J. Phys. Chem. 1989, 93, 926-931
926
way to achieve this is to utilize small “crystallites” of smectite clays rather than extended, ordered arrays. Decreasing the size of the crystallite will also influence the relative efficiency of twoversus three-dimensional (stochastic) flows of the diffusing coreactant to the target molecule. This effect, first formalized by Adam and Delbriick” and referred to as “reduction of dimensionality”, has been documented for reaction-diffusion process taking place within symmetrical and asymmetrical lattice geometries in previous work (see especially Fig. 7 in ref 12). While the insights drawn from Figures 1-5 are of interest, it is clear that a number of further factors need to be taken into account before contact can be made with, for example, the experimental studies of photochemical water cleavage in clays.13-15 (1 1) Adam, G.; Delbriick, M. I n Structural Chemistry and Molecular Biology; Rich, A., Davidson, N., Eds.;Freeman: San Francisco, 1968; p 198. (12) Lee, P. H.; Kozak, J. J. J. Chem. Phys. 1984,80, 705. (13) (a) Ghosh, P. K.; Bard, A. J. J. Phys. Chem. 1984, 88, 5519. (b) Ghosh, P. K.; Bard, A. J. J. Am. Chem. Soc. 1983, 105, 5691. (c) Ghosh, Bard, A. J. J. Electroanal. Chem. 1984, 169, 315. P. K.; Mau, A. W.-H.; (14) (a) Schoonheydt, R. A.; De Pauw, P.; Vlien, D.; DeSchrijver, R. C. J. Phys. Chem. 1984,88, 5113. (b) Schoonheydt, R. A.; Cenens, J.; DeSchrijver, F. C. J. Chem. Soc., Faraday Trans. 1 1986, 82, 281. (c) Viane, K.; Caigui, J.; Schoonheydt, R. A.; DeSchrijver, F. C. Lmgmuir 1987, 3, 107.
Nonrandom distributions of cations (see section I), the clustering of target molecules in the interlayer aqueous environment, multipolar correlations between reactants, and, perhaps most importantly, the influence of layer charge (electrostatic charge density on the clay surface) are factors whose influence has yet to be assessed theoretically. These matters are presently under investigation, and the results will be reported in the near future.
Acknowledgment. This paper is based in part on work done at the Physical Chemistry Laboratory of the University of Oxford, Oxford, United Kingdom, supported by the North Atlantic Treaty Organization under a grant awarded in 1984 to P.A.P.; the kind hospitality of Professor J. S. Rowlinson is gratefully acknowledged. The research described herein was also supported in part by the Office of Basic Energy Sciences of the Department of Energy. This is Document No. NDRL-3097 from the Notre Dame Radiation Laboratory. The authors are grateful to Roberto A. Garza-LBpez for preparing the figures presented in this paper. (IS) (a) Frenski, D.; Abdo, S.; Van Damme, H.; Cruz, M.; Fripat, J. J. J. Phys. Chem. 1980,84,2447-2457. (b) Habti, A.; Keravis, D.; Levitz, P.; Van Damme, H. J. Chem. Soc.,Faraday Trans. 2 1984,80,67. (c) Nijs, H.; Fripat, J. J.; Van Damme, H.J. Phys. Chem. 1983, 87, 1279.
Fourier Transform Infrared Spectroscopic Studies of Microstructures Formed from 1,2-Bls( 10,l2-tricosadlynoyt)-sn -giycero-3-phosphochollne K. A. Bunding Lee* Bio/Molecular Engineering Branch, Naval Research Laboratory, Washington, D.C. 20375-5000 (Received: May 1I , 1988; In Final Form: July 6. 1988)
The interaction of ethanol and methanol with 1,2-bis(lO,12-tricosadiynoyl)-sn-glycero-3-phosphocholineas studied with Fourier transform infrared spectroscopy is reported. This phospholipid forms hollow tubules and long, open helical structures approximately 0.3-1 .O-pm diameter by tens of micrometers length. These tubules and helical structures formed from ethanol-water solvent mixtures are compared spectroscopically to tubules formed by thermal cycling aqueous suspensions. These two microstructures have similar IR signatures; they are both highly ordered microstructures with rigid acyl chain packing and dehydrated interfacial regions. The effect of ethanol on the hydrocarbon vibrations of the lipid microstructures is very slight.
Introduction
It has been shown that 1,2-bis( 10,12-tricosadiynoyl)-snglycero-3-phosphocholine(DC8,9PC)forms hollow tubules having dimensions of approximately 0.3-1 .O-pm diameter by tens of micrometers length.’ There are two procedures by which tubules can be formed, namely, precipitation from mixed solvents and thermal cycling of aqueous suspensions of this lipide2 The resulting morphology of the structures produced by precipitation and thermal cycling varies. As seen by electron microscopy, structures made from mixed solvent precipitation can include tubules with 1-3 bilayers and about 2%open helices, whereas thermally grown microstructures have up to 10 bilayers and are rarely open helices though they can have subtle regular spiral patterns. (See Figure 1.) The mixed-solvent procedure can be more readily adapted to large-scale production of tubules of specific lengths, so it is important to identify any alterations in structural properties of tubules grown by this procedure. We have undertaken an infrared spectroscopic study of the vibrational modes of the structures grown by these two methods in an effort to assess the effect of solvent on the molecular order of these microstructures. Although nonaqueous solvent tends to disorder saturated lipids, the microstructures formed from DC&C *Present address: S. C. Johnson & Son, 1525 Howe St., M.S.056, Racine, WI 53403.
in solvent-water mixtures appear to be highly ordered. Lipid structure, including headgroup charge and composition and diacetylenic constraints on chain packing, plays an important role in tubule microstructure f ~ r m a t i o n . In ~ water, only tubules are formed, but in nonaqueous-water solvent mixtures, a small proportion of helices are formed, suggesting that the nonaqueous solvent causes minor perturbation during microstructure formation. Previous studies of the effect of ethanol on lipids are primarily studies of effect on T,. To our knowledge, infrared studies have not been conducted on lipids in ethanol-water, particularly on tubule-forming lipids. Experimental Section
The 1,2-bis( 10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,gPC) was made by Singh and Herendeen according to their published procedure4 and produced a single spot when analyzed by thin-layer chromatography on silica gel plates developed with 65:25:4 ch1oroform:methanol:water. The method for making (1) Yager, P.; Schoen, P. E.; Davies, C. A.; Price, R.; Singh, A. Biophys. J. 1985, 43, 899. (2) Georger, J. H.; Singh, A.; Price, R. R.; Schnur, J. M.; Yager, P.; Schoen. P. E. J. Am. Chem. Soc. 1987, 109. 616945175. (3) Singh, A., private communication. (4) Schnur, J. M.; Price, R. R.; Schoen, P. E.;Yager, P.; Calvert, J.; Georger, J. H.; Singh, A. Thin Solid Films 1987, 152, 181-206.
This article not subject to U S . Copyright. Published 1989 by the American Chemical Society
The Journal of Physical Chemisfry. Vol. 93, No. 2. 1989 921
FTIR Spectroscopic Studies of Microstructures
Fiyrr 1. Electron micrographs of microslmclures. (A) A tubule formed from temperature cycling. (B)Solvent-formed micrcatmcturcs in which helical and tubule stmctures can be seen. This section of the sample illustrates the stmcture of the various microstmctwes and is not representative of the proportion of helices to tubulus. Both samples are in vacuum and therefore dry, and the electron beam polymerizes the diacetylenes.
microstructures from small unilamellar vesicles (SUVs)is as follows:' SUVs were made by sonicating water suspensions of D C & ' C at 100 mg/mL at temperatures around 50 OC. These are then cooled to -4 O C ; at this temperature the SUVs fuse and multilamellar s h a t s are formed, leaving very few vesicles. When these multilamellar shects are reheated to 50 "C and then m l e d to below 43.3 OC, the chain-melting temperature, Tm,tubules are formed. This method results in almost complete conversion of vesicles to tubules. Samples of "solvent-grown" tubules were prepared by dissolving DC,FC in either methanol or ethanol and adding water such that the final lipid concentration was 0.5 mg/mL and the nonaqueous solvent concentration was 5 0 4 5 % by volume. The mixture turned cloudy as m n as the water was added, and tubule microstructures were observed by optical microscopy. The phospholipid tubule microstructures are a p proximately 0.3-I.O-pm diameter by tens of micrometers length, as observed by electron microscopy. Other solvent and lipid concentrations can be used? Since both methanol and ethanol have IR bands that greatly hinder analysis of the lipid vibrational spectrum, the nonaqueous solvent was removed by either dialysis or pelleting the sample by repeated centrifugation and exchange of the supernatant with water. Centrifugation of the tubules was done at either 1300g for up to 8 h or 2390g for 5 min. Centrifugation dm not microsmaure morphology though it dm result in some tubule breakage. The ethanol seemed to be more difficult to remove completely than expected but can be completely removed by dialysis as judged by the absence of ethanol IR absorbna peak at 2985 and 1046 m-l. The concentration of lipid was limited to the extent it would concentrate by centrifugation, which was between Io and loo mg/mL, compared to 100 mg/mL for thermally grown tubules. The lipid semples were placed between BaFz crystal plates separated by a 25-pm Teflon spacer. Spectra of polycrystalline DCBSPCwere obtained from D C $ C in water, lyophilized, and made into a KBr pellet. Spstra were recorded at 2-m-l resolution on a nitrogen-purged Perkin-Elmer 1800 Fourier transform infrared (FTIR) soectrometer with a DTGS (deuteriated triglycine suliate) detedtor. Two samples scans were taken to one reference scan in a 0.43-min cycle, with a total of 50 cycler averaged. The temperature was controlled by using an external circulating bath and monitored (5) Rudolph. A.; Burke, T.G. Bimhim. Biophy,.
An0
1987. WZ,149-359.
8 %!
lorn
29m
rn
Irn
Iuo
tSXl
12m
??rn
?mo
FREOUENCIIWIMNUUBEwl
at 50 oc kfore z FTIR spectra of D C ~ , ~(A)~ suvs . tepmperature cycling. (B)SUVs at 24 'C that have bcsn transformed to tubules by a temperature cycle; M text for details. The 3000-2800cm-' region is from W&FC in 4 0the ISCC-IWJO-cn-' region is from D C w K in H2O. Spectra have been vertically offset for clarity.
nw
by a t h m o c w l e t a d 10 the ~ F window. Z Temperaturn W m stable to within 2 "C during the approximately 20min total scan time. DzO (Aldrich, 100.0 atom %) and perdeuteriated ethanol (CbCDzOD, Aldrich. 99+%) were also Used to avoid the Obscuration of lipid bands by strong 0-H bands and ethanol alkyl bands. In this case. important regions of the lipid spectra can be analyzed without removing the nonaqueous solvent.
Res& and Discussion FTlR spectra of DC,PC
in the form of SUVs and thermally grown tubules are shown in Figure 2. DClzPC in SUVs in the liquid crsytalline phase at 50 "C is characterized in the IR (see Figure 2A) by two CH2 tending bands at 1466 and 1457 cm-l, broad phosphate stretches arounh 1230 and 1087 cm-', and CH, asymmetric and symmetric stretches around 2925 and 2854 cm-l. respectively. The quaternary amine hcadgroup of phosphatidylcholine is Characterized by a doublet at 1053 and 1087 cm-'! ( 6 ) Nclaon. G. J. Lipid, 1968. 104. 3.
928
Lee
The Journal of Physical Chemistry, Vol. 93, No. 2, 1989
TABLE I: Full-Width at Half-Maximum (fwhm) for the CHI Symmetric Stretch of l,t-Bis( 10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine
lipid prep
temp, 1 2 "C fwhm, f0.3 cm-I
KBr
24
14.0
water thermally grown tubules deuteriated ethanol deuteriated ethanol tubules in 55% CD3CDzOD tubules in 24% CD3CD20D solvent-grown tubules
50
16.5
25
10.3
38 -3
18.9'
25 25
8.3
"he
25
9.5 7.8 7.5
error in this band width is 10.7 cm-I.
The IR of the tubular microstructures formed from temperature cycling of SUVs can be seen in Figure 2B. The changes with respect to SUVs are as follows: the CH, bending band at 1470 cm-I sharpens and shifts up 4 cm-I from the liquid crystalline SUVs; the 1457-cm-I band diminishes to a barely visible shoulder; a sharp CH2 wagging band progression appears in the region 1375-1 177 cm-'; the 1177-cm-' band (attributed to the ester C-O stretch and skeletal vibrations) sharpens; a band a t 11 18 cm-' grows in and the relative intensity of the 1064 cm-' band increases (both are due to skeletal vibrations). The CH stretching region of the tubular morphology is characterized by a shift to lower frequencies of the CH2 asymmetric and symmetric stretches to 2918 and 2850 cm-I, respectively, and narrowing of these bands.' (See Tables I and 11). The band at 2937 cm-' is likely due to the CH2 groups adjacent to the diacetylene moiety. The electron-withdrawing nature of the diacetylene group would be expected to increase the force constant of the adjacent C-H bond, causing the frequency to increase. This attribution is made on the basis of comparison to a similar effect of the carbonyl on the CD2 stretching vibrations of 1,2-dimyristoyl-sn-glycero-3phosphocholine: the frequencies of the CD, stretching vibrations are increased for positions close to the carbonyLs The band at 2895 cm-' becomes sharper as do the CH3 asymmetric and symmetric stretch vibrations at 2957 and 2871 cm-I, respectively.' In general, reduction in band width and increase in peak height indicate a reduction in the mobility of the various functional groupsg Broadening of bands of hydrocarbon chains above T , is due to rotamer broadening, reorientation effects and lattice disorder," which suggests that narrowing of bands is due to increased lattice order and reduced rotation. All of these features indicate a tightly packed, rigid chain and a more ordered structure that is characteristic of lipid material in the gel phase. This same type of structure can also be seen in the spectrum of DC8,9PCin a KBr pellet. The inherent crystallinity of the tubular gel phase is realized when the spectral features are compared to the spectrum of polycrystalline lipid seen in Figure 3B. The phosphate stretching region shows relative intensity differences due to reduced water-headgroup interaction in the polycrystalline sample. The 2937-cm-' band shows a splitting to 2939 and 2934 cm-'. These bands may be due to the CHI groups adjacent to the diacetylene or may be due to Fermi resonance from the band near 1470 cm-'. The spectrum of microstructures grown from solvent precipitation shows the same spectral features that indicate gel phase as those in spectra of microstructures formed from thermal cycling. (See Figure 3.) These solvent-precipitated tubules have had all nonaqueous solvent removed but retain the helical and tubule structure. The CH, bending band at 1470 cm-' is sharp, the CH2-C=0 vibration is at 1417 cm-', the CH, wagging pro(7) Casal, H. L.; Mantsch, H. H. Biochim. Biophys. Acta 1984, 779, 38 1-40 1. ( 8 ) B a n d , R.; Day, J.; Meadows, M.; Rice, D.; Oldfield, E. Biochemistry 1980, 19, 1938-1943. (9) Cameron, D. G.; Mantsch, H. H. Biophys. J . 1982, 38, 175-184. (10) Gordon, R. G. J . Chem. Phys. 1965,43, 1307. Sykora, S. J . Chem. Phvs. 1972. 57. 1795. i l l ) R a b l t ; J. F.; Burns, F. C.; Schlotter, N. E.; Swalen, J. C. J . Chem. P h p . 1983, 78, 946-952. (12) Sturtevant, J. M. Proc. Natl. Acad. Sci. U.S.A. 1982,79,3963-3967.
3000
2900
2800
1500
.4OO
1300
1200
1100
1000
FREOUENCY IWAVENUMBERS)
Figure 3. FTIR spectra of microstructures formed from DC8,,PC. (A) SUVs at 24 OC that have been transformed to tubules by a temperature cycle; see text for details. The 3000-2800-~m-~region is from DC8,9PC in D20, the 1500-1000-cm-' region is from DC8,,PC in H20. (B) Polycrystalline DC8,,PC in KBr. (C) Tubules formed from 55% ethanol-water with all ethanol dialyzed out from the sample. The 30002800-cm-I region is from D(&PC in DzO; the 1500-1000-~m-~region is from DC8,9PCin HzO.Spectra have been vertically offset for clarity.
gression is evident in the 1375-1 177-cm-' region, and the skeletal vibrations at 1177, 1117, and 1063 cm-' are resolved. The CH stretching regiong shows slightly narrower, better resolved bands at 2957, 2937, 2918, 2895, 2870, and 2850 cm-', which indicate more acyl chain order than the thermally grown tubules. The second band in the region of CH2 symmetric stretch at 2985 cm-' has been attributed" to a weak Fermi resonance interaction between CHI symmetric stretching and a binary combination of two CH2 bending modes. The 2937-cm-' band may be due to a second component of the CH3 symmetric stretch fundamental at 2870 cm-' split by Fermi resonance1' or due to the CH, groups adjacent to the diacetylene as mentioned before. We examined the effect of residual ethanol on the DC8,gPC tubules. Spectra of DC6,gPCtubules and helices with residual ethanol have peak positions of the CH2 bending, of the CH2C 4 , of the CH, wagging progression, of the phosphate stretches, and in the C-H stretching region that are virtually the same compared to spectra of tubules with all nonaqueous solvent removed and indicate the rigid packing of gel phase lipid. (SeeTable 11.) Extra vibrations at 2985, 1457, 1387, and 1046 cm-' are attributed to ethanol. These vibrations are of similar relative intensity and within a few wavenumbers of ethanol vibrations of 10% ethanol-water. Disregarding the contributions from ethanol, the spectrum of DC8*9PCwith residual ethanol reveals little effect on the lipid structure compared to samples in which the ethanol has been completely dialyzed. We also examined the effect of 24 and 55% ethanol-water ratio on the DCs,9PC,using perdeuteriated ethanol (CD,CD20D) and D,O to avoid any overlap of ethanol vibrations with DC8,9PC vibrations. We could not avoid the large contributions from C-0 vibrations and therefore were unable to examine the range from 1300 to 1000 cm-', which includes the headgroup vibrational region. The C-H stretching region appears the same with respect to frequency, band width, and resolution of bands for DC8,9PC structures formed in protonated ethanol-water mixtures from which all ethanol was dialyzed out, as for DC8,gPCstructures in 24% perdeuteriated ethanol/water mixtures. The CH stretching region is characteristic of a well-ordered gel phase. The CH2 bending mode is narrow and at 1470 cm-I, also typical of the well-ordered gel phase. There is thus no evidence that the ethanol is dissolved into the hydrocarbon chain region of the DC8,9PCat this concentration. The CH, wagging progression and phosphate stretching region is obscured by solvent vibrations. DC8,gPC in 55% perdeuteriated ethanol/water mixtures does show a slightly broader CH, symmetric stretch (see Table I), which would indicate less rigidity in the acyl chain region than for DC8,9PCin less alcohol, but on the whole the spectrum is that of lipid in wellordered gel phase.
FTIR Spectroscopic Studies of Microstructures
The Journal of Physical Chemistry, Vol. 93, No. 2, 1989 929
TABLE II: IR Vibrational Frequencies for 1,2-Bis( 10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine'
lipid in KBr pellet
assgn6 CH3 (a st)
2957 2939 2934
CHI (a st) CHI (a st) CH2 (a st, split) CH, (S st) CHI (S st)
thermally methanolethanolethanolgrown grown tubules, grown tubules, grown tubules, tubes in 55% SUVs tubules methanol ethanol trace ethanol C0,C020D/ (50 "C) (24 "C) removed (24 "C) removed (24 "C) remains (24 "C) D20, 24 OC 2956
tubes in 24% CD,CO,OD/ D20, 24 OC
2957 2936
2973 2956 2938
2977 2957 2937
2985 2957 2938
2955 2938
2955 2938
2918 2896 2870 sh 2850 2819 1490 1470
2918 2896 2870 sh 2850 2818 sh 1491 1470
2918 2895 2870 sh 2850 2818 sh 1490 1470
2918 2895 2870 2850 2819 1490 1470
2918 2895 2870 2850 2818 1491 1470
2918 2895 2870 2850 2818 1491 1470
1457 sh
1452 1437
1456 1436
1457 1453
1453 sh 1437
1418
1417 1392
1418 1397 1377
1418 1387
1418 1388
1340 1333 1321
1370 1348 1340 1332 1321
2926 2917 2895 sh 2872 sh 2850 2819 1490 sh 1470
CH, (a be) CHZ(be) CHI (be) CHZ(be)
1451 sh
2899
sh
2854 1490 1477 1466 1457 1436 sh 1426
1418 1385
CH, (s def) CH, (s def) CH, (s def)
1377 1368 1348
1371 1343 1333 1322
1322
1305 1288 sh 1277 sh 1271 1250 1239 1229 sh 1213
PO2- (a st) PO4 (a st)
1297 1287 1270 sh 1230
1377 1371
1372 1346
1342 1333 1322
1333 1321
1304
1304
1287
1287 sh
1272 1248 1237 1229 1215
1272 1248 1238 1228 1213
1177
1177
1341 1332 1321 1314 1305 1295 1287 1277 sh 1273 1249 1238 1230 1213
1418 1388
1305 1287 sh 1276 1273 1249 1238 1230 1212 sh
1182 .~.-
C-C and C-0 (st) 1177
1118 1099 sh
1118 1100 sh 1088 1062
1087 1061
1131 1118 1099 sh 1091
Po,- (s st) C-C and C-O (st)
1177 1158 sh 1138 1132 1113 1100 sh
1062
1087 1065
1087 1064
1012
1013sh
1012
1119 1087 1046
a Relative intensities are not given because some base lines are flattened, which distorts relative intensities; "sh" means shoulder. Assignments are tentative and based on saturated DPPC literature as referenced in the text. The CHI wagging band progression is 1380-1190 cm-I. The CH2 rocking band progression is 1150-700 cm-I. Nomenclature: a = asymmetric, st = stretch, s = symmetric, be = bend.
The effect of ethanol on phospholipids with saturated hydrocarbon tails has been studied by many group^.'^.'^ Ethanol was shown to have a biphasic effect on the T, of phosphatidylcholines having acyl chain lengths from 14 to 21 carbons for lipid concentrations of 0.25 to 0.6 mg/mL and ethanol concentrations of up to 50 mg/mL.13 (The concentration of DC8,9PCin our studies is about 0.5 mg/mL of lipid, but the ethanol concentration is 150 mg/mL, 3 times that used in that previous study.) It has been proposed that at lower ethanol concentrations, ethanol interacts preferentially with lipids in the fluid-phase, i.e., the ethanol dissolves into the fluid, hydrophobic region of the bilayer, and at higher ethanol concentration, there is a secondary ethanol-lipid interaction in which ethanol is bound preferentially to the lipid in the gel phase, stabilizing that phase. It was also argued that the elevation in T , may involve specific binding of ethanol a t ~
~~
~
~
(13) Rowe, E. S. Biochemistry 1983,22, 3299-3305. Also: Rowe, E. S. Biochim. Biophys. Acta 1985,813, 321-330. McDaniel, R. V.; McIntosh, T.J.; Simon, S. A. Biochim. Biophys. Acta. 1983, 731, 97-108. (14) Bush, S. F.; Levin, H.; Levin, I. W. Chem. Phys. Lipids 1980, 27, 101-111.
discrete sites to more than one lipid molecule." Although the DC8,9PCstructures resulting from alcohol-water solvent mixtures consist largely of tubules of 1-3 bilayers and some helical ribbons, the IR signature is very similar to that for tubules made of 1-10 bilayers formed from aqueous solutions. What one might expect from ethanol-lipid interaction, on the basis of previous studies of lipid and ethanol, is increased fluidity of the hydrocarbon tail and less order in the headgroup region. This is not observed. The IR spectra of tubules, no matter how they are formed, indicated gel-phase structure. The rigid tubule morphology is relatively stable at room temperatures where other saturated lipids are more liquid crystalline. The DC8,9PCtubule formation immediately from solvent-water mixtures is very much like crystallization. The main driving forces behind this are probably in the headgroup and tial structure with its diacetylenic "kink". As can be seen by the IR, the ethanol does not perturb the hydrocarbon tail gel-phase structure. The IR is not sensitive to the lipidsolvent interactions at the helical edges because these helices make up less than 2%of the mixture and the edges are an even smaller percentage of the lipid. IR of the lipid is also not very sensitive to differences between a few and 10 bilayers
Lee
930 The Journal of Physical Chemistry, Vol. 93, No. 2, 1989
7
W
L t
5W U
L/ y; 3000
2900
3000
2800
FREQUENCY (WAVENUMBERS)
Figure 4. FTIR spectra of DCSpPCin CD3CD20D,50 mg/mL, (A) at 39 "C and (B) at -2 OC. Spectra have been vertically offset for clarity.
because the predominant effect on the lipid is the local waterheadgroup interaction, which should not differ significantly with the number of bilayers. IR is very sensitive to hydrocarbon chain packing, and if there were significant ethanol interaction with the lipid hydrocarbon chain, it would be seen in the IR spectrum. Helices do not form tubules for periods of time up to days even after the nonaqueous solvent has been removed. Though the nonaqueous solvent may be an important factor in the formation of tubules and helices, it is not essential for the morphological integrity of these structures. Because there appeared to be so little interaction of the ethanol with the lipid when it was in the tubule morphology, we also examined the effect of pure CD3CD20D, without water, on DCs,9PCat different temperatures. (SeeFigure 4.) When the temperature is about 45 OC, the lipid is solvated and fluid as can be seen by the C H stretching region, which has broad peaks very similar to those in the spectra of SUVs at 50 O C . DCs,gPCdoes not make tubules in pure alcohol, but if the temperature is made low enough, less than about 8 OC, it does form crystals, as seen in electron microscopy. The CH stretchiqg region of the IR of these crystals looks remarkably like that of both the solvent and thermally grown tubules. (See Figure 5.) Even in pure ethanol, the lipid can crystallize and apparently exclude ethanol from the hydrocarbon tails. The region of the lipid bilayer that includes the carbonyl groups of the esters is referred to as the interfacial region. The two carbonyls of the sn-1 and sn-2 chains can be in different environments due to the molecular conformation, and these environments influence the observed frequency. Though the carbonyl band is usually very broad in hydrated lipid samples, with spectral deconvolution, two bands can be seen for saturated hydrocarbon chain diacyl lipids, the sn-2 at 1742 cm-' and the sn-1 at 1725 cm-1.14915Hydrophobic interactions cause the sn-2 carbonyl to be at higher vibrational frequency, while increasing dielectric constant and H bonding causes the sn-1 carbonyl to be at lower frequency. The carbonyl stretch of the two carboxylic esters of DC8,gPC only can be seen in the absence of H20, which otherwise obscures this region of the spectra. We were able to examine this region in the dried DCs,$C and samples in deuteriated water and ethanol. (See Figures 6 and 7.) These bands are very broad regardless (15) Levin I. W. Adv. IR Raman Specrrosc. 1984, 2.
,
\ 1 I
, , 2900
I
I
,
