1790
J. Phys. Chem. 1984, 88, 1790-1796
between these regions metallographically and compositionally where quantitative characteristic X-ray analyses were performed. However, both the metallographic and quantitative characteristic X-ray analyses have insufficient resolution to distinguish unambiguously between a finely divided mixture of two separate phases or a continuous single-phase region. Since extensive X-ray4 and neutrons diffraction and HRTEM studies have failed to find any structural difference between the purple and blue borides, we conclude they belong to the same structural phase region. Certainly, it is an unusual phenomenon to have a fairly abrupt color change within a nonstoichiometric single phase region. However, it is not without precedent. Both tantalum carbide23 (metallic to yellow) and niobium carbide24(metallic to lavender) undergo color changes with stoichiometry change in a single-phase
region. Also, when carbon diffuses into tantalum, a very thin yellow layer of tantalum carbide forms, which exhibits a very sharp color boundary with the metallic-colored layer, both of which are members of the same single-phase nonstoichiometric tantalum carbide region. Another example is the appearance and disappearance of color centers in NaC12’ as a function of Na activity, temperature, and history. Acknowledgment. We thank Dr. R. Marzke for taking the NMR spectra and for helpful discussions. Partial support by the National Science Foundation through research Grant DMR-8006584 and the Regional Instrumentation Center for High Resolution Electron Microscopy through Grant CHE-79 16098 is gratefully acknowledged. Registry No. La, 7439-91-0;B, 7440-42-8;LaB,, 50647-34-2.
(23) G. Santoro, Trans. Metall. SOC.AIME, 227, 1361 (1963). (24) E. Storms, private communication, 1983.
(25) R. W. Pohl, Proc. Phys. SOC.,London, 49 (extra part), 3 (1937).
Raman Spectroscopic Study of the Melting Behavior of Anhydrous Dipalmitoylphosphatidylcholine Bilayers Timothy J. O’Leary and Ira W. Levin* Laboratory of Chemical Physics, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205 (Received: July 6, 1983)
The temperature dependence of the Raman spectra of anhydrous, polycrystalline dipalmitoylphosphatidylcholine(DPPC) was investigated in the 0-108 OC temperature range. For this temperature interval, spectra of the C-H and C=O stretching mode regions reflect a significant lateral expansion of the lipid matrix and an increased motion of the sn-2 chain within the bilayer interface region, respectively. In the 100-108 OC range, a cooperative phase transition occurs in which further lateral expansion of the bilayer is accompanied both by an increased number of gauche rotamers within the hydrocarbon chain region and by multiple conformers in both the head group and glycerol backbone moieties. When the polycrystalline samples are rapidly cooled, the acyl chain region reorders without a concomitant reorderiqg of the head group and interface regions. For these samples the spectral features characteristic of the lipid head group and interface segments are similar to those of hydrated bilayers. With the exception of specific interface and head group region changes, which are caused directly by the addition of water in contrast to the temperature-induced melting of hydrated bilayers, the melting behavior of anhydrous lipid systems is analogous to the completely hydrated dispersions. The spectral changes accompanying the melting of polycrystallineDPPC bilayers demonstrate the usefulness of investigating anhydrous lipid systems in understanding structural changes characteristic of hydrated systems, in clarifying the role of water in determining membrane bilayer organization, and in assisting in the vibrational assignment of specific spectral features representative of liposomal dispersions.
Introduction The melting behavior of aqueous phospholipid dispersions, or liposomes, has been extensively investigated by using a variety of physical The main lipid phase transition is characterized by a lateral expansion and a thinning of the bilayer and is accompanied by a change in the hydrocarbon “tail” region from an almost all-trans assembly to one in which gauche isomers are predominant. The analogous order-disorder transition in anhydrous phospholipids has received scant attention, although differences in the melting properties of anhydrous and hydrated lipids provide insight into the role of water in determining membrane structure. Certain effects of water in membrane systems are well-known; water depresses the lipid melting temperature and causes structural alterations within the polar head group and glycerol backbone regions of the membrane.4 Raman spectroscopic studies have shown that the changes induced by water in the head group and interface regions of the membrane may be (1) Chapman, D. Q.Rev. Biophys. 1975, 8, 185. (2) Israelachvili, J. N.; Marcelja, S.; Horn, R. G. Q. Reu. Biophys. 1980, 13, 121.
(3) Shinitzky, M.; Barenholz, Y . Biochim. Biophys. Acta 1978, 515, 367. (4) Bush, S. F.; Adams, R. G.; Levin, I. W. Biochemistry 1980, 19, 4429.
reproduced by heating the anhydrous lipid to above its melting point.4 This anhydrous phase transition has been probed to some extent in several spectroscopic investigations. Although Chapman et al. demonstrated that the melting transition of anhydrous and monohydrated dipalmitolyphosphatidylcholine (DPPC) assemblies are characterized by the same chain disorder occurring in aqueous phospholipid dispersions,’ the enthalpy of the transition is only about half (4.3 kcal/mol) of the enthalpy of melting in the fully hydrated liposomes (8.6 kcal/mol). For anhydrous distearoylphosphatidylcholine bilayers, Chapman also noted that, for the first melting of the crystal, the enthalpy of the transition is about twice that of the hydrated crystal; only in subsequent melting and cooling cycles is the enthalpy of the transition lower in the crystal than in the hydrated lipid.’ X-ray diffraction patterns’ and Raman spectra of the C-H stretching mode region6 of several phospholipids indicated that the melting of anhydrous lipids is accompanied by a bilayer expansion analogous to that which occurs in hydrated bilayer forms. Koyama et ale7noted differences between the ~~
( 5 ) Chapman, D.; Williams, R. M.; Ladbrooke, B. D. Chem. Phys. Lipids 1967, I, 445. (6) Bunow, M. R.; Levin, I. W. Biochim. Biophys. Acta 1977, 487, 388. (7) Koyama, Y . ;Tcda S . ; Kyogoku, Y . Chem. Phys. Lipids 1977, 19, 7 4 .
This article not subject to U S . Copyright. Published 1984 by the American Chemical Society
The Journal of Physical Chemistry, Vol. 88, No. 9, 1984 1791
Melting Behavior of Anhydrous DPPC Bilayers spectra of L-DPPC and DL-DPPC which they attributed to alterations in molecular conformation. They demonstrated that, after heating, the DL-DPPC crystals exhibit spectral features similar to L-DPPC. Unfortunately, these authors were unable to investigate the details of the transition. Vibrational Raman spectroscopy provides conformationally sensitive probes specific to the polar head group, interface (which includes the glycerol backbone and carbonyl moieties), and hydrophobic acyl chain regions of phsopholipid assemblies.* In this study we apply Raman spectroscopy to monitor the bilayer changes which occur in anhydrous, polycrystalline dipalmitoylphosphatidylcholine (DPPC) samples as the temperature is increased from -0 OC through melting at 108 OC. This information is used to clarify the assignments of a number of vibrational modes, to elucidate the structural changes occurring in all regions of the phospholipid molecule during melting, and to infer the effects of water on intra- and intermolecular phospholipid rearrangements. Since the enhanced signal/noise characteristics obtained with polycrystalline samples enable the temperature dependence of the frequency, intensity, and line-width parameters of important, but subtle, spectral features to be reliably observed, structural reorganizations are often more easily monitored in anhydrous systems in comparison to hydrated bilayers. In addition, the conformational alterations available to polycrystalline systems occur more slowly as a function of temperature than as a function of degree of hydration.
-
Experimental Section Samples of DPPC were obtained commercially from Sigma Chemical Co., recrystallized from chloroform, dried for 48 h at 10" torr, and sealed in Kimex glass capillary tubes (1.25-mm i.d.). Raman spectra were recorded with a Spex Ramalog 6 spectrometer equipped with holographic gratings. Excitation with 5 14.5-nm radiation from a Coherant CR-12 argon ion laser delivered 200-400 mW at the sample. Spectral resolution of the spectrometer was 2 cm-'. Spectral frequencies, calibrated with atomic argon lines, are reported to kl-2 cm-'. Spectra, acquired with a Nicolet N I C 1180 data system interfaced to the spectrometer, were signal averaged for 1-40 scans, with a uniform scan rate of 1 cm-l/s. Spectra were recorded at approximately 10 O C intervals from 0 to 70 O C and at smaller intervals nearer the melting transition. Sample temperatures below 99 OC were maintained by placing the sample capillary within a thermostatically controlled brass mount, whose temperature was maintained by a flowing solution of ethylene glycol and water. A copper-constantan thermocouple placed adjacent to the capillary within the brass housing recorded the temperature. Above 99 OC, sample temperatures were maintained by a current-regulated, nichrome wire assembly wrapped around the glass capillary. This unit was aligned in the spectrometer within an open sample holder. Temperatures obtained with the latter arrangement were estimated first by linearly extrapolating a standard curve, which was constructed by plotting the spectral peak height intensity ratio Z293J/Z28Boof DPPC as a function of the temperatures determined in the thermostatically controlled system. This information was then combined with a plot of the Raman intensity ratios vs. the nichrome wire current obtained with the open, high-temperature assembly. The resulting standard curve, consisting of a plot of current vs. sample temperature, was linear in the 50-99 O C range. The calibration curve yields temperature estimates within 2-3 OC of those measured by the thermocouple below 99 OC; we expect a similar degree of accuracy from 99 to 110 O C . Results For ease in reporting and integrating the spectral data, the results are given by spectral region. Interpretations of the spectral features in terms of structural changes within specific regions of the lipid molecule will be discussed in the following section, together with the justifications for certain assignment changes. A ~
( 8 ) Levin, I. W. "Advances in Infrared and Raman Spectroscopy"; Clark, R. J. H., Hester, R. E., Eds.; Heyden: London, Vol. 11, in press.
TABLE I: Raman Spectrum a t 0 "C of Anhydrous, Polycrystalline Dipalmitoylphosphatidylcholine in the 2800-3100- and 700-1 800-cm-' Regions frequency, cm-' 3052 3042 3025 2985 2968 295 2 2936
re1 intensi tya
assignment
5
6 8 8 26 33
2902
sh, b d b
2884 2871
100 sh
2865 2850 1739 1722 1461 1437 1370 1344 1336 1300 1296 1250 1236 1221 1175 1159 1141 1130 1106
65 2 2 64 68 4 4 3 sh 100 12 5 5 6 5 7 70 sh
1101 1096
sh 39
1063 1053 1043 1027 1013 96 9 960 95 2 939 909 892 87 7 842 824 762 743 711
71 SI1
choline CH, asym stretch choline CH, sym stretch acyl chain CH, asym stretch CH, sym stretch in FR with 2873-cm-' overtone t acyl chain CH, in F R with CH, sym stretching fundamental acyl chain CH, in k'R with CH, sym stretching fundamental CH, antisym stretch overtone of CH, asym def in F R with 2936-cm-' CH, sym stretch CH, sym stretch C=O stretch;sn-1 chain C=O stretch; SH-2chain acyl chain CH, def CH, syin def head group CH, twist choline CII, sym def (?) acyl chain CH, twist PO; asym stretch head group CH, twist acyl chain CH, rock t CH, rock acyl chain all-trans C-C stretch sn-2 chain, all-trans C-C stretch, k = 1 mode sn-2 chain, all-trans C-C stretch, k = 1 mode glycerol ester C - 0 stretch
SI1
3
9 14 16 Sll 5
head group C-C stretch C-N a\ym stretch C-N asym stretch choline CH, rock
+ C-N
stretch
9 22 26 16 6 4 4 47
acyl chain CH, rock (tC-C stretch) C-N asym stretch glycerol backbone C-C stretch 0-P-0 antisym stretch 0 - P - 0 sym stretch C-N sym stretch
a The relative intensities in the 700-1800-cm-' region are normalized to the 1296-cm-' feature. For the 2800-3 100-cm-' interval the relative intensities are normalized to the 288 1-cm-' feature. Sh, bd and k;R represent shoulder, broad, and Fermi resonance, respectively.
summary of the major spectral features and assignments appears in Table I. 700-1000-cm-'Region. Survey spectra displayed in Figure 1 indicate that no significant frequency changes occur in the spectral transitions in the 700-1000-cm-~ region as the temperature is raised from 0 (Figure 1A) to 99 (Figure 1B) O C . With respect to the head group vibrations, there is no change in the relative intensities of the symmetric and asymmetric choline C-N stretching modes at 71 1 cm-' and at 960 and 969 cm-', respectively. As the temperature increases, the Raman lines at 824,843, 877, 892, and 908 cm-' become superimposed upon a broad pedestal appearing in the 800-910-cm-' region. Thc intensity of
1792 The Journal of Physical Chemistry, Vol. 88, No. 9, 1984 2%
A.
O'Leary and Levin
POLYCRYSTALLINE DPPC
ANHYDROUS DPPC
4 t
B.
1 -
0
20
40
60
80
100
120
T'C
Figure 2. Temperature profile for anhydrous DPPC derived from the ratios. This ratio reflects the number of
11090/11130 peak height intensity
acyl chain conformers in the bilayer matrix.
D.
E?
1
WAVENUMEKE~SFEE~T~M Figure 1. Raman spectra of anhydrous DPPC in the 700-1500-cm-' region: (A) T = 2 OC, (B) T = 99 OC, (C) T = 108 "C (680-1530 cm-'), and (D) T = 104 "C (680-1530 cm-').
the acyl chain terminal methyl rocking mode at 892 cm-' decreases dramatically on increasing the temperature, while the weak 743-cm-' peak disappears. Between 100 and 108 "C (Figure 1, C and D),the symmetric C-N stretching vibration broadens and shifts from 711 to 717 cm-I; the 969-cm-' asymmetric C-N stretching mode decreases in intensity and broadens, becoming a shoulder of the also broadened 960-cm-' asymmetric C-N stretching component. The 763-cm-' 0-P-0 symmetric stretching feature broadens considerably as the temperature increases. Peaks in the latter spectral region are now superimposed upon a broad pedestal, while the initial, smaller pedestal between 800 and 900 cm-' becomes even more prominent in comparison to the vibrational transitions in this region. The 892-cm-l line virtually disappears, as does the 823-cm-' peak, which is observed only as a shoulder. The 843-cm-l glycerol backbone vibration shifts to 849 cm-I, as a result of this increase in background, and becomes much less conspicuous in comparison to either the 717- or 877-cm-' peaks. Rapid cooling of the sample results in maintaining or "locking in" the changes occurring on melting, except for the 892-cm-l acyl chain methyl rocking mode, which returns to nearly its former intensity. Slower cooling restores, in addition, the original frequencies and bandwidths of the C-N stretching vibrations, but not of the other features. 1000-2200-cm-1 Region. As the temperature is raised from 0 to 100 "C, the most conspicuous changes in this region occur in the relative intensities of the 1063-, 1096-, and 1130-cm-' features, which reflect the acyl chain C-C stretching modes for all-trans hydrocarbon sequences. The intensities of the 1063- and
1130-cm-' features decrease dramatically in comparison to the 1096-cm-' feature both because of decreases in the intensities of the former transitions and because of a superposition of the latter line upon a broad peak at -1090 cm-'. This latter spectral transition is associated with the appearance of acyl chain gauche isomers.8 Minor features at 1012, 1043, 1053, 1101, 1106, 1146, and 1159 cm-' remain at the higher temperatures, but a peak at 1175 cm-' disappears by 100 O C . As the temperature is further increased from 100 to 108 OC, the 1063-, 1096-, and 1130-cm-I peaks become small in comparison to the feature reflecting gauche bonds at 1090 cm-I. Weaker features in the spectrum are no longer resolved, with the exception of the 1012- and 1146-cm-' peaks. Changes in the gauche/trans populations, as reflected by the peak height intensity ratios Z1090/Z1130,are shown in Figure ~ 2 for the 8-108 "C temperature interval. When the sample is cooled, all major features in the 1060-1 130-cm-' spectral region are recovered, implying a reordering of the acyl chains into nearly all-trans sequences. 1200-1500-cm-' Region. The two most apparent changes in this region on heating DPPC from 0 to 100 "C are the broadening of the 1296-cm-' methylene twisting modes and the decreased resolution of the 1437- and 1461-cm-' components of the methylene deformation modes. Further dramatic broadening of the 1296-cm-l line, together with a shift of this feature to 1302 cm-I, occurs on heating to 108 "C. At this temperature the CH, deformation modes collapse into a single peak at 1442 cm-', a manifestation of significant interchain disorder. Of the remaining peaks in this region, only the 1340- and 1253-cm-' features are distinctly resolved at this temperature, with both appearing as shoulders on the 1302-cm-' transition. 1700-1800-cm-' Region. As the temperature increases from 3 to 100 "C, no changes occur in the frequencies and relative intensities of the 1721- and 1739-cm-' features, which are assigned to the sn-2 and sn-1 chain carbonyl stretching modes, respectively (Figure 3A,B). A significant broadening of the 1739-cm-' peak is accompanied by an apparent lesser broadening of the 1721-cm-I peak. The result is that, for an increase in temperature, the scattering intensity between the two features at approximately 1730 cm-' increases steadily and significantly relative to that of the 1721- and 1739- cm-' components. Between 100 and 108 "C, the 1721-cm-' feature decreases in intensity relative to that at 1739 cm-' and appears as a shoulder at 108 "C (Figure 3, C and D). When the sample is cooled, the line width of the coalesced features decreases; the reappearance of the 1721-cm-I peak does not occur under either rapid or slow cooling. 2800-3100-~m-~ Region. Significant changes in the 28003 100-cm-' stretching region occur both before and during the
-
Melting Behavior of Anhydrous DPPC Bilayers POLYCRYSTALLINE DPPC
The Journal of Physical Chemistry, Vol. 88, No. 9, 1984
1793
POLYCRYSTALLINE DPPC
1
w
U
I - $ ' . ' '
m
11
1
95°C
1
WAVENUMBER DISPLACEMENT (CM-1) Figure 3. Raman spectra of anhydrous DPPC in the C=O 17001800-~m-~ stretching mode region: (A) T = 11 "C, (B) T = 99 O C , (C) T = 104 "C, and (D) T = 108 "C. melting transition (Figure 4). Relative to the peak height intensity of the 2880-cm-' line, the intensities of the 2850- and -2935-cm-' features increase steadily with increasing temperature. (Actually, the broad background beneath the 2880-cm-' feature decreases as the acyl chains undergo disorder (see, for example, the discussion in ref. 8)) The effect is dramatic in plots of the 1,5/z2880 peak height intensity ratios as a function of temperature (Figure 5). While the increase in the intensity ratio is relatively constant from 0 to 100 OC, an abrupt change occurs between 100 and 108 "C. Cooling to low temperatures restores the original spectral profiles in this region, but subsequent reheating to the 90-100 OC range results in a greater increase in bilayer disorder (Figure 4E) than is observed in the initial heating through this temperature range. Spectral Assignments The assignments of many spectral features (for example, lines at 711,960,969, 1063, 1090, 1130, 1721, 1739,2850,2880, and 2835 cm-I) pertinent to monitoring lipid behavior are well characterized8-12and will not be considered in detail here. Other (9) Bunow, M. R.; Levin, I. W. Biochim. Biophys. Acta 1977, 489, 191.
WAVENUMBER DISPLACEMENT (CM-1) Figure 4. Raman spectra of anhydrous DPPC in the 2800-3100-cm-' C-H stretching mode region: (A) T = 2 "C, (B) T = 99 "C, (C) T = 104 "C, (D) T = 108 "C, and (E) T = 95 "C (second melting).
features have not been well established and are described here because of their potential use in delineating specific conformational changes in the lipid bilayer. 700-1000-cm-' Region. 824-cm-' Transition. Previously, the 0-P-0 antisymmetric stretching mode had been separately assigned to both the 824-7 and 842-cm-' lines.'3 Raman spectra of the model compounds barium dimethyl phosphate and barium diethyl phosphate show a weak feature at 815-820 cm-' that was assigned to the 0-P-0 antisymmetric stretching ~ i b r a t i o n . ' ~In (10) Spiker, R. C.; Levin, I. W. Biochirn. Biophys. Acta 1975, 388, 361. (11) Akutsu, H. Biochemistry 1981, 20, 7359. (12) Gaber, B. P.; Peticolas, W. L. Biochim. Biophys. Acta 1977,465, 260. (13) Bicknell-Brown, E.; Brown, K. G.; Person, W. B. J . Raman Spectrosc. 1981, 11, 356.
O'Leary and Levin
1794 The Journal of Physical Chemistry, Vol. 88, No. 9, I984 ANHYDROUS DPPC
tc
'.O
P
.9
'y .3
0
I
I
20
40
60
I
I
I
80
100
120
T°C
Figure 5. Temperature profile for anhydrous DPPC derived from 12935/12880 peak height intensity ratios. This ratio reflects acyl chain separation and, thus, lateral expansion of the bilayer.
polycrystalline DPPC both the 824- and 842-cm-' features are strong in comparison with the 762-cm-I 0-P-0 symmetric stretching mode, contrary to what would generally be expected for the antisymmetric stretching mode. For this reason, we prefer to assign the 0-P-O antisymmetric stretching mode to the weaker 824-cm-' feature, although we believe that the glycerol skeletal stretching mode may also contribute intensity to this narrow spectral interval. [Neat glycerol exhibits an intense Raman spectral feature at 821 cm-' (unpublished data).] 842-cm-I Transition. As discussed above, this line had been previously attributed to the 0-P-0 antisymmetric stretching mode, but its intensity in the crystal probably precludes this assignment. A glycerol backbone vibration appears to be a possible assignment for this feature, since the neat glycerol system exhibits a strong transition at 849 cm- in both Raman (unpublished data) and infrared spectra.15 877-cm-' Transition. This line was previously assigned both to a component of the C4N+asymmetric stretching mode and to the C2-CI acyl chain C-C stretching mode reflecting the interface region.13 In fluid octadecane a line at 873 cm-l disappears upon freezing of a hydrocarbon (unpublished data). Thus, we may expect a vibrational transition in this region to reflect chain disorder. In DPPC at 0 "C there are few acyl chain gauche linkages, except at the methylene group following the C = O moiety in the 2-chain. However, since the spectrum of dipalmitoylglycerol, a lipid species devoid of the phsophatidylcholine group, shows only a weak line at this frequency, we doubt the attribution in DPPC of a transition of the observed intensity to vibrational modes in either the acyl chain or interface regions. In contrast, carbamylcholine chloride displays a strong feature at 878 cm-I which was assigned to the C-N asymmetric stretching vibration." Since the conformation about the C4-N group probably exhibits nearly tetrahedral symmetry (the solution depolarization ratio for the 71 1-cm-I transition is -O"), the triply degenerate asymmetric C-N stretching mode may be expected to undergo site group splitting. That is, the 877-cm-I feature and the 960-969-cm-' doublet may represent the AI and the split E species features expected for local C3, symmetry for the C4-N moiety. 892-cm-l Transition. This line has also been considered to arise from an acyl chain C-C stretching mode,13 but it clearly reflects the terminal methyl rocking distortion for an all-trans chain. Normal-coordinate calculations, however, indicate weak coupling to C-C stretching modes.16 This spectral feature first loses (14) Shimanouchi, T. Adu. Chem. Phys. 1964, 7, 435. (15) Craver, C., Ed. 'Coblentz Society Desk Book of Infrared Spectra"; Coblentz Society: Kirkwood, MO, 1977; p 138. (16) Snyder, R. G . J . Chem. Phys. 1967, 47, 1316.
intensity and then disappears as the 1-chain and then both the 1- and 2-chain terminal methyl groups are sequentially deuterated.l* Upon melting, this band decreases in intensity for the normal do species of both DPPC and normal alkanes. On hydrocarbon chain melting, new lines appear at lower frequencies. Calculations suggest that these features, assigned to methyl rocking modes, are due to the introduction of gauche conformers along the chain." These rotamers would thus be expected to contribute to the pedestal appearing in the 800-910-cm-' region when lipids (or alkanes) are melted. Other spectral transitions, not only contributing to this pedestal but extending down to approximately 720 cm-I, probably arise from the appearance of infrared-active methylene rocking modes which become weakly allowed due to the loss of local symmetry on chain melting. 952-cm-' Transition. Since this line is present in glycerylphosphorylcholine,' but not in dipalmitoylglycerol (unpublished data), we associate the transition with the head group moiety. With respect to the spectrum of DPPC, lysolecithin, a single-chain species containing the phosphatidylcholine head group, shows an increase in the relative intensity of this feature in comparison to acyl chain vibrations, such as the 892-cm-I peak; however, the intensity of the 952-cm-' line is essentially unchanged in comparison to the C-N stretching modes.10 We, therefore, tentatively associate this vibration to a methyl rocking mode of the choline CH3 groups. 1000-1 200-cm-' Region. 1012-em-' Transition. Since glyceryl phosphorylcholine exhibits a medium-strength Raman line at 1020 cm-' and since only very weak features are observed in the Raman spectrum of dipalmitoylglycerol in this region, we associate the 1012-cm-I feature in DPPC with the phosphatidylcholine head group. Further, we note that the infrared spectrum of dipalmitoylphosphatidylethanolamine displays a strong band at 1020 cm-'. Thus, we assign the 1012-cm-' feature to a choline C-C stretching mode, which may possibly be coupled with C-N stretching mode contributions. 1106-cm-' Transition. Since dipalmitoylglycerol exhibits a Raman feature at 1104 cm-I and glyceryl phosphorylcholine has virtually no features in this region, we attribute the 1106-cm-I component to an acyl chain vibration. We note that the equivalent sn-1 and sn-2 acyl chains are distinguishable not only by chain length, due to the gauche bend at the methylene linkage following the C=C group in the sn-2 chain, but also by crystal packing arrangements because of their different angles of attachment to the glycerol backbone. For this reason, it seems reasonable to expect the k = 1 mode for the sn-2 acyl chain to occur at a slightly different frequency than that for the sn-1 acyl chain. In DMPC polycrystalline systems for which the chain terminal CH3 groups were selectively deuterated, the k = 1 modes were assigned to frequencies at 1094 and 1092 cm-I for the completely hydrogenated do sn-2 and sn- 1 chains, respectively.I6 For the DPPC polycrystalline material in the present study, the k = 1 modes for the sn-2 and sn-1 chains are assigned to the 1106- and 1096-cm-' features, respectively. The disparity in intensity for these two components in Figure 1A presumably arises from the superposition of three features in the 1090-1 1 IO-cm-I interval.
-
Discussion Although the changes which occur in the melting of anhydrous DPPC are concerted, an understanding of these changes is facilitated by separately discussing the different parts of the molecule. For convenience, we have divided the molecule into the polar head group (phosphorylcholine),interface (glycerol backbone and carbonyl moieties), and hydrophobic acyl chain regions. Polar Head Group Region. Changes in the polar head group region are most conveniently probed by the 7 1 1-7 17-cm-I totally symmetric C-N stretching vibration. Heating the anhydrous bilayer from 0 to 100 ' C results in only a small increase in line width (-2 cm-') and in no change in the frequency of this vibrational mode. Melting, however, causes a shift in frequency (17) Zerbi, G. J . Mol. Struct. 1981, 73, 235. (18) Levin, 1. W.; Huang, C . , unpublished data.
Melting Behavior of Anhydrous DPPC Bilayers
The Journal of Physical Chemistry, Vol. 88, No. 9, 1984 1795
TABLE 11: Temperature Dependence of Selected Vibrational Frequencies and Half-Widths (in cm-I) for Anhydrous, Polycrystalline DPPC Bilavers vibrational mode
0 "C
50 "C
9 9 "C
104 "C
108 " C
symmetric C-N stretching CH, twisting C=O stretching
712 (9)" 1296 (7) 1722 (9) 1739 (11)
711 (9) 1296 (8) 1722 (10) 1739 (11)
7 1 1 (11) 1298 (13) 1723 (23) 1738 (14)
7 1 3 (20) 1299 (26) 1723 (nm)b 1737 (nm)
717 (19) 1302 (30) 1739 (28)
" The first entry represents the vibrational frequency in cm-' ; the value in parentheses represents A v , , ~the , half-width, in c n - ' .
nm
implies that the line width is not measurable.
from 71 1 to 717 cm-I, together with a doubling of the line width. Table I1 summarizes these changes. The position and line width of this vibrational mode are unchanged when the sample is rapidly cooled to 0 OC. The small change in line width during heating before melting and the absence of change on cooling indicate clearly that the increased line width is due to a conformational rearrangement of the head group, rather than an increased motional freedom for the choline moiety. Crystallographic studies of DPPC.2H20 show two distinct and slightly inequivalent head group conformations. The Raman spectrum of DPPC.2H20 exhibits a single, narrow peak at 718 crn-I ( A v l j z 10 cm-'). The difference in frequencies between the anhydrous (71 1 cm-') and partially hydrated (718 cm-') forms indicates markedly different environments for the head groups. The large line width in melted, anhydrous DPPC strongly suggests that multiple head group environments exist in the polycrystalline state. The various conformations perhaps involve both arrangements observed in the anhydrous and partially hydrated crystals. Since the intermolecular interactions are quite different for the two inequivalent DPPC molecules in the subcell of DPPC.2H20, and since the C-N stretching mode line width is small in this crystal,19 it appears likely that it is not intermolecular, but intramolecular, reorganizations contributing to the increased line width. It is possible that the intermolecular changes exert equal, but opposing effects. In any event, simultaneous spectral changes involving both the 71 1-cm-' transition and shifts in the 1253-cm-' PO2- stretching frequencies have been presented as evidence for conformational rearrangements within the polar head group.4 Interface Region. The conformational state of the interface region is reflected in the C = O stretching modes at 1721 and 1739 cm-' in anhydrous polycrystalline DPPC. The frequencies of the C=O stretching vibrations are exquisitely sensitive to the local environment and the bonding at adjacent hydrocarbon chain linkages; these effects give rise to the large frequency difference between the 1-chain (1739 cm-') and the 2-chain (1721 cm-') C=O stretching mode^,^^^^ as well as to the spectral pattern observed for the 1-chain or 2-chain modes for the nonequivalent molecules in the DPPC.2H20 subcell.20 In a polar environment the vibrational frequency tends to be lower than it is in a nonpolar enviror~ment.~~ In DPPC.2H20 crystals, crystallographic studies2' indicate that the glycerol backbone exhibits the gauche- (8-) configuration, based upon the Fuson and Prestegard notation.22 Energy-minimization calculation^^^ have demonstrated the existence of a local energy minimum in the gauche' (g+) configuration which is less than 0.5 kcal/mol above that of the crystallographic conformation. Fuson and Prestegard22have suggested that a fast jump between these two conformations is indicated by deuterium N M R order parameters reflecting the 2-methylene group of the DMPC sn-2 chain. The spectra that we have obtained in the 1700-1800-cm-' region strongly support this contention. The line width of the sn-2 C=O stretching mode (1721 cm-') increases slowly with increasing temperature below 100 OC, suggestive of an increased motional freedom in this part of the
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(19) Mushayakarara, B. C . ; Levin, I. W., unpublished data. (20) Mushayakarara, E.; Levin, I . W . Biochim. Biophys. Acta 1982, 686, 153. (21) Pearson, R. H.; Pascher, I . Nature (London) 1979, 281, 501. (22) Fuson, M. M.; Prestegard, J. H. Biochemistry 1983, 22, 1311. (23) MacAlister, J.; Yathindra, N.; Sundaralingham, M . Biochemisfry 1973, 12, 1189. (24) Bush, S. F.; Levin, H.; Levin, I . W. Chem. Phys. Lipids 1980, 27, 101.
molecule. A much srnaller increase in line width is seen in the sn-1 C=O stretching mode. The peak at 1721 cm-' slowly decreases in intensity during the melting transition, but never entirely disappears, as is evidenced by the marked asymmetry of the single C=O stretching mode feature in Figure 3D. This is precisely the behavior which is expected for a conformational transition of the type suggested by Fuson and PrestegardZ2for the hydrated liposomal forms. In the high-temperature case, the sn-2 chain carbonyl group is now in a slightly less poiar environment than when in its initial g- conformation. This structurai change from the g- to g+ conformation would be consistent with an increase in the frequency of the C=O stretching mode. We anticipate this frequency to occur between that of the g- sn-2 chain C=O stretching vibration and that of the sn-1 chain C=O stretching mode, that is, near the observed intensity increase around 1730 cm-I. The asymmetry in the C=O Raman line which we observe in the anhydrous, melted lipid is also present in the fully hydrated lecithin bilayer^,^ supporting the contention that two rotational isomers of the glycerol backbone are present in solution. The temperature dependence of the carbonyl stretching mode frequency and half-width parameters are given in Table I1 with comparisons for the C-N symmetric stretching and CH2 twisting modes. Changes in the C=O stretching region vibrations have been seen on addition of cholesterol to hydrated DPPC bilayer^.^^,^^ These changes may reflect differences in the interaction of cholesterol with g- and g+ conformations for the sn-2 acyl chains. Further investigations are underway in our laboratory to clarify this possibility. Acyl Chain Region. The inter- and intramolecular order of the acyl chains is reflected by numerous spectral features. The peak height intensity ratio Iw2935/I2880 monitors primarily interchain interactions, such as lattice expansion. As the anhydrous lipid is heated from 0 to 100 O C , this ratio increases steadily to the point where, at 99 OC, prior to the crystal melting transition, there is significantly greater chain disorder in the crystal than there is below the phase transition in the hydrated lipid dispersion. This lateral expansion of the lattice on heating has been noted previously in X-ray diffraction s t ~ d i e s .Melting ~ and heating the crystal to 108 OC results in a more disordered system than the fully hydrated dispersion in the liquid crystalline state. The change in lateral disorder upon crystal melting appears to be at least as great as that for the melting of hydrated phospholipid liposomes. Heating the anhydrous crystal to below 100 OC induces a slow, but steady, increase in the number of acyl chain isomers. At the phase transition the number of gauche rotamers, reflected by the 1090-cm-' region of the spectrum, increases rapidly, as occurs in the melting of hydrated bilayers. The number of gauche isomers is greater in the unmelted crystal just below its melting temperature in comparison to the hydrated liposomal gel state at a point prior to its melting temperature. At this premelting temperature, however, the anhydrous crystal exhibits about as many gauche isomers as would be seen for the same degree of lateral expansion in the hydrated gel. The number of gauche isomers is much higher at 108 OC for the anhydrous system than in the liposome above its phase transition. The introduction of gauche isomers is further evidenced by the changes in the 800-900-cm-' region. The 892-cm-' acyl methyl rocking mode decreases in intensity as the number of gauche
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( 2 5 ) Bichell-Brown, E.; Brown, K. G. Biochem. Biophys. Res. Commun. 1980, 94, 638.
1796 The Journal of Physical Chemistry, Vol. 88, No. 9, 1984
isomers increases, while new methyl rocking transitions, due to shorter chain segments with gauche rotamers, appear in the 800-880-cm-' region." In addition, CH2 rocking modes in this region, which are symmetry forbidden in the Raman spectrum of all-trans chains, become weakly allowed on chain disorder. This gives rise to the broad pedestal which spans much of the 7001000-cm-' region. Role of Water in Lipid Melting. One effect of water on the bilayer is to contribute a surface pressure, which shifts the melting transition to lower temperatures. Because we anticipate a contribution on the order of 100 dyn/cm, we might expect the anhydrous system, on the basis of the DPPC monolayer phase diagram,26,27and theoretical calculations,28 to reside in a surface pressure/temperature region in which the transition would not be first order. While we cannot dismiss this possibility, the observed, relatively sharp, melting curves in Figures 2 and 5 are consistent with first-order melting. For this reason, we believe that the role of water is far more complex (vide infra) than a simple contribution of a surface pressure. Chapman noted that the first melting of anhydrous distearoylphosphatidylcholine bilayer is highly endothermic, with a heat of transition of 13.8 kcal/mol.' The second melting results in a much smaller enthalpy, 6.3 kcal/mol. (The enthalpy of melting for the hydrated lipid is 10.6 kcal/mol.) These observations are understandable from the data presented here. When the DPPC crystal melts for the first time, there are three potentially endothermic changes which take place; isomerization of the acyl chains, isomerization of the head group, and isomerization of the glycerol backbone. In the second melting of the sample, there is only acyl chain isomerization, however, because cooling does not generally restore the original head group or the glycerol backbone conformations to their usual, ordered form. Since the second melting results in about the same disordering of the acyl (26) Phillips, M. C.; Chapman, D. Biochim. Biophys. Acta 1968,163,301. (27) Vilallonga, F. Biochim. Biophys. Acta 1968, 163, 290. (28) Nagle, J. F. J . Membr. Biol. 1976, 27, 233.
O'Leary and Levin chains and lattice expansion as the first process, we expect that the enthalpy of head group and interface reorientation is about 7.5 kcal/mol (13.8-6.3 kcal/mol). A critical effect of water on the phospholipid membrane is to disorder and unlock the head group and interface regions. It is clear from our results that this unlocking is not simply a function of lattice expansion, since expanded crystalline systems exhibit frozen head group and glycerol backbone configurations. Why is the enthalpy of melting of the once-heated crystal form less than that of the hydrated liquid? In the hydrated lipid the melting transition starts from a point in which there is much less lattice expansion than that for the crystal just below its melting point. Lattice expansion is thought to contribute about 2 / 3 of the enthalpy of melting.2g While the amount of expansion at the phase transition is about the same in either the hydrated or anhydrous forms, the energy cost of separating the chains is much greater when expanded from the more closely packed hydrated system, particularly since one is on a steeper portion of the r-6 curve for the van der Waals interactions. That is, for the previously heated crystal form, much of the energy change potentially available for the transition has been consumed by the lattice expansion prior to melting. In summary, the structural reorganizations which occur on heating anhydrous, polycrystalline samples of DPPC appear to mimic many of the conformational changes arising in completely hydrated liposomal dispersions. The advantage in monitoring the Raman spectra of anhydrous systems over a large temperature span, rather than observing spectral changes in the hydrated system, lies in the ability both to probe the evolution of structural changes propagated throughout the lipid bilayer and to discriminate spectroscopically between subtle conformational rearrangements which are not necessarily apparent in the liposomal dispersions. Registry No. DPPC,2644-64-6. (29) Nagle, J. F.; Wilkinson, D. A. Biophys. J . 1978, 23, 159.
