-tocopherol and DPPC/α-tocopherol. Cross-relaxation rates corresponding to the protons detected are shown for the α-tocopherol carbons identified in each panel with respect to the protons corresponding to DMPC carbons depicted in ordinates DMPC proton assignments: N(CH3)3 (γ), N-CH2-CH2 (β), CH2-CH2-O (α), O-CH2-CH (G3), CH2-CHCH2 (G2), CH-CH2-O (G1), CH2-CH2-COO (C2), CH2-CH2-COO (C3), CH2=C, CH=CH, (CH2) and CH3. The same protons of DPPC were studied. Mean values ±standard deviations (5 determinations).

Orientation of α-tocopherol into DPPC and DMPC membrane: the tail chain lies nearly parallel to the acyl chains of DMPC and DPPC ATR-FTIR spectroscopy was used in this work to get an estimation of the angle formed by α-tocopherol with the fatty acyl chains of DPPC and DMPC when inserted into the membrane. The tilts of both α-tocopherol hydrophobic chain and phospholipid acyl 29 ACS Paragon Plus Environment

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chains with respect to ATR-FTIR crystal surface were calculated thanks to the different infrared absorption of CH2 and CD2 groups of α-tocopherol and deuterated phospholipids, respectively. The bands close to 2195 cm−1 (antisymmetric CD2 stretching band) and 2090 cm−1 (symmetric CD2 stretching band) were used to assess the lipid orientation, while from the band at 2920 cm-1 (antisymmetric CH2 stretching band), information on α-tocopherol orientation was obtained. In order to calculate the effective angle that CH2 and CD2 chains form with respect to the ATR plate normal, polarized and dichroic spectra were analysed 36. For a qualitative estimation of the chain orientation, dichroic spectra were computed by subtracting the perpendicular-polarized spectra from the parallel-polarized spectra, after multiplication of the perpendicular-polarized spectra by the dichroic ratio of lipid C=O bands, Riso (Fig. 10). It should be taken into account that looking at the absorption of CH2 groups of α-tocopherol means that we are practically looking at the tail chain, since 8 out of 10 are located there: only CH2 groups corresponding to carbons 3 and 4 are located in the chromanol group, whereas those corresponding to carbons 11, 13, 15, 16, 17, 19, 20 and 21 are in the tail. A preferential orientation of both α-tocopherol and phospholipids was revealed by the presence of positive and negative peaks in the dichroic spectra, whereas a flat line would have indicated the absence of orientation 55. In particular, the evident negative peaks of DPPC reflect a strong preferential orientation for this lipid near the normal to the ATR plate. Quantitative information on the effective chain tilt was obtained from the polarized spectra. The order parameters, calculated as previously described

35, 56

, were used to

estimate the effective angle of α-tocopherol and lipid chains according to the following +

equation: 〈 〉 = &3 ()) − 1*, where 〈P2(cosθch)〉 is the order parameter 30 ACS Paragon Plus Environment

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and θeff is the effective tilt between CH2 and CD2 chain of α-tocopherol, DMPC and DPPC, and the ATR plate normal (Table 2). For DMPC membrane, it was calculated that the lipid chains are tilted at an angle of θeff = 38 ± 2°, corresponding to an order parameter of 〈P2(cosθch)〉 = 0.424 ± 0.053, indicating that the membrane is quite well ordered. Inserted in this membrane, α-tocopherol forms an angle θeff of 49 ± 1°, which reflects an effective tilt at 11 ± 2° with respect to DMPC CH2 chains. Similarly, DPPC membrane also shows a good order and orientation, being 〈P2(cosθch)〉 = 0.628 ± 0.018 with a corresponding θeff of 30 ± 1, while the angle that α-tocopherol makes with the ATR plate normal was estimated to be 46 ± 3°. Thus, in DPPC membrane α-tocopherol forms a calculated tilt angle with the DPPC acyl chains of 16 ± 4°. All the angles and parameters including the dichroic ratios (RCH2 and RCD2) are reported in Table 2.

Fig. 10. ATR-FTIR spectra of α-tocopherol inserted into DMPC (left) and DPPC (Right) for parallel (//) and perpendicular (⊥) orientation of the polarizer. The dichroic spectra (// Riso ⊥) were obtained by subtracting the parallel from the perpendicular polarized spectra after multiplication of the perpendicular spectra by the dichroic ratio. All the spectra are shown in the 3000–2880 cm-1 and 2270–2040 cm-1 regions and at the same scale. 31 ACS Paragon Plus Environment

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Table 2. Parameters obtained from the ATR-FTIR spectra of Figure 10. RATR (dichroic ratio), SL and angle of the lipid/tocopherol chains with respect to ATR plate normal at RT. R is the dichroic ratio; P2 represents the order parameter and θ is the effective angle.

Phospholipid

Tocopherol

RCD2 (2195-2090) 〈P2(cosθcd)〉 θeff (°) RCH2 (2920) 〈P2(cosθch)〉 θeff (°) ∆θeff

DMPC-d54/Tocopherol (3:1) 1.388 ± 0.061 0.424 ± 0.053 38 ± 2 1.773 ± 0.006 0.141 ± 0.004 49 ± 1 11 ± 2

DPPC-d62/Tocopherol (3:1) 1.176 ± 0.018 0.628 ± 0.018 30 ± 1 1.665 ± 0.123 0.177 ± 0.088 46 ± 3 16 ± 4


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Discussion

Previous works on the location of α-tocopherol in DPPC and DMPC membranes have been carried out. With respect to DPPC, Katsaras et al. 57 used small-angle X-ray diffraction to conclude that the chromanol ring of δ-tocopherol is in the vicinity of the glycerol backbone–headgroup region. This observation is compatible with model (b) proposed by Fukuzawa et al. 14. The quenching of the intrinsic fluorescence of α-tocopherol in DPPC was studied by using acrylamide and some doxyl stearates and it was observed that acrylamide has a limited access to the chromanol ring in comparison with that in SDS micelles. The highest quenching was observed for 5-doxyl-stearate in comparison with 9doxyl-stearate and 16-doxyl-stearate 24. Again these results may be compatible with model (b). Using FTIR and DSC it was deduced that α-tocopherol in DPPC membranes may position its hydroxyl group near the polar head group of the phospholipid 20. More recently some studies from a group have appeared that claim different locations for α-tocopherol in DPPC. After this group, α-tocopherol will be located in DPPC and also in POPC membranes, with the C5-methyl group well above the hydrophobic/hydrophilic interface with the chromanol group residing in among the lipid choline headgroup 27. They claim a similar location in the case of POPC in a later paper and they also claimed that the tail resides far from the center of the bilayer in the case of POPC 29. We have contradicted the conclusion of the location of the chromanol ring in a recent paper with respect to POPC 30, since we interpreted our results in agreement with a number of previous works concluding that model (b) is the most appropriate one. A very similar conclusion has been reached recently from experiments in vitro and in silico in which α-tocopherol was incorporated in 1,2-dioleoyl-sn-glycero-3-phosphocholine 58. In this work we reach a similar conclusion 33 ACS Paragon Plus Environment

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with respect to DPPC, interpreting the results as α-tocopherol having location type (b) and further we show that the tail is nearly parallel to the bilayer normal, after our ATR-IR results, again in disagreement with the model suggested by Marquartd et al. 29. With respect to DMPC, it was observed, in experiments in which the intrinsic fluorescence of the chromanol ring of α-tocopherol was quenched by acrylamide, that the quenching was poor, indicating that the chromanol group was protected from the water compartment and using quenching by 5-NS, 7-NS, 12-NS and 16-NS it was found that 5NS was the most efficient quencher of both 14, 26. This result is compatible with model (b) proposed by these authors. More recently a different location was located for this type of membrane. It was proposed that DMPC constitutes a remarkable exception to αtocopherol´s membrane presence and that it is located deep in the membrane, with the main axes of the molecule nearly perpendicular to the bilayer normal 28. This is a surprising and striking result. With respect to the location of α-tocopherol in DMPC membranes we are also in disagreement since our results point out again to a location in concordance with model type (b). Nevertheless the results obtained with DMPC using NMR points to the possibility of the chromanol ring occupying more than one position, explaining why it shows a certain proximity to the terminal methyl group of the acyl chains, but this is not supported by the experiments in which the intrinsic fluorescence of this group was quenched. The last experiments showed a good accessibility to acrylamide and a clearly better quenching by 5-doxyl-PC. In any case we conclude that the preferred disposition is with the chromanol ring located in the part of the hydrobophic palisade and close to the lipid-water interface, so that the hydroxyl group binds to water. A similar disposition can be deduced for α-tocopherol in DPPC. 34 ACS Paragon Plus Environment

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Our results with DMPC and DPPC obtained by using SAXD, from which electronic profiles were calculated do not show an increase in electronic density in the hydrophobic part of the membrane. If the localization of α-tocopherol would be deep in the bilayer as suggested 28, one would expect an increase in the electronic density (rho) in the central region of the bilayer, which is not observed. From the electronic profile it was deduced that the main effect of the inclusion of α-tocopherol in both of these membranes was to alter the thickness of the water layer that separates different bilayers, suggesting a perturbation of the lipid-water interface and therefore a certain proximity of α-tocopherol molecule to the surface, in disagreement with the claiming that proposes that α-tocopherol is located deep in the membrane in the case of DMPC 28. The alteration of the thickness of the water layer observed for both DMPC and DPPC may reflect changes in the structure of the polar part of the membrane. Other molecules incorporated in membranes, which are assumed to perturb the lipid-water interface because they possess polar groups that will tend to interact with the water layer, produced an increase of the water layer, as it was observed to be the cases for curcumin 59 or a lipopeptide 60. Changes in the hydration of the polar part of the membrane have been described for α-tocopherol in previous works. In this way it was observed that vitamin E produced a shift towards lower wavenumbers in the infrared spectra of the carbonyl band indicating an increase in the proportion of hydrated phospholipid carbonyl groups 20. It was also shown that α-tocopherol may affect to the Lα to HII phase transition of 1,2-dielaidyl-sn-glycero-3phosphoethanolamine, promoting the destabilization of the membrane, which indicates a perturbation of the lipid-water interface 61. Very interesting with respect to the last work is that α-tocopherol and not α-tocopheryl acetate may promote transition to HII phase, which 35 ACS Paragon Plus Environment

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by itself will not occur when heating this saturated phospholipid underlining the importance of the hydroxyl group of α-tocopherol to perturb the lipid-water interface; α-tocopherol was also shown to increase the hydration of phosphatidylserines, making more difficult their fusion induced by Ca2+, which depends on the dehydration of the polar group of the phospholipids 62 With respect to the possible exceptionality of the location of α-tocopherol in dimyristoyl-phospholipids it is pertinent to remember that it was shown that the binding of 45

Ca2+ to 1,2-dimyristoyl-sn- glycerol-3-phosphoserine was decreased by the presence of α-

tocopherol, so that the number of binding sites was decreased, indicating a perturbation of the lipid-water interface and a higher hydration making more difficult the dehydration necessary for the approaching of the calcium ions 63.These observations pointed to the presence somehow of α-tocopherol in the lipid-water interface. It should be also commented that the increase in the water layer thickness was similar for both DMPC and DPPC; an explanation may come from our previous observation on this system, indicating that the effect of α-tocopherol on the hydration of the carbonyl groups of these phospholipids was especially big below of the phase transition 20. In the cases of both DMPC at 8 ºC and DPPC at 25 ºC a big increase was observed in the thickness of the water layer, going from 12.0 to 21.8 Ǻ for DMPC and from 10.7 to 20.9 Ǻ for DPPC; this remarkable increase can be explained because two effects are added in this case, the fluidification induced by α-tocopherol, as evidenced by the WAXD data and also the incorporation of vitamin E and both of them increase the thickness of the water layer. Note that more moderate, but noticeable, increases were observed for fluid membranes, as


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it was the case of POPC (an increase of 3.6 Ǻ at 20 ºC), DMPC (an increase of 1.9 Ǻ at 32 ºC) and DPPC (an increase of 3.4 Ǻ at 50 ºC). The fluorescence quenching by acrylamide indicated that the chromanol group is not readily accessible from the water compartment in either DMPC or DPPC membrane, in agreement with previous results 14 and the quenching by doxyl-PCs showed a closer proximity to 5-doxyl-PC than to 9-doxyl-PC and to 16-doxyl-PC, in agreement with previous reports 14, 26. These quenching results indicate a location according to model (b) as it was concluded recently for POPC and polyunsaturated phospholipids 30. 1

H-NMR MAS studies showed again in both DMPC and DPPC membranes higher

shifts of the proton resonances bound to the first carbons of the acyl chains and the glycerols of the phospholipids. Cross-relaxations rates obtained by 2D-NOESY spectra 1HNMR-MAS, revealed bigger correlation of protons bound to the chromanol group with the first carbons of the acyl chains. It should be noted that there is a big mobility of the molecules, especially in a fluid membrane. By this reason shifts of all protons of the phospholipids produced by the chromanol group of α-tocopherol are detected, although some are more pronounced than others, indicating the preferred location. Broad distributions in the membrane of bound small molecules are frequently observed 39, 64-65 as it should be expected given the high degree of molecular disorder and structural heterogeneity. What it is defined here for α-tocopherol, on the basis of the NMR experiments, is a maximum of distribution of the molecule in the bilayer, i.e. the average localization. In this context it should be said that the cross relaxation rates obtained between the protons bound to the chromanol ring of α-tocopherol and those to the terminal methyl are larger for DMPC than for DPPC (Fig. 9). This suggests that eventual 37 ACS Paragon Plus Environment

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movements of the chromanol ring of α-tocopherol allowing its relative proximity with the center of the bilayer may be more likely to occur than in the case of the DPPC membrane, although the predominant disposition in both cases is with the chromanol group not very far away from the lipid-water interface. It can be reasoned that since the DMPC membrane is thinner than that of DPPC it could be more probable that the mobility of the chromanol ring could allow it to transiently locate nearer the methyl group than in the case of the DPPC membrane. Finally ATR-IR spectroscopy was used to study the orientation in the membrane of α-tocopherol molecule. Since α-tocopherol is mixed with perdeuterated DMPC and DPPC when looking at the CH2-methylene groups of the vitamin E, we are observing practically only the tail chain since 8 out of a total of 10 of these groups are located in the tail. The conclusion is that the tail chain lies nearly parallel to the fatty acyl chains in both DMPC and DPPC membranes. This conclusion does not agree with the recent claim of 28 that depicts this tail group as nearly parallel to the plane of the membrane. Other authors used DSC to study the interaction of α-tocopherol with DMPC and they claimed that the strong effect observed in the transition with a shift towards lower temperatures of the transition and a considerably widening of the transition peak could be due to the disposition of α-tocopherol in the center of the bilayer with its main axis perpendicular to the fatty acyl chains of the phospholipids 66. However this effect is almost identical to those observed using DSC for DPPC and 1,2-disteraoyl-sn-glycero-3phosphocholine 20, 67. It should be mentioned that when a molecule is located in the center of the bilayer its effect on the phase transition is not so big, as it is case of ubiquinone-10 68. They further claimed a disposition of α-tocopherol with its main axis parallel to the plane 38 ACS Paragon Plus Environment

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of the membrane observing the microscopic dynamics of DMPC by using neutron scattering, however it is clear that α-tocopherol must affect to DMPC dynamic but no specific and direct proof for the location of α-tocopherol was provided 66. The ratio phospholipid/tocopherol used in this work, and in many other studies, is substantially lower than what can be determined for biomembranes. This ratio was examined for a number of original papers in some reviews 4, 69 and it can be in the order phospholipid/tocopherol molar ratio of 65:1, at most with the greatest concentrations in the Golgi membranes and lysosomes 70-71. More recently mass spectroscopy has been used for the study of the presence of tocopherol in neuronal membranes and it has been observed that vitamin E is more concentrated in soma-neurite junction with about 165% of the phospholipid ratio observed across the cell 72. However the use of many physical techniques requires the employ of much higher concentrations of α-tocopherol, although obviously this must be considered as an approach that has its evident limitations. It has also been reported that α-tocopherol in model membranes formed by a mixture of phospholipids preferentially partitions into the most fluid domain 43, 73. In any case the concentrations used in biophysical studies are considerably higher than those so far detected in cells. Our results about the location of α-tocopherol, especially with respect to the DMPC membrane, differ from those previously published in a paper 28. Altough we cannot offer a concluding explanation for this discrepancy, it should be examined whether the mode of making the measurements could be involved. For instance, the neutron diffraction studies carried out in that paper are done on stacked flat bilayers on silicon crystals, which is a set up totally different from those used here in X-ray diffraction, fluorescence spectroscopy, NMR and ATR-FTIR spectrocopies. However we want to stress that, as described above, 39 ACS Paragon Plus Environment

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there is a wealth of evidences indicating that α-tocopherol, when incorporated to 1,2dimyristoyl phospholipids perturbs the lipid-water interface and the hydroxyl group plays an essential role on the effect. Conclusion All these data, together with the previous results described above, led us to conclude that the location of α-tocopherol in DMPC and DPPC membranes is similar to the previously described by us in POPC and polyunsaturated membranes, with the hydroxyl group anchored into the lipid-water interface and with the tail chain nearly parallel to the fatty acyl chains, which will be located (b) as proposed by 14. An illustration of the suggested location of α-tocopherol, according with the results presented in this work and a comparison of the different proposed models is shown in Fig. 11. The importance of the hydroxyl group for the location of α-tocopherol in membranes was illustrated by the comparison with α-tocopheryl acetate which was shown to interact in a markedly different way with DPPC, occupying a much more hydrophobic location 20.


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Fig. 11. Membrane of DPPC plus α-tocopherol (in orange), showing the different localizations suggested for the saturated membranes studied in this work and previously discussed 14.

A molecule that has been described to occupy a central location in the membrane due to be very long is ubiquinone-10 22, 68, 74-76. But α-tocopherol is a molecule not so long. Moreover it has been described that once ubiquinone-10 is reduced to ubiquinol-10 it changes its location in a way that the hydroxyl group goes to the lipid-water interface, making a sort of snorkeling 22, 75. Conflicts of interest. There are not conficts of interest to declare.

Acknowledgements. We thank Ms. Monika Schneider for her help with some preliminary NMR experiments. This work was supported by grant 368 from Universidad de Murcia.


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