J. Phys. Chem. B 1997, 101, 8933-8940
8933
FTIR Spectroscopy Studies of the Conformational Order and Phase Behavior of Ceramides David J. Moore* and Mark E. Rerek International Specialty Products, Skin R&D, 1361 Alps Road, Wayne, New Jersey 07470
Richard Mendelsohn Department of Chemistry, Rutgers UniVersity, 73 Warren Street, Newark, New Jersey 07102 ReceiVed: June 3, 1997; In Final Form: August 26, 1997X
Ceramides, the major lipid component of the stratum corneum (SC), provide many of its unique physical properties. Surprisingly, only a few biophysical studies of hydrated ceramides have been reported. The current Fourier transform infrared (FTIR) spectroscopy investigation provides the first detailed study of intermolecular and intramolecular chain and headgroup interactions in hydrated non-hydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) ceramides. Information about NFA and HFA ceramide chain subcell structure and conformational order is derived from the temperature dependence of the methylene stretching, scissoring, and rocking mode frequencies. At low temperatures, NFA ceramide is highly ordered and packed in an orthorhombic subcell structure that undergoes a solid-solid phase transition to a conformationally ordered hexagonal phase at 60 °C. A second transition to a conformationally disordered non-bilayer-like phase occurs at 80 °C. The lipid chains of HFA ceramide undergo a single transition from a conformationally ordered, orthorhombic subcell, phase to a conformationally disordered phase at 76 °C. In NFA ceramide the amide I and II modes of the headgroup are each split into two bands, indicating strong intermolecular headgroup coupling between two NFA ceramide headgroups in a factor group perpendicular to the bilayer plane. In contrast, splitting is not observed for either amide mode in HFA ceramide. However, the presence of strong H bonding indicates an interaction between molecules in the bilayer plane. The contrasting behavior of the headgroups in NFA and HFA ceramide suggests that these molecules make distinct contributions to the structural integrity of the stratum corneum. The implications of these findings to the recently proposed domain mosaic model of the stratum corneum lipid barrier are discussed.
Introduction Ceramides have generated much interest recently in large part because of their role as signal transducers in the regulation of several fundamental biochemical processes including cell proliferation, cell differentiation, and apoptosis.1-3 In addition, ceramides are the major lipid component of the outermost layer of skin, the stratum corneum (SC). As such, they play a fundamental role in mammalian physiology.4-6 The skin retards water loss to the environment and thereby helps maintain the homeostasis necessary for other organs to function.7 This “barrier function” of skin resides in the lipids of the SC.6 The SC consists of corneocytes (large flattened cells filled with keratin) embedded in a hydrophobic lamellar lipid matrix of ceramides, cholesterol, and fatty acids. The lipid component of SC is unique among cell membranes in having a very high ceramide content. In addition, one-third of SC lipid acyl chains are C22 or longer. As such, these skin lipids are in fact waxes.8 Whereas most membrane lipids in vivo exist in a liquid crystalline phase through which water can pass, the majority of SC lipids are in gel or solid phases and present a barrier to water passage.9,10 In 1989 the proposed stacked lamellar bilayer structure of SC lipids was confirmed by direct observation with transmission electron microscopy (TEM).11 Biophysical techniques including X-ray,12-14 NMR,15,16 DSC,17 AFM,18 and FTIR19-21 have been applied to further characterize the structure and phase behavior * Corresponding author: e-mail
[email protected]; Tel 973-6283221; Fax 973-628-3886. X Abstract published in AdVance ACS Abstracts, October 1, 1997.
S1089-5647(97)01810-5 CCC: $14.00
of SC lipids. From these studies, a picture is emerging of a highly complex heterogeneous organization containing solidand gel-state lipids. Of the aforementioned biophysical techniques, FTIR provides extensive information on both the conformational order and molecular organization of the highly ordered lipid states which dominate the structure under physiological conditions. A widely used model for the SC lipids is a three-component equimolar system of bovine ceramide III, palmitic acid, and cholesterol. NMR,22,16 X-ray,14 and AFM18 studies have all indicated the presence of ordered phases and domains within model SC lipids under physiological conditions. We recently reported that the palmitic acid and ceramide components of model SC exist in separate ordered domains with orthorhombic subcells under physiological conditions.21 The ceramide and palmitic acid were further demonstrated to remain segregated as the palmitic acid undergoes a thermotropic transition to a disordered state. The current studies of hydrated non-hydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) ceramide were undertaken as part of an ongoing project to characterize the biophysical properties of SC and to aid in the interpretation of data from the three-component model SC. Human ceramides 2 and 4 are members of the NFA and HFA ceramide families, respectively, and constitute the majority of ceramide in human SC.18 Although there exists a substantive and detailed biophysical literature on sphingomyelin and cerebrosides (the parent molecules of ceramide) and model SC lipid mixtures containing ceramide, there appears to be only a few studies of pure hydrated ceramides.18,23-28 To our knowledge there have been no detailed FTIR biophysical studies of the conformational order and molecular organization of hydrated NFA ceramides. © 1997 American Chemical Society
8934 J. Phys. Chem. B, Vol. 101, No. 44, 1997
Figure 1. Chemical structures of NFA and HFA ceramide (bovine ceramide types III and IV, respectively) used in this study.
Experimental Section Materials. Bovine brain ceramides (N-acylspingosine) type III, the non-hydroxy fatty acids (NFA), and type IV, the R-hydroxy fatty acids (HFA), were purchased from Sigma (St. Louis, MO) and used without purification. The amide-linked fatty acids of ceramide III consist primarily of octadecanoic (stearic) acid (C18:0) and cis-15-tetracosenoic (nervonic) acid (C24:1,cis15). Bovine brain ceramide III is widely used in model lipid systems of human SC. The amide-linked fatty acids of ceramide IV consist primarily of octadecanoic (stearic) acid (C18:0), docosanoic (behenic) acid (C22:0), tetracosanoic (lignoceric) acid (C24:0), and cis-15-tetracosenoic (nervonic) acid (C24:1,cis15). The structures of both ceramides are shown in Figure 1. Sample Preparation for FTIR Spectroscopy. Bovine ceramide III was dried from chloroform solution onto a ZnSe ATR crystal mounted in a trough plate and placed in a temperature-controlled, horizontal ATR setup (Spectra-Tech, Shelton, CT). The trough was filled with buffer (150 mM NaCl in H2O or D2O) covering the ceramide film. A cover plate was placed over the trough to prevent buffer evaporation. These “hydrated” ceramide samples were left for 16-18 h at room temperature before spectra were acquired. ATR samples of bovine ceramide type IV were prepared in the same manner, except that ceramide IV was dried from a mixed chloroform/ methanol solution. As a general experimental method this ATR setup appears to be a good model for the stratum corneum. The ceramides are adjacent to, and in contact with, an aqueous environment (as is the stratum corneum in human skin), yet as is shown below, only a small amount of water penetrates through the ordered ceramide bilayers. Thus, at physiological temperatures, the ceramides are fully hydrated with, however, a low percent of water compared to other membrane models. Samples of anhydrous ceramide and fatty acids were prepared as pressed KBr disks. Spectra of NFA ceramide in CHCl3 were acquired at room temperature using a 0.1 mm fixed path length liquid transmission cell (International Crystal Laboratories, Garfield, NJ). FTIR Spectroscopy. All FTIR spectra were acquired on one of two Mattson RS1 spectrometers equipped with broad- and narrow-band MCT detectors, respectively. Spectra were generated by coaddition of 512 interferograms collected at 2 cm-1 resolution under continuous dry air purge. The temperaturecontrolled horizontal ATR setup allowed the temperature of the ATR plate to be precisely controlled and monitored. Spectra in the ATR experiment were routinely acquired at 2 or 3 °C intervals from 30 to 85 °C. The transmission measurements (KBr and chloroform) were conducted on samples at room temperature.
Moore et al.
Figure 2. (A) Thermotropic response of the symmetric CH2 stretching mode frequency of NFA ceramide showing transitions at 60 and 80 °C. (B) Thermotropic response of the symmetric CH2 stretching mode frequency of HFA ceramide showing a single transition at 75 °C.
Figure 3. (A) Frequencies of the CH2 scissoring bands for NFA ceramide as a function of temperature. (B) Inverted second-derivative spectra from 30 to 85 °C of the scissoring region for NFA ceramide.
IR Data Analysis. All IR spectra were analyzed off-line. Data reduction protocols such as the generation of derivative spectra or difference spectra, curve fitting, and peak picking were performed using software written at the National Research Council of Canada. Results Figure 2 shows the thermotropic response of the CH2 symmetric stretching frequency of NFA ceramide (Figure 2A) and HFA ceramide (Figure 2B). As discussed below, the temperature dependence of the CH2 symmetric and asymmetric stretching frequencies in hydrocarbon chains provides a qualitative measure of changes in conformational order and/or chain packing. In NFA ceramide the C-H stretching mode data clearly reveal two transitions. The first, characterized by a frequency increase of ∼1 cm-1, occurs with a midpoint of about 60 °C from a low-temperature value of ∼2848 cm-1. As the temperature is increased, a second highly cooperative transition (from 2849 to 2853 cm-1) centered at 80 °C is observed. The CH2 stretching modes for HFA ceramide show a single transition from a highly ordered phase (2847 cm-1) to a disordered phase (2853 cm-1) at 76 °C. In Figure 3 the CH2 scissoring mode frequencies of NFA ceramide are plotted (Figure 3A) along with the inverted second-derivative spectra (Figure 3B) showing the splitting in the scissoring band as a function of temperature. At lower temperatures the mode is split with components near 1464 and 1471 cm-1. The splitting collapses at 60 °C to a single peak at 1467 cm-1. The frequency then decreases at 80 °C to ∼1466 cm-1. The spectra showing the CH2 scissoring and rocking modes for hydrated HFA ceramide are displayed in Figure 4; these spectra were acquired at 30 °C. The top spectra in Figure 4 are original (left) and inverted second-derivative (right) spectra of hydrated HFA ceramide before heating. The
FTIR Sudies of Ceramides
Figure 4. (A) Original spectra on the left show the scissoring region of HFA ceramide at 30 °C under three conditions: before heating (top); spectrum taken immediately after heating to 88 °C and cooling to 30 °C (middle); spectrum taken 96 h after heating to 88 °C and cooling to 30 °C. The spectra on the right are the corresponding inverted secondderivative spectra. (B) Original spectra on the left show the rocking region of HFA ceramide at 30 °C under three conditions: before heating (top); spectrum taken immediately after heating to 88 °C and cooling to 30 °C (middle); spectrum taken after 96 h after heating to 88 °C and cooling to 30 °C. The spectra on the right are the corresponding inverted second-derivative spectra.
middle spectra are original and derivative spectra of HFA obtained immediately after cooling a sample back to 30 °C from 88 °C. The lower spectra were obtained 96 h after heating to 88 °C and cooling to 30 °C. The scissoring bands in the top spectrum exhibit two peaks near 1468 and 1462 cm-1. The former is shifted down by several wavenumbers from values expected of typical orthorhombic phases. After heating and cooling, a metastable hexagonal phase is formed characterized by a single peak (1467 cm-1) in the original and secondderivative spectra (middle spectra of Figure 4). The HFA ceramide appears to have converted to an ordered orthorhombic phase after 96 h and displays typical scissoring band positions of 1471 and 1462 cm-1. The original (left) and inverted secondderivative (right) spectra of the same samples in the 700-750 cm-1 region are displayed in Figure 4B. The CH2 rocking modes near 720 cm-1 show a splitting in the preheated sample (top spectra) of 721 and 726 cm-1. Immediately upon cooling (middle spectrum) only one band is observed at 721 cm-1 which, as with the scissoring modes, splits (720, 727 cm-1) after 96 h. The CH2 wagging modes couple in all-trans chains to produce progressions such as those clearly visible in the 30 °C difference spectrum (a) of NFA ceramide plotted in Figure 5. This spectrum was generated by subtracting the spectrum of NFA ceramide at 85 °C from that at 30 °C. Reference spectra for pure nervonic (b) and stearic (c) acids (the major fatty acid components of NFA ceramide) are included in the figure. Figure 6 shows the spectral region 1330-1390 cm-1 for NFA ceramide in D2O which includes the conformationally sensitive CH2 wagging modes. In disordered phases these features arise from particular, localized, two- or three-bond conformational states, as discussed below. The lower spectrum (a) in Figure 6, taken at 30 °C prior to heating, reveals a band at 1365 cm-1 which arises from C-OH bending (vide infra). This band overlaps the conformationally sensitive CH2 wagging bands at 1341, 1353, 1362, and 1368 cm-1. Spectrum (b) is NFA ceramide in D2O buffer at 30 °C, after the sample was first heated to 88 °C on the ATR crystal and then cooled back to 30 °C. When the NFA ceramide undergoes its phase transition at 60 °C, all exchangeable hydrogens are replaced by deuterons.
J. Phys. Chem. B, Vol. 101, No. 44, 1997 8935
Figure 5. The upper spectrum (a) is a difference spectrum of NFA ceramide at 30 °C showing the coupled CH2 wagging progression of NFA ceramide. The middle spectrum (b) shows the progression band in a KBr disk of nervonic acid. The lower sample (c) is a KBr sample of stearic acid showing the same spectral region.
Figure 6. Spectra of the CH2 wagging region for NFA is a variety of conformationally disordered phases, as follows: (a) NFA ceramide in D2O, spectrum acquired at 30 °C prior to heating. Note the feature at 1365 cm-1 arising from C-OH in-plane bending. (b) NFA ceramide in D2O, spectrum acquired at 30 °C after the sample was heated to 88 °C and cooled. The C-OH hydrogen is exchanged; the interfering band at 1365 cm-1 is mostly eliminated. (c) As (b), acquired at 73 °C. (d) As (b), acquired at 85 °C. (e) NFA ceramide dissolved in CHCl3 (isotropic or inverted micellar phase).
The C-OH bending mode at 1365 cm-1 is shifted in frequency as a result of H f D exchange. Removal of this spectral feature simplifies the spectral region and permits detailed analysis of the conformationally sensitive CH2 wagging modes in NFA ceramide. Spectra c and d show the CH2 wagging region of hydrated NFA ceramide at 67 and 85 °C, respectively. The top spectrum e in Figure 6 is of NFA ceramide dissolved in CHCl3, where it presumably exists as a monomeric species or possibly an inverted micelle. The 1500-1800 cm-1 spectral region contains the amide I and amide II modes of ceramide. Figure 7 shows a thermotropic series of spectra for NFA ceramide in H2O buffer. From ∼28 to 62 °C the amide II mode is split into two components at 1545 and 1568 cm-1. At 64 °C the amide II region collapses to a single peak centered at 1551 cm-1 which broadens into the baseline at ∼80 °C. The amide I mode of NFA ceramide is also split into a doublet giving peaks at 1619 and 1645 cm-1. This splitting does not collapse at the 60 °C transition, as the amide II does, but the two peaks broaden with a slight frequency increase in the lower component and then coalesce to form a single broad band at ∼80 °C. Equivalent spectra for NFA ceramide in D2O buffer are displayed in Figure 8. Again, both the amide I (1619 and 1645
8936 J. Phys. Chem. B, Vol. 101, No. 44, 1997
Figure 7. These absorbance spectra show the 1500-1800 cm-1 spectral region of NFA ceramide in H2O buffer from 30 to 88 °C. Both the amide I (∼1650 cm-1) and amide II (∼1550 cm-1) modes are split into two bands at lower temperatures. From ∼60 to 80 °C only the amide I mode appears split. At 80 °C this broadens to a single band.
Figure 8. Amide I and II spectral region of NFA ceramide in D2O buffer. At lower temperatures, both modes are split. At the first phase transition the amide II modes disappears due to deuterium exchange, and the amide I modes shift. Again the amide I mode remains split until ∼80 °C.
cm-1) and II (1546 and 1568 cm-1) modes are split at low temperatures. Between 64 and 70 °C the amide II bands disappear as a result of H f D exchange. Concurrent with the disappearance of the amide II modes, the components of the amide I bands each shift to lower frequency (1645 f 1632 cm-1 and 1619 f 1611 cm-1) as expected upon deuterium exchange. Again, at high temperature the amide I mode broadens into a single contour. Figure 9 shows spectra of HFA ceramide in D2O buffer as a function of temperature. There is no splitting in the amide I or II modes of HFA ceramide, although the occurrence of two amide I bands is suggested by the asymmetry of the peak. The temperature dependence of the contour suggests that the two components arise from the normal and H f D exchanged amide I. With increasing temperature, there is increasing H f D exchange as evidenced by increased intensity in the lower frequency component of the amide I band and the loss of the amide II band. Complete H f D exchange of these modes does not occur until the onset of the phase
Moore et al.
Figure 9. These absorbance spectra show the 1500-1800 cm-1 spectral region of HFA ceramide in D2O buffer from 33 to 88 °C. Unlike NFA ceramide, these modes do not show splitting. Complete deuterium exchange does not occur until the phase transition at 80 °C.
Figure 10. Absorbance spectra showing the 1500-1800 cm-1 spectral region of anhydrous samples of NFA and HFA ceramide. As in the hydrated samples, splitting is observed in both modes for NFA ceramide but not for HFA ceramide.
transition, indicating that HFA ceramide remains highly ordered (therefore the amide bonds are not completely solvent accessible) until this transition. Spectra of NFA and HFA ceramide in the solid state (nonhydrated) are shown in Figure 10. For NFA ceramide in the solid state both amide modes are split and occur at approximately the same frequencies (amide I: 1620, 1647 cm-1; amide II: 1547, 1569 cm-1) as in the hydrated samples. In contrast, HFA ceramide shows no splitting of the amide modes even in the nonhydrated solid state (amide I solid ) 1631 cm-1, amide I buffer ) 1629 cm-1, amide II solid ) 1535 cm-1, and amide II buffer ) 1531 cm-1). Finally, spectra of NFA ceramide in Figure 11 in the 9301000 cm-1 region show the out-of-plane C-H bending mode of the trans CdC bond in the spingosine chain at the expected frequency of 960 cm-1. In NFA ceramide but not HFA ceramide (data not shown for HFA ceramide) this mode splits into two components approximately 20 cm-1 apart. In Figure 11 the temperature response of this splitting can be observed in the spectra (Figure 11A) and in the plot of frequencies as a
FTIR Sudies of Ceramides
Figure 11. These absorbance spectra show the temperature dependence of trans CdC-H out-of-plane bending mode of the spingosine chain in hydrated NFA ceramide. Below ∼60 °C this mode shows a 20 cm-1 splitting into two bands which increases to 25 cm-1 from 60 to 80 °C and then collapses to a single peak at 80 °C.
function of temperature (Figure 11B). At 60 °C there is an increase in the splitting of the 960 cm-1 band from 20 to 25 cm-1. This is followed by a collapse to a single peak at 80 °C. The temperatures of these transitions correlate with those observed from the acyl chain stretching and bending modes. Discussion The power of IR spectroscopy for the study of lipid phases and molecular interactions is clearly demonstrated from the current experiments. The following discussion is based upon the extensive and detailed spectra-structure correlations available for chain modes, in large part due to the studies of alkanes by Snyder and his collaborators.29-34 The numerous extensions of these investigations to phospholipid phases have been reviewed.35-37 Although IR studies of gangliosides have been reported,38,39 to our knowledge no detailed IR reports of hydrated NFA ceramide structure and organization have appeared. Recently, a DSC and FTIR study on the interaction of cholesterol sulfate with HFA ceramide was published.25 Comparison of the current IR measurements with low-angle X-ray diffraction and DSC studies of NFA and HFA ceramide26,27 provides a reasonably complete picture of the molecular conformation and organization of the NFA and HFA ceramide phases. CH2 Stretching Frequencies. The temperature dependence of the CH2 stretching frequencies provides a qualitative indicator of changes in chain packing and conformational order. Although the dependence on conformational order is wellestablished, the effects of altered chain packing are not as widely recognized. Nevertheless, Snyder and co-workers33 clearly
J. Phys. Chem. B, Vol. 101, No. 44, 1997 8937 showed that the frequencies of the antisymmetric CH2 stretching (d- in their notation) and the symmetric CH2 stretching (d+ in their notation) modes for a polyethylene crystal with all-trans chains depend on both packing geometry and density in a temperature-dependent fashion. Thus, changes in both conformational disorder and packing can alter the d- and d+ frequencies. These effects can be distinguished on the basis of the actual measured values of the frequencies. For the d+ mode, which ranges from ∼2847 to 2856 cm-1, shifts are assumed to arise from packing changes if the band position is below ∼ 2850 cm-1. The introduction of conformational disorder is suggested if the frequencies exceed this value. In a recent study Snyder and co-workers40 used the d+ mode to identify solidsolid phase transitions in aqueous dispersions of disaturated long-chain phosphatidylcholines. In the current study, the d+ mode of NFA ceramide clearly reveals (Figure 2) two transitions as the temperature is increased from 30 to 90 °C. The first is a solid-solid phase transition at 61 °C in which the d+ band increases by ∼1 cm-1; the second, centered at 80 °C, is a transition to a conformationally disordered phase. In contrast, HFA ceramide shows a single transition to a disordered phase centered at 76 °C. This Tm agrees well with a recent IR and DSC study of HFA ceramide and cholesterol sulfate.25 The Tm values observed in the current work may also be compared with the DSC-derived values of Shipley and coworkers.26,27 They observed a single, reversible transition at 80.0 °C for fully hydrated HFA ceramide, in good accord with the current work. For NFA ceramide, they observed two transitions, at 72.7 and 81.1 °C. The discrepancy between their temperatures and the current values may be explained by differences in the composition of the amide-linked fatty acids (Figure 1) in the two studies. The current sample consists primarily of stearic (C18:0) and nervonic (C24:1,cis15) acids. In the DSC study the principal fatty acids were C24, C22, and C18. CH2 Scissoring Bands. While the thermotropic response of CH2 stretching frequencies provides a useful measure of phase transition temperatures, the observed band positions cannot unambiguously identify the nature of the phases present. The CH2 scissoring bands are very useful in this regard. The scissors band region (1460-1475 cm-1) reveals two components for orthorhombic subcell chain packing.29,30,37 In the current work, the temperature dependence of the scissors bands for NFA ceramide (Figure 3) shows a clear splitting (1464, 1471 cm-1) which collapses at ∼60 °C. The splitting unambiguously identifies the low-temperature phase as possessing an orthorhombic perpendicular subcell packing. A similar conclusion arises from the observations of the CH2 rocking frequencies (not shown). At 59 °C, a phase with a scissors band frequency at 1468 cm-1 appears. This frequency is suggestive of hexagonal chain packing, perhaps associated with chain orientational disorder. As the main transition at 80 °C is passed, the scissors band increases in width by 3 cm-1 and decreases in frequency by ∼1 cm-1, suggestive of a conformationally disordered chain. We recently reported the presence of orthorhombic domains of NFA ceramide in a ternary lipid model stratum corneum.21 Previous X-ray41 and IR42 studies of mammalian stratum corneum have also reported the presence of orthorhombic phases up to ∼60 °C. Unusual behavior of the scissoring bands is observed for HFA ceramide upon initial hydration. The observed splitting (1462, 1468 cm-1) exhibits an atypical high-frequency component, which usually appears between 1470 and 1474 cm-1. The effect could be related to the presence of the R-hydroxyls in the chain, which likely perturbs the packing from an ideal orthorhombic
8938 J. Phys. Chem. B, Vol. 101, No. 44, 1997 perpendicular subcell. An alternative explanation is that the sample as initially prepared contains a mixture of orthorhombic and hexagonal phases. The latter might give rise to the higher frequency component of the doublet and mask the 1471 cm-1 band, which only appears upon annealing. These atypical scissoring frequencies are also observed in the spectra of anhydrous HFA ceramide. The single scissoring mode at 1468 cm-1 at the highest temperatures reveals the presence of hexagonally packed chains. Shipley and co-workers have used X-ray diffraction to identify the observed transition as Lβ-HII (inverted hexagonal).27 In the current experiment the chains remain hexagonally packed in a metastable bilayer gel state after cooling the sample. Over several days this converts to an orthorhombic phase exhibiting the expected scissoring mode frequencies of 1462 and 1471 cm-1 (Figure 4). CH2 Wagging Modes. Further information about the conformational states of the acyl chains may be deduced from the CH2 wagging modes. In ordered phases (all-trans chains), these modes couple and split to produce band progressions characteristic of the number of CH2 groups in the chain.29,43 For unsaturated chains of the form CH3(CH2)mCdC(CH2)nR, where R is a polar functional group such as a methyl ester, Chia and Mendelsohn showed that the progressions arise from the n CH2 groups between the CdC bond and the polar moiety.44 This observation results from the fact that the progression intensity originates in the polar headgroup (rather than in the methylene groups), since the normal mode involved contains substantial contributions from the C-O stretch. Most of the dipole moment change during the vibration arises from this internal coordinate. The CdC bond destroys the coupling between the two halves of the chains. The wagging mode progressions at 31 °C for NFA ceramide, nervonic acid, and stearic acid are shown in Figure 5. The progression in NFA ceramide is weaker than observed in phospholipids, presumably a consequence of altered primary structure and smaller transition dipole moment of the C-N bond. The spectrum for the NFA ceramide progression is to a first crude approximation a weighted sum of the contributions of the two main-chain components. The observation of the progression alone reveals the presence of a conformationally ordered phase. The sphingosine chain does not contribute substantially to the progression since coupling of the progression bands to the C-O stretch is disrupted by the intervening trans CdC bond. The progression vanishes at ∼80 °C. This indicates that a large population of the NFA ceramide acyl chains remain in the all-trans conformation until the main transition at 80 °C. The observation provides further evidence for the identification of the 60 °C transition as arising from interconversion between two solid phases. In disordered phases, the coupling that produces the wagging mode progressions is destroyed by the presence of conformational disorder. The progression bands are superseded by localized CH2 wagging modes arising from particular two- or three-bond conformational states as follows: 1341 cm-1, endgauche (eg states); 1353 cm-1, double gauche (gg states); 1368 cm-1, kink + gtg states. As NFA ceramide shows a strong vibration at 1367-1368 cm-1 with significant contribution from the O-H in-plane bending mode (Figure 6a), it was necessary to exchange the sample in D2O, to eliminate spectral interference from this feature. The results are shown in Figure 6b-e. Exchanged NFA ceramide at low temperature (Figure 6b) shows a weak band at 1341 cm-1, arising from eg states, along with the temperature-invariant methyl symmetric deformation (“umbrella”) mode at 1378 cm-1. A broad feature on the lowfrequency side of the umbrella mode may represent unexchanged
Moore et al. -OH. As the temperature is increased to 73 °C (Figure 6c), the eg marker band increases in relative intensity and a new feature appears at 1361 cm-1. The acyl chain conformation associated with this band is obscure. Mendelsohn and coworkers45,46 have observed a feature at this position in the spectra of liquid 5-cis-decene as well as in disordered (Lβ or HII) phases of phospholipids. We therefore associate this feature with a particular but undetermined conformation about the CdC bonds. In the high-temperature phase (Figure 6d) dramatic additional increases occur in the intensity of bands at 1352 and 1367 cm-1. As noted previously, these arise from gg and the sum of (kink + gtg) states, respectively. It is of interest to compare the spectra of NFA ceramide in its high-temperature phase with that of the molecule dissolved in CHCl3 (Figure 6e). The latter presumably produces an isotropic or inverted micellar phase, with maximal disorder in the acyl chains. The relative intensity of the disorder bands (measured by peak heights) in the hightemperature phase is about 75-80% of those in the isotropic phase. In particular, the intensity of the gg marker bands is relevant in this context, since gg conformational states are known to be constrained in bilayer phases. Prior studies of conformational disorder in phosphatidylcholines and phosphatidylethanolamines have shown that the number of gg states per chain is reduced by 45% (DPPC) and 80% (DPPE) in going from an isotropic to an LR phase.47 In the current study, the number of gg states per chain is reduced by only 20-25% under the same conditions, suggestive of much more conformational freedom in the high-temperature phase of NFA ceramide than in the LR phase of phospholipids. This suggestion of a more liquidlike phase for NFA ceramide is in excellent accord with the X-ray diffraction data of Shipley and co-workers.27 At high temperatures they observe the diffuse 4.6 Å reflection characteristic of melted chains. The IR data directly reveal a level of disorder that approaches the disorder of an isotropic state. Taken together, the temperature dependence of the chain modes as discussed above suggests the following sequence of phases for NFA ceramide. At low temperature the chains are packed in a conformationally ordered (all-trans) form, in an orthorhombic perpendicular subcell. At 59 °C, the chain packing becomes hexagonal (possibly) with some orientational disorder. The chains are still conformationally ordered as indicated by both the low frequency of the CH2 stretching mode and the continued observation of the coupled CH2 wagging modes. The main transition (∼80 °C) is to a state with liquidlike chain disorder, possibly an isotropic phase. In particular, the presence of most of the theoretical maximum number of gg conformers suggests the absence of a bilayer structure. HFA ceramide shows a transition from a phase containing an orthorhombic perpendicular subcell to a phase that is LR- or HII-like in terms of the conformational disorder (data not shown). Amide I and II Modes. A major advantage of IR, compared with other physical methods, and in addition to the structural analysis of the hydrophobic regions, is the availability of structural information from the polar region of the ceramides. The amide I and II vibrations near 1650 and 1550 cm-1 arise from (mostly) CdO stretch and mixed N-H in-plane bend and C-N stretch, respectively. A temperature-dependent splitting is observed in each of these bands for NFA ceramide (Figure 7). The splitting for the amide II mode vanishes at the solidsolid (orthorhombic-hexagonal chain packing) phase transition, while the amide I splitting is reduced. The spectral assignments are confirmed, and additional features of interest are revealed from spectra of NFA ceramide in D2O (Figure 8). At the lowest temperatures, the amide I and II are again each split into a doublet. When the temperature
FTIR Sudies of Ceramides is raised, each of the amide I modes develops a feature on its low-frequency side so that at ∼60 °C additional amide I features are noted at 1632 and 1611 cm-1. Simultaneously, the amide II modes each gradually diminishes in intensity prior to disappearing at the orthorhombic-hexagonal chain interconversion. Similarly, the high-frequency component of each amide I doublet disappears at this temperature. At 80 °C, the amide I contour collapses to a broad single band. The assignments of the 1619, 1645 cm-1 doublet to amide I are confirmed from these experimental data. The presence of the 1611 and 1632 cm-1 peaks as the temperature is increased is consistent with H f D exchange of the amide group, producing frequency shifts of 8 and 13 cm-1 in the components, respectively. Similar shifts have been observed in protein spectra. Thus, the possible presence of a band due to the CdC stretch (known to occur in this spectral region) is definitively eliminated by the observation of a sensitivity in the frequency to H f D exchange. The presence of strong hydrogen bonds to the amide group of NFA ceramide is evident from comparison of the spectrum of the low-temperature phase with that of CHCl3 solution (data not shown). The amide I and II frequencies of the latter are 1657 and 1505 cm-1, respectively, and are characteristic of weakly hydrogen-bonded systems. In the orthorhombic state, the lowering of the amide I and the increase in the amide II frequency are consistent with strong hydrogen bonds to the amide group. The amide II mode of NFA ceramide in D2O disappears completely at the temperature of the orthorhombic-hexagonal transition, as expected upon H f D exchange of the amide bond. The observation is consistent with a much better packed structure in the orthorhombic phase which is not very susceptible to exchange. The amide II mode is a mixture of N-H in-plane bend and C-N stretch, so that H f D exchange shifts this mode to a frequency (1450 cm-1) which cannot be easily detected in the current experiment due to the confounding presence of the H-O-D bending (solvent) and chain scissoring modes close to that frequency. The origin of the splitting of the amide I and II modes in NFA ceramide and the absence of same in HFA ceramide is of interest. Splitting of the amide I and II modes is predicted and observed for proteins48,49 containing extended secondary structures but has not to our knowledge been observed for isolated amide groups. The exact relationship between the structure of the polar region and the spectral splittings of the amide I and II modes is difficult to ascertain. It is relevant to note that in the spectra of the anhydrous ceramide samples both amide modes were split for NFA ceramide but neither were split for HFA ceramide, exactly as in the hydrated samples. Studies by Pascher and his colleagues of ceramides and related molecules reveal that the carbonyl carbon and the hydrogen atom on the secondary sphingosine carbon are in a nearly syn-periplanar relationship.50 As a result, the conformation of the amide group plane is nearly perpendicular to the axis of the sphingosine chain. A similar eclipsed orientation of the amide group appears in the parallel β-pleated sheet structure of polypeptides. The observation of splitting in the amide I and II modes in hydrated and anhydrous NFA ceramide reveals that an interaction between amide groups, which is lacking in HFA ceramide, takes place in this molecule. At the solid-solid phase transition of NFA ceramide, the bands at ∼1120 and 1140 cm-1 (see Figure 12A), suggested to contain a large contribution from the C-O stretching internal coordinate, shift significantly as does the C-O-H bending mode at ∼1365 cm-1 (see Figure 12B). These shifts are evidence of hydrogen bond rearrangements
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Figure 12. (A) These spectra show modes in the 1120-1160 cm-1 region containing contributions from C-O stretch for NFA ceramide in H2O buffer as a function of temperature (top, 30 °C; bottom, 88 °C). A significant shift down in frequency occurs at ∼60 °C, indicating hydrogen bond rearrangement at the orthorhombic-hexagonal chain transition. (B) The same spectra in the 1350-1390 cm-1 region show a shift from ∼1365 to 1385 cm-1 of the C-O-H in-plane bending mode at the 60 °C solid-solid transition. The mode at 1378 cm-1 arises from the symmetric methyl deformation (umbrella) mode.
occurring as NFA ceramide undergoes a solid-solid transition from orthorhombic to hexagonal chain packing. HFA ceramide contains additional hydroxy groups that provide hydrogenbonding possibilities not available in NFA ceramide as well as add steric bulk to the headgroup region. In the hydroxy fatty acid ceramide, the fatty acid chain hydroxyl group points away from the molecule and is available for intermolecular interactions. Hydrogen bonding between these OH groups and the amide group CdO and N-H moieties would not be expected to produce a splitting of the amide I and II modes. In NFA ceramide, the absence of the fatty acid chain hydroxy moiety is suggested to permit intermolecular interactions between amide groups perhaps located in different layers in the structure. Such interactions would result in a splitting arising from intermolecular coupling of similar vibrational modes. The vibrational situation is analogous to an isolated polypeptide chain in an extended sheet structure. The difference between NFA ceramide and the isolated polypeptide chain is that in the current case only intermolecular interactions are possible whereas in the isolated polypeptide chain only intramolecular interactions can occur. The presence of two molecules in the “factor group” produces two components for each amide band. The observed frequencies correspond to (using the notation of Fraser and Macrae)51
υ(0)| ) υ0 + D1 υ((π)⊥ ) υ0 - D1 with υ0 ) the unperturbed frequency at 1632 cm-1 and D1 ) the intermolecular interaction constant ) (13 cm-1. The ambiguity in the sign of the latter arises since assignment of the observed frequencies to particular transitions is not known. For antiparallel sheets in proteins, the interchain interactions constants are
