J. Phys. Chem. 1992,96, 10543-10547
10543
CH, Wagging Modes of Unsaturated Acyl Chains as I R Probes of Conformational Order In Methyl Aikenoates and Phospholipid Bilayers Nian-Chemg Chia and Richard Mendelsob. Department of Chemistry. Newark College of Arts and Sciences, Rutgers University, 73 Warren Street, Newark, New Jersey 07102 (Received: July 16, 1992; In Final Form: September 2, 1992)
CH2wagging progressions have been monitored in the IR spectra of a series of methyl alkenoate solid phases and diunsaturated phosphatidylcholine (PC)aqueous gel phases. The acyl chain structures are of the form CH3(CHJ,C=C(CHJ,,CO(-o)cH,. Band assignments for each series are verified from dispersion curves constructed with the assumption that the n CH2 groups between the C - C and ester moieties are solely responsible for the observed progressions. The utility of the progression intensities as a marker of acyl chain conformational order in the phapholipids is established through studies of thermotropic phase transitions in 1.2-dinervonoyl phosphatidylcholine (DNPC), 1,2-dierucoylPC (DErPC), and 1,24elaidoylPC (DEPC). Finally,the thermotropic behavior of the wagging progression intensities in diunsaturated PC/cholestcrol mixtures is compared with that in 1,24ipalmitoylPC (DPPC)/cholesterol. The unsaturated PC's exhibit a response at 33 mol 4% cholesterol that differs from DPPC. In particular, the 'liquid-ordered" phase present in DPPC/cholesterol is absent for the unsaturated phospholipids under similar conditions of temperature and sterol concentration and is replaced by a substantially more disordered state. Implications of these results for the structure of biological membranes are discussed.
Introduetioa
TABLE I: Methyl Esters Used in the Cwent Work of the Form
It is well documented that methylene wagging vibrations in the IR spectra of o r d e r e d alkanes couple to produce band progressions characteristic of a particular all-trans chain length.14 Several structural applications utilizing these progressions have appeared. Umemura et al.' demonstrated the existence of cis and trans isomers in fatty acid dimer crystals, while Naselli et al.6 monitored twodimensional melting in Langmuir-Blodgett monolayer filmr of cadmium arachidate. In systems chosen as models for biological membranes, Fringeli and Gunthard' noted the occurrence of the wagging modes in the IR spectrum of 1,l-dipalmitoylphaephatidykholine (DPPC), while Cameron et al.8 have observed intensity changes dwing the thermally-induced sequence of phases L8: 7Pk. L, in the same molecule. Finally, Senak et al.9 have utd~zadintensity changes in these progressions to semiquantitatively probe (with the aid of a simple model for progression intensity) conformational order in the series of saturated PC bilayers with chain lengths ranging from diC13 to diC20,as well as in DPPC/cholesterol mixtures. The hydrocarbon chains found in native biological membranes umtain substantial levels of unsaturation. For example, the lipids of the human aythrocyte membrane arc about 50% unsaturated.1o Thus, to extend the utility of the CH2 wagging progressions as probes of amformational order to applications involving biological membranes, it is necessary to establish spectral assignments and spcctradructurc correlations for this mode in unsaturated acyl chains. Studies of alkenes and related molecules were initially reported some 40 years ago, mostly by R.N. Jones and his coworkers,4.11J2although no detailed assignments of wagging vibrations were undertaken. The current study first reports an analysis of the wagging progressions in a series of methyl alkenoata, the appropriate model compounds for unsaturated phospholipid acyl chains. The information gained from the resultant dispersion curves is used to corroborate band assignments for the 1180-1 34O-cm-l region in the gel phases of several diunsaturated PC bilayers. Finally, it is demonstrated that the intensity of these bands offers novel insight into the interaction of cholesterol with unsaturated PC's, a model system of importance in membrane biophysics.
~ 3 ( ~ 2 ) m ~ ( ~ Z ) $ ( - o ) O c H J
-
Expdo~eatdseetloll Nohtioo. The methyl alkenoates studied in the current work are of the general form: CHdCHz)&=C(CHz)tPOW
II
0
common name m n cis or trans 10 4 trans petroselaidic 10 6 cis cis-8-eicosenoic 3 1 trans myristelaidic 5 1 cis palmitoleic 5 1 trans palmitelaidic 1 1 cis oleic 1 1 trans elaidic cis-10-heptadecenoic 5 8 cis cis-10-nonadecenoic 7 8 cis 5 9 trans-vacccnic trans 5 9 cis-vaccenic cis 7 9 cis-11-eicosenoic cis 3 10 cis cis-12-octadecenoic cis- 13-eicosenoic 5 11 cis 7 11 erucic cis 1 13 cis nervonic 'The methyl esters of the indicated molecules were examined. The notation adopted for easy delineation of the relevant primary structural features will be (m,n:x) where x will be c or t to indicate cis or trans. The molecules studied are listed in Table I. Materials Unsaturated PC's were obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). These materials were used without further purification. Cholesterol (Sigma Chemical Co., St. Louis, MO)was of stated purity greater than 99% and used without further purifcation. Unsaturated fatty acid methyl esters, also obtained from Sigma Chemical Co., were stated to be of >99% purity. These chemicals were used without purification, although purity was verified in some instances by gas chromatography. 1,2-Dinervonoyl (DNPC) purity was evaluated by differential scanning calorimetry. Transition temperatures for 1,2-dierucoylPC (DErPC) and 1,2-dielaidoylPC (DEPC) were each 11 OC as measured by R - I R , in good accord with Caffrey and Feigen~0n.l~ DNPC showed a narrow transition (half-width of 1 OC) centered at 27.0 OC as seen by both DSC and FT-IR. The measured transition temperature was 3 deg higher than that noted previ~usly.'~ Methods. Phospholipids and phospholipid-1 mixtures, dried from CHCI3 solutions, fmt under a stream of N2 and then under high vacuum, were dispersed in double-distilled H 2 0 in sealed ampules (usually 4 1 (w/w) H,O/lipid or H,O/lipid-steroid) at temperatures well above T,. Samples were incubated for at least 2-3 h with intermittent agitation to ensure complete hydration. For FT-IRthermotropic studies at temperatures greater than 0
0022-3654/92/2096-10543$03.00/0Q 1992 American Chemical Society
10544 The Journal of Physical Chemistry, Vol. 96, No. 25, 1992
Chia and Mendelsohn
OC, the aqueous dispersions were contained in a thermostated
transmission cell (Hanick Scientific, Ossining, NY)equipped with CaF2 windows and a Teflon spacer of 6-pm thickness. Temperature control was achieved with a Haake circulatingwater bath. Temperature was monitored with a digital thermocouple placed adjacent to the point where the IR radiation was focused. Temperature precision is M.10 O C ; temperature accuracy is estimated at f0.5 OC. For temperatures in the range -100 to 0 OC, spectra were acquired in a liquid N2dewar equipped with external AgCl IR windows. The sample was contained in a separate brass cell between CaF2windows. Both the brass cell and external windows could be individually heated. Temperature regulation was achieved with controlled boil-off of liquid N2 Temperature was monitored with a digital thermocouple (Physitemp Instruments, Inc., Clifton, NJ). Temperature variation during a run is f 3 OC. FT-IRspectra were acquired on a Digilab FTs-40spectrometer that was equipped with a DTGS detector. Spectra were obtained at 4-cm-' resolution, under N2purge, by coaddition of 256 scans. The resultant interferogram was apodized with a triangular function and Fourier-transformed with one level of zero-filling to yield data encoded every 2 cm-'. Data from two or three independent preparations were acquired for most samples. Data Rcductioa. Prior to isolation of the wagging progressions, spectra were corrected for any minor residual absorption bands due to water vapor, using spectra of the latter recorded under the same amditionsof instrument aperture and resolution. Subtraction factors were determined from water vapor bands in the 19001980-cm-' region. For quantitative analysis of the CH2 wagging modes in the region 1350-1 170 cm-', subtraction of the underlying PO2- antisymmetric stretching band at 1230 em-' was required. The phosphate region of the spectrum acquired at the highest temperature for that sample (usually at least 30 deg above T,) was used for this purpose. Subtraction factors were chosen by maximizing the band heights of the progression and choosing a consistent shape for the baseline of the residual contour of wagging progression components. A flattened baseline was generated for all samples for the k = 0 through k = 4 or 5 progression bands by selecting appropriate spectral minima. Band area measurement was accomplished by transferring spectra to an off-line microcomputer employing software supplied by D. Moffatt of the National Research Council of Canada. The same end points chosen for baseline leveling were used for band area determination. Progression intensities were estimated by ratioing the intensity of either the sum of all the observed progression components or k = 1 component alone to that of the underlying phosphate band, as judged by the subtraction factors. k r y
Methylene wagging modes in the all-trans conformation are described through a coupled oscillator model for which the eigenvalues of the vibrational secular equation are given by 4dv2 Ho
+ 22HmCOS (mt$k)
where the 4's are the phase dif€erences between adjacent oscillators as given by & = k r / ( n 1) ( k = 1, 2, 3, ,.., n)
+
where n is the number of oscillators in the chain. When the integers k are correctly identified for an observed band progression, a smooth curve results from a plot of v k vs & Such a procedure was used extensively by Snyder in his assignments of alkane vibrations2'' and more recently by Koyama and Ikedals in studies of the Raman spectra and conformations of cis-unsaturated fatty acyl chains. Extension of this protocol to the current problem requircs elucidation of the effect of the mbon-carbon double bond on the coupling between smaller chains each containing a sequence of CH2 groups. in a molecule of primary structure CH3(CH2),C-C(CH2),C(-O)OCH,,one might expect as a first approximation that the C.-C bond eliminates the coupling between the sets of -mr and "nr methylene groups. Thus two distinct progressions corresponding to chain
- I \
I
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wave numbe r (cm" )
Figwe 1. FT-IRspectra of several methyl alkenoates: (a) nervonic acid (7,13:c) methyl ester; (b) erucic acid (7,ll:c) methyl ester; (c) elaidic acid (7,7:t) methyl ester; (d) petroselaidic (10,4:t) acid methyl ester.
TABLE Ik W8&
Mode Proprcssioas io Methyl Alkenoatecr
k m,n:x' 10,6:c 3,7:t 5,7:t 5,7:c 7,7:t
7,7:c 5,8:c 7,8:c 5,9:t 5,9:c 7,9:c 3,lO:c 5,l l:c 7,ll:c 7,13:c
Oh
1181.3 1180.7 1180.0 1173.9 1181.5 1180.5 1182.3 1181.8 1178.9 1173.7 1174.2 1173.0 1173.2 1173.4 1173.2
1 1213.9 1209.4 1210.4 1212.6 1211.3 1214.4 1206.9 1209.6 1205.1 1205.4 1205.9 1201.3 1201.5 1202.0 1199.7
2 1269.8 1252.8 1253.4 1255.5 1253.2 1254.2 1251.2 1247.0 1234.8 1237.9 1238.2 1226.4 1226.3 1226.7 1218.4
3 1320.8 1293.2 1293.5 1296.5 1294.5 1299.7 1284.4 1282.8 1269.3 1272.2 1273.7 1255.5 1255.4 1256.5 1243.2
4
5
6 ~~
1331.5 1331.9 1332.5 1332.9 1335.1 1319.4 1320.3 1303.6 1304.3 1306.4 1283.7 1284.3 1284.6 1268.9
~
1360.5 1360.5 1332.9 1332.9 1333.6 1309.8 1333.1 1310.3 1312.0 1334.2 1292.1 1315.3
Structural formula CH3(CH2),,,C=C(CH2),C(=O)OCH3, c = cis, mode was occasionally overlapped with another vibration of uncertain origin, possibly involving C-O stretching. We believe the true k = 0 mode to occur in the 1170-1 175-cm-' range. t = trans. "his
lengths, of m and n CH2units, would be anticipated. However, the current results suggest that most if not all of the progression intensity arises from coupling of the wagging mode to the C-O bond of the polar ester moiety. The only progmsion to be expected in this instance (and indeed noted in the current case) arises from the sequence of n methylenes between the C - C and ester functional groups.
Results (A) Methyl Akewrtes. Spectra typical of the CH2 wagging region for solid-phase methyl alkenoates are shown in Figure 1. Regular progressions typified by those for the 7,13:c, 7,11:t, or 7,7:t species (the notation in use is described in the Experimental Section and the molecules are listed in Table I) are normally observed. These progressions vanish in the liquid state, indicating their origin in the all-trans conformational state of the chains. For chains with n < 5, such as for the methyl ester of petroselaidic (10,4:t) acid (Figure Id), no clear cut progressions were noted, and spectra with irregularly spaced,highly overlapped features are observed. The series of wagging bands for chains with n > 5 are listed in Table 11. Of substantial import is the fact that molecules with the same values of n but differing values of m produce similar band frequencies. This observation lends decisive support to the idea that the n methylenes between the c--C bond and the ester moiety generate the progression. The dispersion curve plotted from these data is constructed (Figure 2) under the assumption that the lowest band in each progression arises from the k = 0 mode. Other assumptions produce dispersion curves with much more scatter. For example, two alternative assignment schemes (either k = 0 or k = 1) are possible for the lowest frequency component in each progression. We have statistically
CH2 Wagging Modes of Unsaturated Acyl Chains
-
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TABLE III: Wagging Mode Progressions in Dimsaturated PCs
k 0 1171.3 1172.9 1174.0 1175.9 1176.5 1175.4 1174.2 1174.3
structure di3,7:tPC di5,7:tPC di5,7:cPC di7,7:tPC di7,7:cPC di7,g:cPC di7,ll :cPC di7.13:cPC
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The Journal of Physical Chemistry, Vol. 96, No. 25, 1992 10545
0
1 1210.3 1209.9 1209.6 1209.8 1212.0 1205.0 1198.8 1195.1
2 1250.6 1251.8 1252.3 1251.1 1250.5 1237.0 1227.2 1218.8
3 1296.8 1290.8 1297.4 1294.8 1298.0 1269.9 1254.1 1243.1
4 5 1331.4 1332.7 1358.3 1334.3 1360.0 1334.0 1358.8 1304.9 1334.3 1282.2 1334.5 1265.3
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5
5,7:c (*); 7,7:t (*); 7,7:c (A);$ 8 : ~(V);7,8:c ( 0 ) ;5,9:t (0);5,9:c (A); 7,9:c (V); 7,13:c (m); 7,ll:c (0);5,ll:c (-). Note that molecules with the same value of n have similar frequencies so that there is extensive overlapping on the plot. The notation in use is discussed in the Experimental Section. The trivial names for the molecules are given in Table I. (X);
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(A) CH2 wagging mode progression for DNPC: evident in the FT-IRspectrum at 4.3 OC but absent in the spectrum at 50.6 OC. (B) Isolation of the CHI wagging progression. The spectrum shown is the difference spectrum (4.3-50.6 "C) of the two spectra shown in Figure 3A.
analyzed the dispersion curves arising from the observed complete set of bands according to each of these possibilities. For the set of 86 bands (Table 11) observed for the methyl alkenoates, a cubic polynomial function was used to fit the observed frequencies vs 6/II. The sum of the squares of the residuals according to the fmt assignment scheme (lowest band arising from the k = 0 mode) was 9.34 X lo2 ,while the sum of the squares of the residuals according to the second assignment scheme (lowest band arising from the k = 1 mode) was 3.45 X 10'. Thus the chosen (first) assignment scheme gave about one-fourth the scatter of the alternative scheme, as judged from this statistical measure. Alternative choices for assignments yielded even poorer fits. We note that the k = 0 band at 1172-1 175 cm-'is overlapped in some casea with a mode ascribed to C-0 stretch of the ester moiety and produce an averlapped band contour positioned at 5-74311-' higher frequency (Table 11). Overall, the regular monotonic behavior and overall similarity to the dispersion curves for alkanes and for the acyl chains of saturated pc's lendsg additional confidence that the selected assignments (Table 11) are correct. For values of n < 5, the methylene wagging modes are presumably coupled to other vibrational features. Sources of scatter in the plotted dispersion curves include crystal lattice effects and coupling of particular vibrations to either the ester moiety C - 0 stretch, or
to the =C-H in-plane bend (expected in this spectral region for molecules with trans disubstitution). (B)PhoephatidylchoMnes. Spectra of the CHI wagging region for DNPC below and above T, (27 "C) are shown in Figure 3A. The strongest feature in each spectrum a r k from the broad PO,asymmetric stretching mode near 1230 cm-'. Below T,, a series of weak bands appears superimposed as weak shoulders on this main feature. To isolate these modes, the highest temperature spectrum (well above T,,,) is subtracted from all lower temperature data sets. The procedure succeeds because the band shape of the v, POT mode is relatively temperatwinvariant. A typical result of the subtraction process (Figure 3B) reveals a well-defined wagging progression. Estimates of the relative intensity of the progression bands as a function of temperature are accomplished as discussed in the Experimental Section. The observed bands and their assignments to particular k values for several diunsaturated gel-phase phospholipids are listed in Table 111. For several of the systems studied, T, < 0 OC, so that some gel-phase spectra were acquired in an ice environment. A dispersion curve (Figure 4), again constructed under the assumption that the n methylene groups between the C-C and the ester functionalities generate the observed progression, closely matches that for the methyl alkenoates (Figure 2). Phosphatidylcholinesundergo a well-studied thermotropic gel liquid crystal phase transition during which the hydrocarbon chains acquire conformational freedom, with concomitant alterations in many other bilayer properties. (For thorough reviews, see refs 16 and 17). The effects of the gel-liquid crystal phase transition on two IR spectral parameters for DNPC are shown in Figures 5 and 6. The frequency of the acyl chain symmetric CH2 stretching mode near 2850 cm-' has been widely adopted as a qualitative probe of acyl chain configuration.'* Increased CH2 stretching frequencies imply greater conformational disorder.19 The phase transition in the three pure diunsaturated PC's for which T, > 0 OC is clearly evident as a sharp discontinuity in plots of the frequency vs temperature as typified by the data for DNPC in Figure 5 (data for DErPC and DEPC are not shown). Of interest is the observation that,below T,, the change in frequency with temperature is minimal. Quantitative aspects of the relationship between frequency changes and extent of
-
10546 The Journal of Physical Chemistry, Vol. 96, No. 25, 1992
Chia and Mendelsohn 0.045 x
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Figure 6. Intensity of the k = 1 component of the CH2wagging progression for 2:l DNPC/cholesterol (A)and for pure DNPC (*).
conformational disorder have been developed elsewhere in a two-state model.20 Included in Figure 5 is a melting curve for a 2: 1 DNPC/cholesterol sample. A sfcond IR parameter, namely, the intensity of the CH2 wagging mode progression (as characterized by the k = 1 component), is plotted in Figure 6 for DNPC and for DNPC/ cholesterol 2 1. This parameter shows a decnase prior to the main chain melt for the cholesterolantaining sample and approaches zero near T,,,for the pure phospholipid. In contrast, the intensity sharply goes to zero for pure DNPC at T,. The differing response of the two IR parameters used to characterize acyl chain conformation may be understood from the simple model for band progression intensity utilized in our previous report? The pro&on intensity is assumed proportionalto the continued product of trans probabilities at each chain methylene, Le., I a II(pJi, where the product is taken over the i methylenes and p is the trans probability. The acyl chains are assumed to be in the all-trans conformation at the lowest temperature studied, and all intensities in the ensuing discussion are referenced to that state. It can be seen that a small average gauche probability @# = 1 - p t ) is sufficient to induce a great intensity decrease. In the current case with 13 CH2groups contributing to the dinervonoyl progression intensity, a trans probability of 0.95 at each position produces an intensity drop of -4996, componding approximately to the obrved intensity loss for DNPC/cholesterol betweeen 2 and 19 OC. This gauche probability corresponds to 0.6 or 0.7 gauche states out of the 12 C-C bonds (1 3 CHI p u p s ) giving rise to the progression. Thus, the observation of a progression is in itself a strong indication of a highly ordered acyl chain. Above T,, if the gauche probabilities increase to about 0.2 (b analogy with our experimentally determined value for DPPCz), the progression intensity will decrease to 0.8,') or 5-696 of its initial value. This diminished intensity is probably near the current limit of our ability to detect the
+ C)
7. Plots of k = 1 component intensity vs reduced temperature for the wagging mode progression of DPPC (+), DNPC (*), and DErPC (A) all complexed with cholesterol at 2:l PC/cholesterol molar ratios. The abscissa scale is T - T,, where T, is the gel-liquid crystal phase transition temperature.
progression, given the weakness of the bands relative to the underlying phosphate mode. In unsaturated systems, we thus have developed a semiquantitativeprobe of acyl chain conformational order in relatively ordered chains, in regions of the chain between the double bond and the ester. To illustrate the utility of the (C) PC/c"l CH2wagging progression intensity as a probe of conformational order, the interaction of PC's with cholesterol, a major component of mammalian cell membranes, has been undertaken. PC/ cholesterol interactions have been widely studied (for a review see PrestiZ2). However, most of the reported work has involved disaturated derivatives such as DPPC. Nevertheless, there have been reports in the literature that the mode of cholesterol interaction with unsaturated PC's differs substantially from the satIR evaluation of the effect of urated c a ~ e , * ~A- comparative ~~ cholesterol in PC's with unsaturated and saturated acyl chains is thus indicated. The effects of cholesterol at 33 mol % ' on the intensities of the methylene wagging progression for DPPC, DNPC, and DErPC are shown in Figure 7. To compensate for the different melting temperatures of the pure lipids (T, values: DPPC, 41 O C ; DNPC, 27 OC; DErPC, 11 "C), the data are replotted on a reduced temperature scale (T"= T - T,) as the abscissa. Striking differences are evident between the saturated (DPPC) molecule and the two unsaturated systems. The progression intensity in DPPC/cholesterol persists to at least 58 OC, well above T, for the pure phospholipid (41 "C). In contrast, the CH2 wagging progressions for the unsaturated derivatives vanish prior to or at the main transition temperature of the pure phospholipid. These data will be considered below.
Disccussion The discussion will focus on two aspects of the current work. First, implications of the data in Figure 7 for PC/cholesterol interaction in model systems and in native membranes will be considered. Second, the advantages of the CH2 wagging progression intensity as a probe of acyl-chain conformational order will be evaluated. In studies of the interaction of cholesterol with DPPC, a phase diagram has been produced (from NMR spectroscopy) by Vist and Davis,% while a detailed theoretical analysis of the diagram has been given by Ipsen et al.27 The most interesting feature is the existence under physiologically revelant conditions of temperature and composition of a "liquid-ordered" phase, Le., a phase simultaneously possessing low positional order with high conformational order. In our previous studies of DPPC/cholesterol interaction, we were able to quantitatively test those parts of this description that depict relatively high conformational order. Whereas the liquid crystalline (L,) state of DPPC was found to possess about 3.6 gauche states/chain, incorporation of 33 mol % cholesterol into the bilayer reduced the level of diporder to about
CH2 Wagging Modes of Unsaturated Acyl Chains
1 gauche bond/chain.21s28 This reduction in conformational disorder compared with the L, phase allows detection (Figure 7) of the CH2wagging progression at temperatures well above T,,, for DPPC. The current experiments reveal a very different mode of interaction between the unsaturated PC's and cholesterol. The vanishing of the progression at or below T,,, for DNPC/cholesterol and DErPC/cholesterol (Figure 7) indicates an average gauche probability of about 0.15-0.20 (based on estimates of our ability to detect the progression). This translates to a conformational disorder of 2-2.5 gauche bonds in the upper portion of the DNPC chains and 1.8-2.2 gauche bonds in the upper portion of the DErPC chains. Additional disorder would be expected toward the bilayer center in each case. Thus much more acyl chain disordering in the presence of cholesterol (at temperatures >T, for the pure phospholipid) is observed for DNPC and DErPC than for DPPC. There is no sign of a "liquid-ordered" phase for the unsaturated PC/cholesterol systems studied. Although reports of the interaction between cholesterol and unsaturated phospholipids are sparse, some previous studies involving ESR techniques have been reported. Pasenkiewicz-Gierula et al.24*25 monitored the rotational diffusion of an androstane spin-label in dioleoyW: (DOPC)/cholesterol membranes and observed only a modest effect on the motional freedom of phospholipid analogue spin probes. They concluded that the ordering effect of cholesterol in systems with acyl chain unsaturation would not be as efficient in DPPC, in good accord with the current direct IR experimental evidence. To explain their data,a conformational mismatch between DOPC and cholesterol is postulated. That is, the cis C - C of the oleate chains at a depth in the bilayer comparable to the positioning of the rigid sterol rings prevents efftcient packing of cholesterol with the acyl chains. In the current case, the cis C - C bonds are at a much greater depth in the bilayer and hence produce a much smaller effect on packing near the rigid sterol nucleus. Thus the marked reduction in the ability of the sterol to order the phospholipid acyl chains in the current work may be traced to the length mismatch between the nervonoyl or erucoyl side chains and the cholesterol molecule, which would favor PC/PC and sterol/sterol interactions rather than PC/sterol interactions. This would clearly lead to nonideal mixing between the phospholipid and sterol, with the possible occurrence of phase separation in certain regions of the phase diagram. The IR melting curve for DNPC/cholesterol constructed from the frequency of the CH2 symmetric stretching mode (Figure 5) shows perturbation of the main-chain melt, as revealed by a broadening of the transition and shift to lower temperature of its midpoint. The observation of a broadened transition width suggests that, if phase separation occurs, the domain size is small. In any case, the magnitude of the perturbations is less than thw eviously noted by calorimetry for DPPC/cholesterol mixtures.2 g r To consider the relevance of the current observations to native membranes, we note that there is much evidence for domain formation in both native and model membrane systems (for a review, see ref 30). The wide distribution of phospholipid chain lengths and levels of unsaturation in plasma membranes may be (in certain instances) a consequence of the different response to cholesterol produced by saturated compared with unsaturated chains. Particular microenvironments neaded to create domains of characteristic conformational and fluidity characteristics as required for specific membrane functions may be induced rather simply by altering unsaturation levels in the phospholipid acyl chains.
The Journal of Physical Chemistry, Vol. 96, No. 25, 1992 10547 The IR methods proposed here and in our earlier s t ~ d i e s ~ , ~ ' . ~ ~ provide a comprehensive means of measuring membrane conformational order as a function of depth in the bilayer and in the presence of additional membrane components. The CH2wagging mode progressions discussed here and in ref 9 are sufficiently intense so that they may be detected (D. Moore and R. Mendelsohn, in preparation) in intact cells and native membranes. This spectral parameter thus constitutes an effective complement to previous studies,21.28which utilized CD2 rocking modes of specifically deuterated phospholipids as quantitative probes of acyl chain conformational order. The CD2 rocking modes are extremely weak and are located in a spectrum difficult to access experimentally so as to make them difficult to detect in native membranes.
Acknowledgment. This work was supported by a grant from the U.S.Public Health Service (GM 29864) to R.M.
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