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Structure of Mixed Multilayers of Palmitoyloleoylphosphatidylcholine and Oligo(oxyethylene glycol) Monododecyl Ether Determined by X-ray and Neutron Diffraction G. Klose,*,† A. Islamov,‡ B. Ko¨nig,†,§ and V. Cherezov‡ Department of Physics, University of Leipzig, Linne´ str.5, 04103 Leipzig, Germany, and Laboratory of Neutron Physics, Joint Institute of Nuclear Research, Dubna, Russia Received May 17, 1995. In Final Form: August 10, 1995X The structure of oriented multilayers of the zwitterionic phospholipid palmitoyloleoylphosphatidylcholine containing nonionic surfactants (oligo(oxyethylene glycol) monododecyl ethers, C12En with n ) 2, 4, and 6) was studied by X-ray and neutron diffraction. Investigations were done at T ) 20 °C in the lamellar liquid-crystalline phase with surfactant/lipid molar ratios RA/L ) 0.5 and 1 and hydration levels corresponding to 85 and 97% relative humidity. The transbilayer distribution of the surfactant was determined by neutron diffraction using selectively deuterated C12En. The incorporation of surfactant causes a decrease of the repeat distance and of the thickness of the hydrocarbon core of the membrane. The effect increases with the number of oxyethylene units and with the concentration of the surfactant as well as with the relative humidity. The R-methylene group of the surfactants is anchored near the boundary of the hydrophobic core. The oxyethylene moieties are mainly located in the polar membrane/water interface region. The impact of molecular disorder on the diffraction data is discussed.
Introduction Ternary mixtures of phospholipid, nonionic surfactant, and water form a variety of mesophases (see e.g. refs 1-4 and literature cited therein). Recently, the phase diagrams of palmitoyloleoylphosphatidylcholine, di- and tetra(ethylene oxide) dodecyl ethers, respectively, and water have been studied in detail by using phosphorus and deuterium nuclear magnetic resonance techniques.3,4 Although the chemical shift anisotropy of the phosphorus of the lipid and the deuterium quadrupole splitting of 2 H2O as well as deuterium-labeled moieties of the surfactant did provide some insight into the structure of mixed lipid/surfactant/water complexes formed, the information obtained is rather indirect. In addition, fluorescence techniques were used to estimate the area per surfactant molecule in the membrane/water interface for the lamellar liquid-crystalline phase.5,6 Previous data suggest that structural changes of the bilayer in the liquid-crystalline lamellar phase are induced by changing molar surfactant/ lipid ratio, water concentration, and oxyethylene chain length.3-8 However, the detailed structure of the mixed bilayers is not known yet. Neutron and X-ray diffraction on oriented multilamellar membrane stacks are well-established methods for the determination of the bilayer structure perpendicular to the membrane surface. Both X-ray and neutron diffraction †
University of Leipzig. Joint Institute of Nuclear Research. § Current address: Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892. X Abstract published in Advance ACS Abstracts, November 15, 1995. ‡
(1) Sadaghiani, A. S.; Khan, A.; Lindman, B. J. Colloid Interface Sci. 1989, 132, 352. (2) Ma¨dler, B.; Klose, G.; Mo¨ps, A.; Richter, W.; Tschierske, C. Chem. Phys. Lipids 1994, 71, 1. (3) Funari, S. S.; Klose, G. Chem. Phys. Lipids 1995, 75, 145. (4) Klose, G.; Eisenbla¨tter, S.; Ko¨nig, B. Colloid Interface Sci. 1995, 172, 438. (5) Lantzsch, G.; Binder, H.; Heerklotz, H. J. Fluoresc. 1994, 4, 339. (6) Lantzsch, G.; Binder, H.; Heerklotz, H.; Wendling, M.; Klose, G. Biophys. Chem., in press. (7) Ko¨nig, B. Ph.D. Thesis, University of Leipzig, Germany, 1992. (8) Ko¨nig, B.; Klose, G. Prog. Colloid Polym. Sci. 1993, 93, 279.
0743-7463/96/2412-0409$12.00/0
data have been recorded because of the quite complementary sensitivity of the methods for different structural features of the membrane.9 In addition, neutron diffraction allows one to map out regions of interest in the membrane profile by deuterium labeling. Measurements of the hydration force between mixed lipid/oligo(ethylene oxide) surfactant multilayer stacks are currently performed in our laboratory. The proper interpretation of the results of these experiments requires a detailed knowledge of the structure of the lipid/surfactant membrane and in particular of the polar membrane/water interface.7 In this paper we report on some structural properties determined by neutron and X-ray diffractions of mixed palmitoyloleoylphosphatidylcholine and oligo(ethylene oxide) monododecyl ether membranes at different compositions. Materials and Methods Substances. The synthetic lecithin 1-palmitoyl-2-oleoyl-snglycero-3-phosphatidylcholine (POPC) was purchased from Enzymatics/GB and checked for purity using thin layer chromatography. The oligo(ethylene oxide) dodecyl ethers of the general type C12H25O(CH2CH2O)nH (C12En) with n ) 2, 4, and 6 were provided by Nikko Chemicals, Tokyo, Japan, and were used as obtained. The partially deuterated surfactants C11H23C2H2O(CH2CH2O)nH (C12En-d2) and C12H25O(C2H2C2H2O)nH (C12En-d4n) with n ) 2, 4, and 6 were synthesized by etherification.10 Perdeuterated oligo(oxyethylene glycols) and CH3(CH2)10C2H2Br were both synthesized as described in ref 11. Nondeuterated oligo(oxyethylene glycols) and dodecyl bromide were purchased from Merck (Germany). Oriented Multilayers. A POPC/C12En dispersion in ethanol (5% by wt) was spread on a clean quartz slide (24 × 24 mm2). After initial evaporation of the solvent at T ) 35 °C, a vacuum of about 5 Pa was applied for 2 h to remove the remaining solvent. Overnight storage of the sample at high relative humidity (RH) improved the orientation. The final mosaic spread was typically 0.5° (full width at half-maximum, fwhm), as judged from X-ray rocking curves. Samples were mounted in the X-ray or neutron (9) Wiener, M. C.; White, S. H. Biophys. J. 1992, 61, 434. (10) Wrigley, A. N.; Stirton, A. J.; Horward, E. J. Org. Chem. 1960, 25, 439. (11) Schnabel, R. J. Labelled Compd. Radiopharm. 1992, 31, 91.
© 1996 American Chemical Society
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beam, respectively, in sealed and thermostated chambers containing a reservoir of a saturated aqueous solution of KCl (RH ) 85%) or K2SO4 (RH ) 97%) to control relative humidity. All experiments were done at 20 °C. The samples were allowed to equilibrate for at least 8 h prior to each measurement. Diffraction Experiments. Neutron diffraction measurements were made at the DN-2 diffractometer at the pulsed reactor IBR-2 in Dubna (Russia). Data were recorded in the time-offlight mode, and a one-dimensional, position sensitive detector was used (for details see ref 12). X-ray diffraction experiments were conducted with nickel-filtered Cu KR radiation at a PW 3020 diffractometer (Philips, Germany) operated in the θ-2θ mode. The divergency of the line-shaped primary beam was 0.25°. The diffracted intensity was recorded in stepwise mode by means of a proportional counter. All diffraction patterns were typical for lamellar structures. Four (for X-rays) or five (for neutrons) orders were detected. The repeat distance d of the membrane stack was derived from the position of the Bragg peaks with a precision of better than (0.5 Å. The modulus of the structure factors F(h) of the membrane unit cell is equal to the square root of the corrected integral intensities I(h), where h is the diffraction order. Their precision was (5%. The neutron data were corrected for absorption effects,13 and an inverse Lorentz factor correction of h was applied.14 The X-ray intensities had to be corrected for the geometrical mismatch of the sample size against the effective beam cross section at very low angles. The inverse Lorentz factor correction was sin 2θ.15 The phase angles for centrosymmetric structures are either 0 or π so that F(h) ) (|F(h)|. The phases were determined using the isomorphous H2O-2H2O exchange technique (neutron data only)16 and the swelling method.15 Data Analysis. The one-dimensional scattering density profile F(z) of the unit cell on an arbitrary relative scale, where z is the coordinate normal to the membrane plane, is given by hmax
F(z) )
∑ F(h) cos(2πhz/d)
(1)
h)1
The neutron scattering length density profiles for one set of samples (identical composition but C12En differently deuterium labeled) were scaled to the same arbitrary relative scale. A correction factor k was determined for each sample prior to the neutron measurements by X-ray diffraction, assuming identical electron density profiles for the sample set. The locations of the specifically deuterated surfactant moieties in the unit cell F1(z) were obtained by difference Fourier analysis: hmax
F1(z) )
∑ [F (h) - kF D
h)1
H
(h)] cos
2πhz d
(2)
where D and H denote lipid/surfactant mixtures with partially deuterated and nondeuterated surfactants, respectively. The mean position and distribution of the R-C2H2 moiety of C12En were determined by fitting a Gaussian peak to the data in reciprocal space as proposed by Bu¨ldt et al.17 In addition, a strip-function approach18 was applied to model the distribution of the deuterium-labeled groups F1(z). Model profiles Ftheor(z) were assembled from one (R-C2H2 moiety) or two ((C2H2C2H2O)n moiety) pair of strips, each one characterized by its position ((zi), width (2∆zi), and height (pi). Ftheor(z) is zero outside the strips. The structure factors of this model are as follows (see, for example, ref 18): (12) Klose, G.; Ko¨nig, B.; Gordeliy, V. I.; Schulze, G. Chem. Phys. Lipids 1991, 59, 137. (13) Arndt, U. W.; Willis, B. T. M. Single Crystal Diffractometry; Cambridge University Press: Cambridge, 1966. (14) Buras, B.; Gerward, L. Acta Cryst. 1975, A31, 372. (15) Franks, N. P. J. Mol. Biol. 1976, 100, 345. (16) Zaccai, G.; Blasie, J. K.; Schoenborn, B. P. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 376. (17) Bu¨ldt, G.; Gally, H. U.; Seelig, A.; Seelig, J.; Zaccai, G. Nature (London) 1978, 271, 182. (18) Worthington, C. R. Biophys. J. 1969, 9, 222.
Ftheor(h) )
2d πh
∑
pi cos
2πhzi d
i
sin
2πh∆zi d
(3)
The strip-function parameters were fitted to the experimental data in reciprocal space by minimizing the crystallographic R factor:
R)
∑|F
theor
(h) - Fexp(h)|
∑|F
exp
(h)|
(4)
The final value of R was always smaller than 0.06.
Results The neutron scattering length density profiles of POPC/ C12En, POPC/C12En-d2, and POPC/C12En-d4n membranes with surfactant/lipid molar ratios RA/L ) 0.5 and 1.0 as well as the difference profiles corresponding to the distribution of the R-CH2 or (CH2CH2O)n moieties of the surfactant, respectively, are shown in Figures 1 and 2. [Increasing the surfactant concentration or/and reducing the relative humidity, especially in cases of C12E2 and C12E6 causes other phases to come into existence (paper in preparation).] The electron density profiles are represented together with the strip-function profiles calculated on the basis of the neutron diffraction data in Figure 3. The major peaks in the X-ray profile of hydrated phospholipid multilayers correspond to the electron-dense phosphate groups, whereas the hydrogen-deficient fatty acid ester groups give the major peaks in the neutron profile.19 Even though we have studied mixed POPC/ C12En membranes, the moderate scattering density values expected for nondeuterated C12En should shift the major peaks only marginally. The distance of the major peaks from the membrane center (z ) 0) is referred to as the phosphate distance dP (X-ray scattering density profile) or fatty acid ester group distance dE (neutron scattering density profile). The values of dP together with the repeat distance d are collected in Table 1. The distance between the phosphate group and the fatty acid ester group in the same leaflet (dp - dE) was found to be 4 ( 1 Å in all cases studied, regardless of whether the surfactant was present in the membrane. Worcester reported a similar value of 4-5 Å for egg phosphatidylcholine.20 The repeat distance d and the phosphate distances dP of the mixed membranes are smaller than those of the pure POPC membrane. The thinning is more pronounced for RA/L ) 1 than for RA/L ) 0.5. Both d and dP decrease with increasing length n of the oxyethylene chain at constant RA/L and RH. An increase of the relative humidity from 85 to 97% does enlarge the repeat distance of a given POPC/C12En mixture while there seems to be little or no effect on dP. Table 1 also contains the mean distance dR from the center of the membrane as well as the 1/e half width ν of the distribution of the R-CH2 group of C12En as obtained from the Gaussian fit. The R-methylene group of the surfactant is located closer to the membrane center than the phosphate group. The distance between these moieties (dP - dR) amounts to about 6.0 ( 1.2 Å. Only for C12E6 (RA/L ) 1, RH ) 97%) does it seem to be smaller. The results of the strip-function modeling are shown in Table 2. The parameters a1 and a2 correspond to the total width of the strips with nonzero scattering length density (19) Franks, N. P.; Lieb, W. R. J. Mol. Biol. 1979, 133, 469. (20) Worcester, D. L. In Biological Membranes; Chapman, D., Wallach, D. F. H. Eds.; Academic Press: London, 1976; Vol. 3, p 1.
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Figure 1. Neutron scattering length density profiles of POPC/C12En (- - -), POPC/C12En-d2 (- · -), and POPC/C12En-d4n (‚‚‚) membranes as well as the R-C2H2 group (s) and the (C2H2C2H2O)n moiety (-‚‚-) in membranes with RA/L ) 0.5 hydrated at RH ) 85% (left) and 97% (right) at T ) 20 °C. In regions where curves overlap, only one is drawn.
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Figure 2. Neutron scattering length density profiles of POPC/C12En (- - -), POPC/C12En-d2 (- ‚ -), and POPC/C12En-d4n (‚‚‚) membranes as well as the R-C2H2 group (s) and the (C2H2C2H2O)n moiety (-‚‚-) in membranes with RA/L ) 1 hydrated at RH ) 85% (left) and 97% (right) at T ) 20 °C. In regions where curves overlap, only one is drawn.
used to model the distribution of the R-C2H2 group and the (C2H2C2H2O)n moiety, respectively, of C12En in one half of the unit cell (cf. also Figure 3). A1 and A2 are the total areas of the strips which gave the best fits for the distribution of the R-C2H2 group and the (C2H2C2H2O)n moiety, respectively. If a strip function is a geometrically appropriate model for the distribution of the deuteriumlabeled moieties in the membrane, the area of a strip, A ) 2∆zipi, is proportional to the number of deuterium atoms represented by this strip. To make sure that the area values obtained for different sample sets (C12En with different n but same RA/L) are on the same scale, the raw
values were corrected with respect to the number of surfactant molecules in the samples. The distribution widths a1 and a2 of the labeled moieties strongly increase with both surfactant concentration in the membrane and relative humidity. A prolongation of the length of the oxyethylene chain not only increases a2 but also a1. The strip-function area A1 is about 2 for samples with RA/L ) 0.5 and about 3.8 for samples with RA/L ) 1 (except for C12E6 at RH ) 97%). The agreement between the ratio of these area values and the surfactant concentration ratio
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Figure 3. Electron density profiles of POPC/C12En membranes and strip functions representing the R-CH2 group and the (CH2CH2O)n moiety of the surfactant in POPC/C12En membranes with RA/L ) 0.5 (above) and 1 (below) at T ) 20 °C. Relative humidity was 85% (left column) and 97% (right column). Only one half of the unit cell is shown.
is surprisingly good, considering the experimental uncertainties involved. The ratio A2/A1 should be 4, 8, and 12 for C12En with n ) 2, 4, and 6, respectively. We found approximately these values for most of the samples with RA/L ) 0.5. However, the A2/A1 values obtained for samples with RA/L ) 1 mostly do not satisfy the prediction.
Discussion The particular strip-function approach used here to model the neutron scattering length density difference profiles F1 requires that the deuterium-labeled groups are excluded from the membrane center. This condition is essential to adjust the zero level of the otherwise arbitrary scattering length density scale. It is certainly satisfied,
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Table 1. Structural Parameters of POPC/C12En Membranesa RA/L
n
d (RH ) 85%) (Å)
dp (RH ) 85%) (Å)
dR (RH ) 85%) (Å)
ν (RH ) 85%) (Å)
d (RH ) 97%) (Å)
dp (RH ) 97%) (Å)
dR (RH ) 97%) (Å)
ν (RH ) 97%) (Å)
0 0.5
0 2 4 6 4 6
52.2 51.2 49.4 49.0 47.5 46.3
20.3 19.6 18.6 17.5 17.5 16.5
13.8 13.3 12.0 12.4 11.6
6.0 5.6 5.0 5.4 5.4
54.2 53.2 51.7 51.6 50.1 49.5
19.7 18.5 18.2 17.5 17.0 16.5
12.3 9.8 11.9 12.0 12.9
6.5 6.0 5.6 5.6 5.6
1
a Variables: d, repeat distance; d , distance of the maximum X-ray scattering profile from the membrane center; d and ν, distance of p R the R-methylene group of the surfactant from the membrane center and its distribution parameter, respectively.
Table 2. Parameters of the Strip-Function Modela RA/L
n
a1 (RH ) 85%) (Å)
a2 (RH ) 85%) (Å)
A1 (RH ) 85%)
A2/A1 (RH ) 85%)
a1 (RH ) 97%) (Å)
a2 (RH ) 97%) (Å)
A1 (RH ) 97%)
A2/A1 (RH ) 97%)
0.5
2 4 6 4 6
3.5 4.0 6.0 6.0 8.0
5.9 8.8 12.4 12.1 14.6
2.1 1.9 2.0 3.8 3.8
4.15 8.8 10.8 9.3 5.7
4.0 5.7 6.3 7.0 10.0
9.5 13.2 16.8 15.0 16.5
2.0 1.8 1.9 3.7 2.0
3.4 8.9 3.4 3.1 1.8
1
a Variables: a and A , total width of the distribution and total area of the strip function for the R-methylene group of the surfactant, 1 1 respectively; a2 and A2, the same quantities for the ethylene oxide moiety.
if the thickness of the label free region exceeds the canonical resolution (d/2hmax) of the data set of about 5 Å. The surfactant R-C2H2 groups are expected to be located closer to the membrane center than the polar (C2H2C2H2O)nH moieties. The dR values determined (Table 1) are much higher than 5 Å. This strongly suggests a deuterium label free membrane core of more than 10 Å thickness. The failure of the strip-function approach at RA/L ) 1 is most likely the result of increased disorder of the membrane stack. In general, two kinds of disorder have to be distinguished.21 Disorder of the first kind is the appropriate model for a membrane stack with a perfect long-range order of the individual bilayers but with thermal fluctuations of atoms or moieties within the bilayer. It influences the intensity of the Bragg peaks, but not their width. Also there might be low-intensity diffuse scattering in addition to the Bragg peaks, if the fluctuations in different unit cells are correlated with each other.22 Noncorrelated thermal fluctuations show up in the reconstructed scattering density profile as a smearing out of molecular details. However, they cannot account for the discrepancies encountered during our strip-function analysis. Disorder of the second kind is characterized by the absence of perfect long-range order. In distinction from the first kind of disorder, the Bragg peaks broaden as their order h increases. Disorder of the second kind has to be explicitly taken into account by using an appropriate model in order to calculate a correct scattering density profile of the membrane unit cell from the diffraction data. While a perfect long-range order was observed in studies on pure lipid model membranes,23,24 disorder of the second kind was detected in stacks of natural membranes (see refs 25 and 26 for reviews). Our diffraction curves do not directly allow for distinction between the two kinds of disorder. The observed (21) Hosemann, R.; Bagchi, S. N. Direct Analysis of Diffraction by Matter; North-Holland Publishing Co.: Amsterdam, 1962. (22) Lambert, M. Diffuse scattering. In Neutron and Synchrotron Radiation for Condensed Matter Studies; Springer-Verlag: Berlin and Les Editions de Physique Les Ulis, 1993; Vol. 1, p 223. (23) Wiener, M. C.; White, S. H. Biophys. J. 1991, 59, 162. (24) Smith, G. S.; Safinya, C. R.; Toux, D.; Clark, N. A. Mol. Cryst. Liq. Cryst. 1987, 144, 235. (25) Blaurock, A. E. Biochim. Biophys. Acta 1982, 650, 167. (26) Franks, N. P.; Levine, Y. K. Low-angle x-ray diffraction. In Membrane Spectroscopy; Springer-Verlag: Berlin, 1981; p 437.
width of the Bragg peaks was entirely due to the geometry of the incident beam. The diffraction pattern did not show diffuse scattering peaks in between the Bragg peaks. However, the partial failure of the strip-function approach strongly suggests the presence of some sort of disorder of the second kind in the affected sample sets. We basically envision two possible sources of the second kind of disorder in POPC/C12En membrane stacks. First, the presence of the oxyethylene units in the polar membrane/water interface region is very likely to influence the force balance, adjusting the distance between adjacent bilayers in the stack (ref 27 and unpublished results). Fluctuations both in the local surfactant concentration and in the conformation of the oxyethylene chains should result in small variations of the water layer thickness (random stacking disorder). If present, this effect is expected to increase with the length of the oxyethylene group. Second, a fragmentation of individual bilayers into flat aggregates with a large C12En concentration in their highly curved edges would result in locally different scattering density profiles for bilayer patches versus edge regions (substitutional disorder). A similar fragmentation has been reported for C12E6/water samples in the LR phase in the vicinity of transitions to nonlamellar phases.28 NMR measurements of the deuterium quadrupolar splitting of POPC/C12E4-d24 and POPC/C12E6-d2 at RA/L ) 1 and hydration levels corresponding to RH ) 85 and 97%3 gave spectra characteristic for a lamellar structure. If fragmentation is present, the bilayer patches consequently have to be rather extended. The same conclusion can be drawn from the observed linear dependency of the modulus of the structure factors F(h) on the isotopic composition of the water. The most serious impact of bilayer fragmentation on the neutron scattering length density profiles would be expected for POPC/C12En-d4n with large n. Even though our diffraction data do not allow us to characterize the disorder in POPC/C12En stacks in detail, the strip-function approach presented is useful to screen the sample sets for indications of disorder of the second kind. We conclude from the data shown in Table 2 that all samples at RA/L ) 1 and the set POPC/C12E6 (1:0.5) at RH ) 97% are affected. The value of the corresponding scattering density profiles is seriously questioned by the currently inevitable (27) Claesson, P. M.; Kjellander, R.; Stenius, P.; Christenson, H. K. J. Chem. Soc., Faraday Trans. 1 1986, 82, 2735. (28) Rancon, Y.; Charvolin, J. J. Phys. Chem. 1988, 92, 6339.
Structure of Mixed Multilayers
lack of a quantitative consideration of the disorder in the data treatment. However, the impact on several features of the profiles seems to be only minor. The constancy of the distance between the major peaks in the X-ray and neutron scattering density profiles (dP - dE) for all the sample compositions studied justifies the interpretation of dP and dE in the usual way despite the disorder. The mean thickness d of the unit cell is not affected at all. The following discussion of parameters different from d, dP, and dE is explicitly restricted to sample compositions for which we did not detect indications of disorder of the second kind. The incorporation of C12En into POPC membranes causes a decrease of the repeat distance d as well as of the distance of the phosphate group from the membrane center dP (cf. Table 1). The R-CH2 group of C12En is located about 6 Å closer to the membrane center than the phosphate group of the lipid, that means near the boundary of the hydrophobic core of the membrane. Nevertheless, there is a mismatch of the lengths of the acyl ester chains of the lipid and the hydrocarbon chain of the surfactant. The mismatch must be compensated by higher disorder or/and larger motional freedom of the acyl ester chains of the lipid compared with those in pure POPC membranes which causes the thinning of the membrane. Obviously, this compensation is thermodynamically favored compared with a deeper penetration of the surfactant into the membrane core. The main reason for this is the hydrophilicity which hinders further penetration of oxyethylene units into the hydrophobic membrane core. The conservation of the distance between the R-CH2 group of the surfactant and the phosphate group of the lipid indicates a strong anchoring of the R-methylene group near the boundary of the hydrophobic membrane core probably caused by the interplay of hydrophobic and hydrophilic interactions. Another aspect of the thinning of the membrane core is the lateral repulsion between the polar headgroups of the lipid and the surfactant both due to sterical and hydration effects. This repulsion increases with the growing number of oxyethylene units n (unpublished results), and in this sequence dP decreases (Table 1). This mechanism is also known from hydration studies on lipids at low water content (see, for example, ref 29). (29) Parsegian, V. A.; Fuller, N.; Rand, R. P. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 2750.
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The mean position of the R-CH2 group of the surfactant as determined by the Gaussian fit (dR) and by the stripfunction approach (z1) agrees very well (Figure 3). The distribution width increases with the relative humidity and the length of the oxyethylene chains of the surfactant as reflected by the parameters ν and a1. This indicates a rising thermal mobility of the labeled group, probably due to the increasing disorder or/and larger motional freedom of surrounding hydrocarbon chains of the lipid or small fluctuations of the surfactant molecules normal to the membrane plane. The (CH2CH2O)n moieties extend from the location of the R-CH2 group toward the polar membrane/water interface (Figure 3). The partial overlap of the strips is again a result of thermal fluctuations. The distribution width a2 increases with the number of oxyethylene groups per molecule and the water content of the membrane. It is not possible to decide whether the latter effect is solely due to the enhanced thermal fluctuation amplitude or really reflects a more extended chain at higher water content. An increase of the mobility of the oxyethylene units with their distance from the hydrocarbon membrane core was confirmed by 2H NMR order parameter measurements on POPC/C12En-d4n (unpublished results). The length per oxyethylene monomer is known to be 3.5 Å in the fully extended conformation of the chain.30 Our data surprisingly suggest a penetration of about one oxyethylene group into the hydrocarbon region beyond the fatty acid ester group of the lipid. An intrusion of polar oxyethylene moieties into the hydrocarbon core of micelles was reported previously.31 In the case of POPC/C12En bilayers the effect is owed most likely to the mismatch of the hydrocarbon chain length of lipid and surfactant as discussed. The remaining oxyethylene moieties are incorporated in the polar membrane/water interface region. Acknowledgment. We thank the staff of the DN-2 spectrometer at JINR in Dubna, Russia, for allocation of beam time. We gratefully acknowledge helpful discussions with Prof. G. Bu¨ldt. This work was supported by the Bundesministerium fu¨r Forschung und Technologie and by the Deutsche Forschungsgemeinschaft (SFB 294). LA950383S (30) Tanford, C.; Nozaki, Y.; Rohde, M. F. J. Phys. Chem. 1977, 81, 1555. (31) Elworthy, P. H.; Patel, M. S. J. Pharm. Pharmacol. 1984, 36, 116.
