J . Phys. Chem. 1987, 91, 5298-5301
5298
An FTIR Linear Dichroism Study of Lipid Membranes Allan Holmgren, * Lennart B.-A. Johansson, and Goran Lindblom Department of Physical Chemistry, University of Umeb. S-901 87 Umei, Sweden (Received: March 18, 1987)
Infrared linear dichroism (LD)was measured for bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine/water, l-octanoylsn-glycerol/water, and 1-oleoyl-sn-glycerolin H20and D20.Order parameters were determined for vibrational modes in the acyl chain region of the bilayer (symmetric CH2 stretch and olefinic CH=CH stretch), the interface region (C=O stretch), and the head group region (antisymmetric PO, stretch). The results show no significant differencebetween the order parameters calculated for 1-oleoyl-sn-glycerol in H 2 0 as compared with D20. However, the two acyl chains in 1,2-dioleoyl-snglycero-3-phosphocholineseem to be more ordered than the single chains in 1-octanoyl-sn-glyceroland I-oleoyl-sn-glycerol. We conclude that FTIR LD spectroscopy is a sensitive method for studying lipid lamellar liquid crystalline phases.
Introduction The study of molecular orientations in large aggregated systems such as biological membranes, lyotropic and thermotropic liquid crystals, and polymers is of great importance for the understanding of their structural and dynamical properties. A number of different spectroscopical methods are available for such investigations, among which FTIR is one. Hitherto as far as we know, Fourier transform infrared (FTIR) spectroscopy has not been utilized previously for measurements of molecular dichroism. However, ordinary IR has been used previously for determination of molecular ~rientation.’-~Here we report FTIR determinations of the dichroic ratio of three membrane lipids in macroscopically aligned lamellar liquid crystals. The method is developed in accordance with the well-known linear dichroism spectroscopy technique, which is and has been applied in many investigations of the orientation of chromophores absorbing UV-visible light. (For reviews see ref 4 and 7.) Although the FTIR method is less sensitive than UV-visible linear dichroism spectroscopy, it can be more generally applied since every molecule contains intrinsic vibrational modes. A critical analysis of the FTIR linear dichroism technique will be given. Experimental Section 1,2-Dioleoyl-sn-glycero-3-phosphocholine, DOPC (>99%), was synthesized at the Department of Physiological Chemistry, BMC, Uppsala, according to Gupta et aL6 1-Octanoyl-sn-glycerol (monooctanoin) was synthesized by Syntestjanst at Kemi-centrum, University of Lund. 1-0leoyl-sn-glycerol (monoolein) was purchased from Nuchek ( U S A . ) and was used as received. Water was distilled in an all-quartz apparatus, and D 2 0 (99.75%) was obtained from Merck. The lamellar phases were prepared by mixing the lipids with appropriate amounts of water. The monooctanoin/water and monoolein/water systems contain 15 wt % water, and the DOPC/water system contains 20 wt % water. The samples align spontaneously between calcium fluoride windows separated by a 6-pm spacer. The birefringence of the CaFz plates was examined when these were placed between two crossed polarizers so that light propagates perpendicular to their planes. No significant change of the transmitted intensity was observed for a rotation of the plates about their surface normal. Hence, (1) Akutsu, H.; Kyogoku, Y.; Nakahara, H.; Fukuda, K. Chem. Phys. Lipids 1975, 15, 222. ( 2 ) Wallach, D. F. H.; Verma, S. P.; Fookson, J. Biochim. Biophys. Acta
the depolarization of the linearly polarized light caused by the windows can be neglected. The infrared spectra were recorded on a Bruker Model 1 13 V FTIR spectrometer equipped with a liquid-nitrogen-cooled mercury-cadmium-telluride (MCT) detector. A special sample chamber unit was used to allow for vacuum in all the instrument but the sample chamber, which was purged continuously with nitrogen. Polarization of the infrared beam was achieved with a rotatable grid polarizer of aluminum deposited on BaF2, placed in front of the sample cell. Another polarizer was placed after the cell (cf. Figure 1) in order to make sure that the transmitted light is reflected through the mirror system of the instrument in the same way before reaching the detector. All spectra were obtained at 26 O C and 2-cm-’ resolution (512 scans). At this temperature all of the lipids investigated are in the liquid crystalline state.
Theoretical Prerequisites The absorption coefficient, E(?), of an infrared band is where L(?) denotes the bandshape as a function of the wavenumber ?. The magnitude of the transition is determized by the interaction between the electrid field vector of light ( E ) and the transition dipole, ii, which is given by
aji/dQq describes the change of the permanent dipole moment with respect to the normal coordinate Qqof the nuclei. 4, and 4f are vibration wave functions of the initial and final states, respectively. Since is fixed in the molecule, eq 1 shows that the absorption coefficient depends on the orientation of the molecule when it interacts with the electric field of light. In order to obtain the absorption coefficient of a macroscopical ensemble of absorbing molecules, eq 1 must be averaged with respect to the orientational angular distribution. An angular distribution density function, f(O), describes the orientation of the molecule-fixed vector with respect to a laboratory-fixed frame X , Y , Z (cf. Figure 1). The procedure of averaging eq 1 is identical with that for electronic transitions induced by linearly polarized light.’ For a uniaxial orientational distribution around the laboratory Z axis, the dichroic ratio ( D ) and the linear dichroism (LD)are given by7
1979 -.. - , -559 - ., 1.-5 -1.
(3) Fringeli, U. P.; Giinthard, H. H. In Membrane Spectroscopy; Grell, E., Ed.; Springer-Verlag: New York, 1981. (4) Hofrichter, J.; Eaton, W. A. Annu. Reu. Biophys. Bioeng. 1976.5, 5 1 1. Norden, B. Appl. Specfrosc. Reo. 1978, 14, 157. Thulstrup, E. W.; Michl. J. J . Phys. Chem. 1980, 84, 82. (5) Fringeli, U. P. Z . Narurforsch., C: Biosci. 1977, 32C, 20. (6) Gupta, C. M.; Radhakrishnan, R.; Khorana, H. G. Pror. Natl. Arad. Sci. C‘.S.A. 1977, 74, 4315.
( 7 ) Johansson, L. B.-A.; Lindblom, G.
0022-3654/87/2091-5298$01.50/00 1987 American Chemical Society
Q. Rec. Biophys. 1980, 13, 6 3 .
The Journal of Physical Chemistry, Vol. 91. No. 20, 1987 5299
FTIR LD Study of Lipid Membranes
P
0
200
p
I
08
0 - P" - 0
0
,601
X
V I
01 Sample
I
,
02
03
,
04
1
I
05
06
I
+ coszw Figure 2. A typical plot of (A,/A,) - 1) vs. cos2 w for the antisymmeric stretching vibrations of PO2- and CH=CH in DOPC.
Figure 1. Experimental setup for the FTIR measurements. Light with the polarization directions either w or I impinges on a macroscopically aligned lamellar sample. The transmitted light is detected (D) after passing the sample and two polarizers (P).
A is the absorbance,_and the subscripts w' and I denote the polarization of light ( E ) forming an angle w or 90' to the Z axis or the optical axis, as illustrated in Figure 1. pL2) are irreducible second-rank tensor components of a transition dipole moment tensor. These are related to the Cartesian components of a molecular frame (x,y,z) by
TABLE I: Order Parameters ( S ) Calculated from the Dichroic Ratio Measured on Different Macroscopically Aligned Lamellar Liquid Crystals at 299 Kn o/cm-' 3006 2853 1750-1710 1232 assignment D,(CH2) O(C=O) D,,(CH=CH) D,,(POT)
order parameter in DOPC/H20 -0.22 (20 wt 5% H 2 0 ) monoolein/H20 -0.12 ( 1 5 wt % H20) monoolein/D20 -0.13 (I5 wt % D 2 0 ) monooctanoin / H 2 0 (15 wt 70' H2O)
-0.15
-0.22
-0.10
-0.17
-0.10
-0.16
-0.09
-0.18
-0.31
O S has been calculated at four different vibrational transitions by means of eq 8 and by putting n = 1.4. S is reproducible within f O . O 1 .
The Wigner irreducible spherical tensors, D::*(a,p,y), describe the angular transformation from the molecular to the laboratory frames. a, P, and y are Eulerian angles.Ig The angular ensemble averages
are called order parameters and characterize the average orientation of the molecules. If, for a particular vibrational transition, one chooses the z axis of the molecular frame to coincide with the transition dipole, eq 3 becomes
D=1+
3s cos2 w'
(6)
1-s
The order parameter S = ( D i t ) ( @ )was ) originally introduced by Saupe8 and can be written S = ' / 2 ( 3 cos 2P - 1 ) . Equations 3 and 6 do not account for the fact that light impinging at an angle w with respect to the optical axis is refracted when it reaches the absorbing molecules (see Figure 1). The measurable angle w and the true angle w' are, however, related through Snell's law according to sin
(5
-w) =
n sin
(5
-a!)
(7)
where n is the refractive index of the lamellar liquid crystalline phase. Since this phase is uniaxially anisotropic or birefringent, it is characterized by both ordinary (n,) and extraordinary (n,) (8) Saupe, A. Z . Naturforsrh., A : Astrophys., Phys. Phys. Chem. 1964, 19A, 161.
refractive indexes. It was recently shown for electronic absorption20 that for less birefringent systems such as lyotropic liquid crystals n = (n, 2 n , ) / 3 , and eq 6 may be rewritten as
+
D=1+
3s cos2 w (1
- S)n2
The IR dichroic ratio measured as a function of w gives support for using eq 8 as can be seen in Figure 2. We find that D is linear with cos2 w and has an intercept equal to unity as is predicted by eq 8. In the derivation of eq 8 we have neglected any contribution from reorientational relaxation to the observed bandshape. This means that the tumbling motion of the lipids is assumed to be much slower than the spinning motion around the molecular axis. We find that the half-widths of L&j) and L,(t) are equal, and therefore it seems reasonable to ignore the influence of the reorientational relaxation. Results and Discussion We have by using FTIR investigated LD of our lyotropic lamellar liquid crystals that were macroscopically aligned as is schematically illustrated in Figure 1. The lamellar phases were composed of DOPC/H20, monoolein/H,O, monoolein/D20, and monooctanoin/H20 in the proportions given in Table I. The order parameters of vibrational transitions in the head group, interface, and acyl chain regions have been calculated by applying eq 8 and assuming n = 1.4. The results are summarized in Table I. Head Group Region. Infrared-active modes of the moieties found in the head group of membrane lipids have been discussed e l s e ~ h e r e . ~The ~ ~dichroic ~ ~ * ~ratio ~ was determined at different (9) Bellamy, L. J. The Infrared Spectra of Complex Molecules, 2nd ed.; Chapman and Hall: London, 1958. (IO) Casal, H. L.; Mantsch, H. H. Biochim. Biophys. Acta 1984, 779, 381.
5300
The Journal of Physical Chemistry, Vol. 91, No. 20, 1987
Holmgren et al,
01 Y
c
a n L
0
al
v)
n
a
Wavenum b e r s / c m - '
U
C
m
n
L
0
vl
n
a
Figure 3. Infrared absorption spectrum of DOPC bilayers (upper spectrum). The incident angle, 90 - w , of the perpendicularly polarized light ( A , ) is 52.2'. The LD spectrum ( A , - A , ) of DOPC is shown below.
angles ( a / 2 - w ) between incident light and the optical axis. Plotting D - 1 as a function of cos2 w yields a straight line (see Figure 2). From the slope we obtain an order parameter. For the antisymmetric PO2- stretching vibration (Cas = 1232 cm-l) in DOPC we obtain an order parameter of S = -0.31. The dichroic ratio D(C) of this absorption band is constant within f 1 5 cm-' from the absorption maximum at 1232 cm-'. The LD spectrum (Figure 3, LD A, - A , ) shows a positive linear dichroism in the 1100-1040-cm~~ region. The dominant spectral feature in this region originates from the symmetric PO2stretching vibration, C,(POT), at 1088 cm-I and from a vibration characteristic of the R-0-P-0-R' group," appearing at 1065 cm-I. The dichroic ratio in the spectral region 1100-1050 cm-I is not constant with wavenumber. This behavior will appear if the infrared transitions are not pure, Le., if transition moments of different directions are responsible for the observed absorption bands. Contributions from, e.g., P-0 single band vibration and the C-0 symmetrical stretching vibration of the ester group are possible. This makes the interpretation of LD spectra extremely complicated. For the same reason we cannot determine the order parameters of the C-0 vibrations at the sn-2 and the sn-3 positions in monoolein and monooctanoin. However, assuming that the transition moment vector of the symmetric POT stretching vibration has a positive LD, the head group of phosphatidylcholine tends to be oriented parallel to the plane of the bilayer. Interface Region. The most useful infrared-active region in lipid molecules is the carbonyl stretching vibration of the ester group. In the DOPC/water system, the absorption in the carbonyl region seems to consist of two bands with their maxima at 1736 and 1733-'. This is due to an absorption peak of water vapor at 1734 cm-l which results from a minor difference in purging level of the sample chamber. As shown by Levin et al.,12s13the absorption bands in polycrystalline dipalmitoylphosphatidylcholine at 1739 and 1721 cm-* are due to the C=O groups in the sn-1 and sn-2 chains, respectively. The wavenumbers ranging from about 1727 to 1744 cm-I reflect a nearly trans conformation while wavenumbers between 1716 and 1728 cm-' are characteristic of (1 1 ) Arrondo, J. L. R.; Coni, F. M.; Macarulla, J. M. Blochim. Biophys. Acta 1984, 794, 165. ( 1 2 ) Mushayakarara, E.; Levin, I. W. J . Phys. Chem. 1982, 86, 2324. (13) Levin, I . W.: Mushayakarara, E.; Bittman. R. J . Raman Speclrosc. 1982, 13. 23.
1800
1750
1700
Wavenum b e r s /cm-l Figure 4. Infrared spectra from the C=O stretching mode region of monooctanoin, monoolein, and DOPC including the dichroic ratios, D, of the absorption bands. a gauche conformation of the C-C bond adjacent to the ester group. The single carbonyl band in DOPC then indicates that the two acyl chains have similar conformations. The calculated order parameter S = -0.22 is a mean order parameter for the carbonyl transition moments of the two acyl chains. The dichroic ratio, D ( t ) , in the region 1745-1720 cm-l is constant within fO.O1 absorbance unit. This suggests that the two transition dipoles are parallel and furthermore that the order parameters of these are very similar. The polarized absorption spectra and D(?)of the monooctanoin and monoolein systems are displayed in Figure 4. The spectral region of the carbonyl group clearly shows two peak maxima at 1738 and 1724 cm-I for monooctanoin and at 1737 and 1723 cm-l for monoolein. This could not be explained by the presence of different conformations of the acyl chains at sn-1 and sn-2. Intramolecular hydrogen bonding seems to be the major reason for this spectral feature.I4 The order parameters for the C=O transition moments are S = -0.17 and S = -0.18 for monoolein and monooctanoin, respectively. These values are obtained by using the peak absorption coefficients at 1738 and 1737 cm-'. If D,O is substituted for H 2 0 in the monooleinlwater system, the corresponding S value is -0.16. This small difference in the order parameter may reflect a difference in hydrogen-bonding interaction and/or a measure of the experimental reproducibility. The negative order parameters indicate that the C-0 bond is preferentially oriented in the plane of the lipid bilayer. The constant dichroic ratio between 1745 and 1705 cm-' is somewhat unex(14) Holmgren, A,; Johansson, L. for publication.
B.-A.;Lindblom, G., to be
submitted
FTIR LD Study of Lipid Membranes
The Journal of Physical Chemistry, Vol. 91, No. 20, 1987 5301
pected since it strongly suggests that neither the orientation nor the direction of the transition dipole moment is significantly influenced by hydrogen bonding to the C=O moiety. Our results for the interface region can be compared to those of FringeL5 He finds that, for dry samples of dipalmitoylphosphatidylcholine (DPPC), five Lorentz bands are needed in order to get a reasonable fit to the measured C=O band. The dichroic ratios for these bands varied from 0.94 for the most dominant band at 1730 cm-' to 1.40 for a band at 1738 cm-I. If we use D = 0.94 in eq 8, along with an angle of incidence w = 45" and a refractive index n = 1.4, we obtain that S = -0.085, while D = 1.40 yields S = +0.34. This difference in polarization of the components of the carbonyl band is not observed in the present investigation, where the dominant absorption bands are more homogeneously polarized. One possible explanation to this discrepancy is that the orientational distribution of DPPC is not uniaxially symmetric in the dry samples. This might cause a varying polarization of light with wavenumber. Acyl Chain Region. In the acyl chain region we have used the olefinic 4 - H stretching mode at 3006 cm-' and the symmetric methylene stretching vibration at 2853 cm-' to probe the order of the unsaturated and saturated parts of the lipid tails, respectively. A typical plot of D - 1 vs. cos2 w for the C-H stretching vibration in CH=CH is shown in Figure 2. The transition moment, which is directed along the C=C double bond, has an order parameter S = -0.22 for DOPC and S = -0.12 for monoolein (Table I). These figures indicate that the two acyl chains in DOPC are more ordered than the single C,8 chain in monoolein. The negative sign of the order parameters imply that the C=C double bond is parallel rather than perpendicular to the bilayer surface. However, this differs from what is found for the C=C band on a lamellar phase of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and water." Here Seelig and Waespe-SarceviS. have calculated that the C=C bond is tilted by 7-8" with respect to the normal of the bilayer. This result is based on the data given
by Fringeli' for an egg lecithin bilayer of unspecified hydration. The order parameter for the symmetric methylene vibrations of monooctanoin and monoolein are very similar, viz. S z -0.10. A rather low order is also expected since the order of the methylene groups has been shown to decrease toward the terminal methyl group of the acyl chains.Is Our value of S is just an arithmetic mean value of all the methylene groups along the hydrocarbon chain. The sign of the order parameter indicates that the C2 axis of a methylene group tends to be oriented perpendicular to the normal of the lipid bilayer. An order parameter of the methylene groups of dipalmitoylphosphatidylcholine has been reported by Akutsu et al.' Their value of S = -0.1 1 is close to our results on monoolein and monooctanoin but also similar to the value of -0.15 obtained for DOPC. Unfortunately, the antisymmetric CH2 stretching mode is too strong for an accurate determination of S.
(15) Pimentel, G. C.; McClellan, A. L. The Hydrogen Bond; W. H. Reeman: London, 1960. (16) Holmgren, A.; Fontell, K.; Lindblom, G. Acta Chem. Scand., Ser. A 1986, A40, 299. (17) Seelig, J.; Waespe-SarceviE, N. Biochemistry 1978, 17, 3310.
(18) Seelig, J.; Seelig, A. Q. Reu. Biophys. 1980, 13, 19. (19) Brink, D. M.; Satchler, G. R. In Angular Momentum; Oxford University Press: London, 1968. (20) Johansson, L. B.-A.; Davidsson, A. J . Chem. SOC.,Faraday Trans. I 1985, 81, 1375.
Concluding Remarks It has been shown by the present study that FTIR spectroscopy is a promising method to measure the order of lipid molecules forming bilayers separated by water. Different regions of the lipid may be probed by a single LD spectrum. Overlapping bands and choice of base lines may introduce some ambiguity in the determination of order parameters. The olefinic C H = C H stretching vibration at 3006 cm-I, the symmetric methylene mode at 2853 cm-l, the C=O stretching vibration at 1750-1710 cm-I, and the antisymmetric phosphate group vibration at 1232 cm-I seem to be useful vibrational modes to probe the orientation of lipid molecules. Such information provides information important for the understanding of molecular organization of lipid membranes. Acknowledgment. This research is supported by grants from the Swedish Natural Science Research Council. Registry No. DOPC,4235-95-4; monooctanoin, 109785-17-3; monoolein. 34487-30-4. ~
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