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Fourier-transform infrared spectroscopic evidence for a novel lyotropic phase transition occurring in dioleoylphosphatidylethanolamine
CPL CHEMISTRY AND
ELSEVIER
Chemistry and Physics of Lipids 82 (1996) 191 198
PHYSICS OF LIPIDS
Short communication
Fourier-transform infrared spectroscopic evidence for a novel lyotropic phase transition occurring in dioleoylphosphatidylethanolamine Walter Pohle*, Carsten Selle Department ~! Biophysical Chemistry, Institute ~[ Molecular Biology, Friedrich-Svhiller UniversiO' Jena, Winzerlaer Str. 10, 07745 Jena, Germany
Received 4 December 1995; revised 3 May 1996; accepted 24 May 1996
Abstract
The hydration of 1,2-dioleoyl-sn-glycerophosphoethanolamine (DOPE)* has been studied by Fourier-transform infrared spectroscopy applied to macroscopically oriented films in comparison to related phospholipids (DPPE and DPPC). DOPE differs from the other lipids mainly in one respect, since it displays a number of relatively drastic, correlated spectroscopic changes within a distinct very narrow range of water activities at an ambient relative humidity of ~ 50%. These striking alterations can be observed especially for a set of infrared bands due to the headgroup (phosphoethanolamine) moieties whose parameters are simultaneously changed at a certain degree of hydration estimated to be less than one in the water-per-DOPE molecular ratio. This unique spectral behaviour being much more dramatic than, for instance, that observed for the main transition of lipids seems to reflect the occurrence of considerable structural reorientations within the polar part of DOPE molecules and may be explained as indicating the existence of a lyotropic phase transition. Discussion of the peculiar nature of this transformation results in a tentative assignment of the participating phases as belonging to the non-lamellar aggregations enclosing most probably the inverse hexagonal phase (at higher hydration) and the inverse fluid ribbon phase (at lower hydration). Keywords: Dioleoylphosphatidylethanolamine; Hydration; FTIR spectroscopy: Lyotropic phase transition: Non-bilayer phases; Dipalmitoylphosphatidylcholine
Abbreviations: DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamineor dioleoylphosphatidylethanolamine; DPPC, dipalmitoylphosphatidylcholine; DPPE, dipalmitoylphosphatidylethanolamine: IR, infrared; FTIR, Fourier-transform infrared; RH, relative humidity; H., inverse hexagonal phase; L~, liquid-crystallinephase; P~t, inverse ribbon phase; P~, partly ordered inverse ribbon phase; P~, inverse fluid ribbon phase; AoH, absorbance of the OH stretching-vibration band of water: vC = O, carbonyl stretching vibration; v~,~PO2~, antisymmetric stretching vibration of the PO) moiety; v~PO~, symmetric stretching vibration of the PO~ moietyy; v~sPOC, antisymmetric stretching vibration of the P-OC moiety; v~CCN,symmetricstretching vibration of the CCN + moiety; vsCH2, symmetric stretching vibration of methylene CH bonds; n,~,water-to-lipid molecular ratio: nw,, water-to-lipid molecular ratio at phase transition. * Corresponding author. Tel.: + 49 3641 657566.
0009-3084/96/$15.00 ,~ 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0009-3084(96)02586-8
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W. Pohle, C. Selle / Chemisto, and Physics oj Lipide' 82 (1996) 191 198
1. Introduction Phospholipids forming major constituents of biological membranes are distinguished by a very pronounced polymorphism. Its specification is realized, in each case, via the consideration of different, partly contradicting influences which arise from a number of intrinsic and external factors. Amongst the latter, a particular role is played by water, not only because of its omnipresence in living systems, but also since it delivers the physical-chemical background for asserting the amphiphilicity of lipids as the essential for their ability to self-organize into various supramolecular structures. The phase behaviour of lipids is mostly abstracted in the form of phase diagrams, which are usually established based on the results of physicochemical methods which provide data of a more or less integral character, such as differential scanning calorimetry and X-ray scattering. A valuable methodical supplement or extension for these methods is given by Fourier-transform infrared spectroscopy. This method can be likewise used to follow phase transitions in model membranes, but has further merits especially because of its capability to elucidate more detailed structural implications due to the different forms of lipid self-assembling. These as well as a number of further advantages have accounted for the fairly frequent application of FTIR spectroscopy in this field [1-5]. Nevertheless, one of the principal conclusions in the most recent of these reviews [4] is that hitherto the potentialities of FTIR spectroscopy have not been exhaustively utilized, This statement holds, for instance, for a certain lack of investigations by vibrational spectroscopy devoted to the gradual hydration of lipids; such measurements which are potentially appropriate to study the lyotropic polymorphism ofphospholipids were performed up to now only in several specific cases [6-8]. Therefore, we have been developing a technique based on FTIR spectroscopy to conduct these investigations in order to apply it to a whole series of phospholipids [9]. In this short communication, a first set of exciting data, focussing on DOPE, are reported and compared to those obtained for the related, common lipids DPPC and DPPE. DOPE was chosen since this molecule is of
considerable interest as being a paradigm for a lipid with a great preference to form non-bilayer aggregates, and, in particular, to adopt the inverse hexagonal (Ha) phase as the apparently most prominent non-lamellar phase [10] - - over a wide range of parameters including those vouching for physiological conditions [11-15]. DOPE is also able to form, under certain conditions, an inverted cubic phase [15,16]. Non-bilayer aggregations of lipids were first observed and defined by Luzzati and co-workers [17,18], and they are suspected to have also some implicit functional meaning, for instance in the process of cell fusion [19]. Actually, looking at the IR-spectroscopic behaviour of DOPE in the course of stepwise hydration/dehydration results in the observation of extraordinarily strong variations of many of the spectral parameters and may be, after all, suitable to shed new light on some peculiarities of the complex phase behaviour of this molecule.
2. Experimental procedures Samples from different lots of DOPE purchased from Sigma Co. (Munich) as pure chloroformic solutions (20 mg/ml)have been investigated. DPPC and DPPE were obtained from Bachem Biochemica (Heidelberg). All these compounds were used without further purification since uniformity was confirmed by thin-layer chromatography. Chloroform (UV-grade) from Baker (Deventer, Netherlands) was used as a solvent for DPPC and DPPE. Films of these lipids were prepared by casting the chloroformic solutions (10 mg/ml in the case of DPPC and DPPE) on IR-transparent ZnSe windows and completely evaporating the solvent whilst simultaneously stroking the solution unidirectionally by a spatula. Dehydration-rehydration cycles have been performed with these samples in situ; the water content of the films was varied changing the relative humidity between 98 and 0% in the cells by saturated solutions of different appropriate salts. Reaching equilibrium was assured by applying sufficiently long waiting times of at least 24 h (which were determined in pre-experiments). Reproducibility of the measurements was excellent, and hystereses along the dehydration-rehydration cycles could not be observed.
W. Pohle, C. Selle / Chemistry and Physics q/' Lilfids 82 (1996) 191 198
Infrared spectra were recorded, for each R H step adjustment, by means of an IFS-66 F T I R spectrometer from Bruker (Karlsruhe) equipped with a shuttle device under standard conditions and at a temperature of about 28°C. Thirty-two scans have been performed at a resolution of 2 cm t and using a zero-filling factor of 2. Data processing was done by the OPUS software package (Bruker). Wavenumbers of the peak maxima were determined by the OPUS peak-picking routine in the standard mode resulting in an accuracy of 0.1 cm ~. Fig. 4 was prepared by use of the Grams/386 programme (Galactic Industries Corp., Salem, N H , USA).
193
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In Fig. 1, comprehensive IR spectra of D O P E obtained at very low (A) and high (B) R H values are depicted. Already a glance reveals drastic differences between these t w o c a s e s with respect t o a set of spectral features. As a matter of course, the water content of D O P E indicated by the intensity of the O H stretching-vibration band near 3400 cm ~ is much higher in the wetted sample (spectrum B); the integral absorbance of this band, A o u , c a n be used to follow the consecutive sorption of water by the lipid film. Not so self-evidently and therefore worth mentioning is the finding of AOH = 0 at zero R H (spectrum B) implying that D O P E does not already contain ' ' , .......
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Fig. 2. Wavenumber displacements of the bands due to the symmetric PO 3 stretching vibration (A) and the antisymmetric P OC stretching vibration (B) compared for DOPE (left ordinate axis, dots are circles) and DPPC (right ordinate axis, dots are triangles), respectively, in dependence on the relative humidity surrounding the tilms.
some residual, tightly bound water after drying the film under gentle conditions; the latter type of water is known to occur in DPPC [20], as could be confirmed also by us [9]. A second and even more striking aspect refers to the observation that there are several more or less dramatic wavenumber displacements of lipid bands arising in the course of D O P E hydration (for details, see below). This behaviour is remarkable in so far as it is not quite usual for cephalins: for instance, infrared spectra of DPPE as a saturated-chain phosphatidylethanolamine (which are not shown h r
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w. Pohle, C. Selle / Chemistry and Physics ~/ Lipids 82 (/996) 19/ 198
194
changes within very narrow hydration intervals are observed. This is illustrated by Fig. 2 where the peak-wavenumber displacements are exemplarily compared for DOPE and DPPC in terms of R H by two relevant vibrational modes, namely those represented by the symmetric PO) and antisymmetric P - O C stretching vibration bands, respectively. Although the sign of frequency shift is in both cases the same, the steepness of the typical D O P E 'jumpings' is in a striking contrast to the essentially smooth and continuous type of wavenumber shifting found for DPPC. An interesting approach, enabled by F T I R spectroscopy, is to study more intricate features of how a lipid responds to the influence of hydration on a submolecular level. Fig. 3 shows a respective comparative picture for DOPE involving the regions due to the apolar acyl chains, the polar/apolar interface, and the polar headgroup represented by the bands assigned to the symmetric CH 2 stretching vibration near 2850 cm ~ (A), to the
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carbonyl stretching vibration at ~ 1740 c m l ( B ) and to the symmetric CCN + stretching vibration around 915 cm 1 (C), respectively. The behaviour of the pertinent wavenumbers reveals a typical diversity and can be taken as to reflect the involvement of each of the lipid sub-regions into the molecular processes induced by hydration in a qualitative (is there a response or not) and in a quantitative (order of magnitude of the response, if present) respect. The invariance of the wavenumbers due to both the symmetric methylene stretch (Fig. 3A) and other acyl-chain bands (not shown) indicates that: (i) the apolar tail region is, according to rough expectations, not directly affected by water molecules; and (ii) and more importantly, there is no change in the state of ordering of the hydrocarbon chains. Enhanced acyl-chain fluidity as caused by, e.g. increasing temperatures, is evidenced via significant blue shifts of several of the C - H stretching vibration bands [1,2] ascribed to an enlargement of the fraction of gauche conformers along the chains. In a qualitatively variant manner, the frequencies of the bands arising from the functional groups of the polar region are more or less changed. There appears to be, however, a quantitative difference in the size of wavenumber shifts between the carbonyl band on the one hand and 'headgroup' bands on the other. In the RH region of drastic changes, the wavenumber of the vC = O band decreases only very slightly during DOPE hydration (by about 0.5 c m 1, see arrow in Fig. 3B), whilst as in the example in Fig. 3C the "headgroup' bands are much more strongly influenced. For the sake of obtaining a better survey about the headgroup region, in Fig. 4 the FTIR spectra of DOPE were plotted in the range of 1300 700 cm ' in a three-dimensional manner with hydration as the third axis. This kind of illustration is instructive in elucidating that many of the spectral alterations observed, or, in other terms, all the dramatic ones, are correlated with each other since they occur just at one and the same relative humidity, namely at about 50% RH (cf. Fig. 2. corresponding to AOH = 0.2, see Figs. 3 and 4). Marking in black in Fig. 4 is to label the edge which indicates, in this contour plot, that defined locus on the hydration scale (according t o
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o "~ 912 , , o, , , e, , o, , , , ,~, , 0.0 0.5 1.0 1.5 %, Fig. 3. W a v e n u m b e r s of characteristic DOPE bands due to (A) the symmetric methylene stretch, (B), the carbonyl stretch, and (C), the symmetric C C N + stretch, depicted vs. AoH which is a measure of the water content of the films (cf. text) and RH; arrows indicate the abscissa where wavenumber j u m p s occur,
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Chemistry and Physics ~/ Lipids 82 (1996) 191 198
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AoH = 0.2) at which major structural reorientations in the headgroup and, presumably to a lesser degree, also in the carbonyl regions (according to the data in Fig. 3B) of DOPE molecules seem to occur. In order to roughly estimate the stoichiometry of water-per-lipid molecules for this critical hydration level, nwt, one can simply correlate a series of Aon values obtained spectroscopitally for several lipids with the corresponding water-per-lipid ratios determined gravimetrically at the same RH. This estimation based on the data t¥om different gravimetric studies [22-24] yields n~t = 0.6 _+ 0.1. In view of both the multitude and magnitude of the spectral changes found, it is suggestive to assume that these rearrangements, supposed to occur for DOPE, do affect the mode of lipid assembling or that they are a consequence of an hydration-induced change of the latter. In other words, these spectral alterations appear to indicate, by virtue of their distinctness, the existence of a lyotropic phase transition between two supermolecular assemblies with sufficiently variant geometrical characteristics. Making such a far-reaching assumption is promoted by the fact that the spectral effects observed in this case are much more dramatic than even those which are known to accompany, e.g., the main transition of lipids [1,2]. Once having accepted the idea that the experimental findings presented here reveal a lyotropic
195
phase transition, interpretation in terms of its nature is, however, not quite so easy in view of the present state of knowledge. As already mentioned above, DOPE belongs to the particular class of lipids that have a great propensity to form non-bilayer phases [10 16]. According to the most extensive and recent phase diagrams, DOPE exists under conditions near room temperature as used in the present experiments in the inverse hexagonal phase over a very wide range of water contents [15]. Let us start imaginarily with hydrated DOPE as being in the HH phase, and then continue to stepwise diminish the w a t e r c o n t e n t of the sample. When reaching a certain critical threshold (at a water-to-lipid ratio of ~ 0.6, s e e above), the H H structure persisting so far collapses and is substituted, apparently in a cooperative process, by another one (as spectroscopic findings reported here suggest), which will be called the X phase in the following. What could be the nature of this second phase of DOPE? First, the likely idea of X being the lamellar L~ phase should be considered. Actually, the spectroscopic findings for the proposed phase transition of DOPE (Figs. 2-4) are markedly different from the respective pattern which was reported for the L ~ - H u phase transition [25~27] that consisted essentially of slight wavenumber increases of the v~CH2 band of the methylene groups due to the acyl chains by not more than 1 cm - ~, the vC = O band by approximately 3 cm ~, and, sometimes, also the v~PO2 band by 3 - 4 cm ~ (only in [27]). Nevertheless, we have also recorded temperaturedependent FTIR spectra of DOPE, and the relevant results for the L~ HH transition qualitatively reproduce the literature data mentioned above [25 27] instead of the data presented here for DOPE hydration (Figs. 2-4), In particular, the wavenumber of the v~CH, band due to the methylene groups of the acyl chain of DOPE in the films investigated is hydration-independently situated at about 2853.6 cm ~, i.e. at a value typical for the HH phase with a relatively large degree of chain disorder and, thus, significantly higher than for phospholipids in the L~ phase, where generally ~ 2852.5 cm- ~ are found ([1 4], own data, not shown). Furthermore, the poorly hydrated X phase of DOPE is maintained in a
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W. Pohle, C. Selle / Chemistry and Physics o[ Lipids 82 (1996) 191 198
largely unvaried fashion down to zero water fraction. This represents, however, a state of sample, where the existence of L~ is highly improbable for any lipid and nearly to be excluded for cephalins, as can be proved by the inspection of relevant phase diagrams [17,18,28-30]. It is, moreover, very improbable from molecular-physical grounds to expect, under the experimental conditions as described above, a change from the HH to the L~ phase, since a decrease of the water content in the headgroup region of a molecule like DOPE with a cross-section area of its headgroup significantly smaller than for its apolar tails should rather deteriorate the prerequisites for a lamellar phase to exist, Therefore it becomes quite clear that the phase transition postulated here for D O P E cannot be identic with the L~-H~. transition. Nevertheless, the X phase must be suggested to be sufficiently different with respect to the Hli phase. Defining the former has to proceed from the experimentally ensured facts: this 'superstructure' contains very little or no water, has highly fluid hydrocarbon chains, and upon its formation from the H,. phase the lipid headgroups are strongly influenced, Taken together, these three or four specifics appear to be best realized by taking into account that X is given by another non-lamellar phase, Then two alternatives arise: (i) the cubic phase already descibed as to occur for DOPE [15,16] or (ii) the inverted form of the so-called ribbon phase denoted as Pn [10] which is thought to be constructed by strains of headgroups-centered lipid assemblies having an ellipse as the cross-section, This phase has been reported primarily to exist in a region of the phase diagrams of certain lecithins which is characterized by very low water content and higher temperatures [17,18,30] and, later on, similarly also for DPPE at somewhat lower ternperatures [29]. In either of these cases, the P phase under debate is specified by suffix ci representing a partly ordered state of lipids [17,18,29,30]. Our data demonstrate, however, a strong prevalence of largely disordered hydrocarbon tails in the assumed P phase with a fraction of gauche conformers as high as in the more strongly hydrated H H or H~ phase; therefore we are inclined to term the phase observed in dried fihns of DOPE as P~
differing from the P s phase defined on the basis of previous X-ray scattering data [17,18,29] by including a more fluid apolar part. It should be noted that, to the best of our knowledge, nonlamellar PI. phases have, up to now, not been discussed to occur in DOPE; likewise, we found no mention of a P~ phase in the relevant literature. Remarkably, the poorly hydrated X phase of DOPE is maintNned in a largely unvaried fashion down to zero water fraction (as already stated above). This fact is very much in favour of our idea to assign the X phase to the fluid ribbon phase: all the relevant phase diagrams of different lipids (lecithins [17,18,30], DPPE [29]) show that the P,~ phase occurs only at extremely low water contents of n~. < ~ 0.5, with the highest vertical extension of the related area at n,,, = 0 (along the temperature axis). These nw boundaries of the P,~ phase fit fairly well the existence region of the X phase found by us to be stable at nw < ~ 0.6. Thus, P~ phase is in a principal contrast to L~ phase generally not touching the y-axis of phase diagrams at all (cf. above). A direct consequence of the absence of water in the ribbon phase is that the opposite headgroups (which are more or less separated by solvent molecules in the H,~ phase) come there inevitably into an immediate contact with each other. This particular superstructural element that we would like to mark with the term 'headgroup interdigitation' can be suggested to account very well for the apparently high quality of geometrical rearrangements spectroscopically indicated as to proceed precisely in the headgroup region. Moreover, the HH-P~ transition seems to be not very expensive from a 'superstructural' point of view: upon gradually detaching water from the array of DOPE molecules being in the HH phase, a critical point will be reached (at a ratio of ~ 0.6 water per lipid, see above), where the radius of the central 'water cylinder' inside the H H tubes becomes too small to maintain the overall structure underlying this phase. Thus, the facing headgroups are induced to stick together, whereby a great deal of them is displace& from sterical demands, such that the cross-section (according to one headgroup layer of the strains formed) is now
W. Pohle, C. Selle / Chemistt3' and Physics of Lipids 82 (19961 191 198
given by a rod or ellipse, as shown in the pictures in Fig. 7 of ref. [18] or in Fig. 5b of ref. [10], instead of the circle for H , . F r o m these last lines of argumentation, we tend to strongly prefer an assignment of X to the P~ phase vs. the cubic phase, which, however, cannot be quite excluded to exist in water-depleted D O P E by spectroscopic means,
Eventually identifying the real nature of this phase transition will be facilitated by a more detailed analysis of the spectroscopic data arising from extended measurements as well as by the use of other structural methods, above all X-ray diffraction studies, which are underway.
4. C o n c l u s i o n s
The lyotropic phase behaviour of dioleoylphosphatidylethanolamine has been probed by F T I R spectroscopic investigations of the successive hydration and dehydration of cast lipid films. At room temperature and at a certain high lipid content, drastic and simultaneous spectral changes occur which are interpreted in terms of indicating the existence of a reversible lyotropic phase transition. The latter is tentatively ascribed to a Hu-X transformation (going from high to low water contents) with X being represented most probably by a highly fluid ribbon phase, denoted as P~, possibly also by one of the cubic phases, but by no means by the L~ phase,
Acknowledgements The authors are indebted to the Thuringian Ministry for Science ( T M W F K ) for a grant and to Prof. H. Fritzsche for a critical reading of the paper.
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ELSEVIER
Chemistry and Physics of Lipids 82 (1996) 191 198
PHYSICS OF LIPIDS
Short communication
Fourier-transform infrared spectroscopic evidence for a novel lyotropic phase transition occurring in dioleoylphosphatidylethanolamine Walter Pohle*, Carsten Selle Department ~! Biophysical Chemistry, Institute ~[ Molecular Biology, Friedrich-Svhiller UniversiO' Jena, Winzerlaer Str. 10, 07745 Jena, Germany
Received 4 December 1995; revised 3 May 1996; accepted 24 May 1996
Abstract
The hydration of 1,2-dioleoyl-sn-glycerophosphoethanolamine (DOPE)* has been studied by Fourier-transform infrared spectroscopy applied to macroscopically oriented films in comparison to related phospholipids (DPPE and DPPC). DOPE differs from the other lipids mainly in one respect, since it displays a number of relatively drastic, correlated spectroscopic changes within a distinct very narrow range of water activities at an ambient relative humidity of ~ 50%. These striking alterations can be observed especially for a set of infrared bands due to the headgroup (phosphoethanolamine) moieties whose parameters are simultaneously changed at a certain degree of hydration estimated to be less than one in the water-per-DOPE molecular ratio. This unique spectral behaviour being much more dramatic than, for instance, that observed for the main transition of lipids seems to reflect the occurrence of considerable structural reorientations within the polar part of DOPE molecules and may be explained as indicating the existence of a lyotropic phase transition. Discussion of the peculiar nature of this transformation results in a tentative assignment of the participating phases as belonging to the non-lamellar aggregations enclosing most probably the inverse hexagonal phase (at higher hydration) and the inverse fluid ribbon phase (at lower hydration). Keywords: Dioleoylphosphatidylethanolamine; Hydration; FTIR spectroscopy: Lyotropic phase transition: Non-bilayer phases; Dipalmitoylphosphatidylcholine
Abbreviations: DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamineor dioleoylphosphatidylethanolamine; DPPC, dipalmitoylphosphatidylcholine; DPPE, dipalmitoylphosphatidylethanolamine: IR, infrared; FTIR, Fourier-transform infrared; RH, relative humidity; H., inverse hexagonal phase; L~, liquid-crystallinephase; P~t, inverse ribbon phase; P~, partly ordered inverse ribbon phase; P~, inverse fluid ribbon phase; AoH, absorbance of the OH stretching-vibration band of water: vC = O, carbonyl stretching vibration; v~,~PO2~, antisymmetric stretching vibration of the PO) moiety; v~PO~, symmetric stretching vibration of the PO~ moietyy; v~sPOC, antisymmetric stretching vibration of the P-OC moiety; v~CCN,symmetricstretching vibration of the CCN + moiety; vsCH2, symmetric stretching vibration of methylene CH bonds; n,~,water-to-lipid molecular ratio: nw,, water-to-lipid molecular ratio at phase transition. * Corresponding author. Tel.: + 49 3641 657566.
0009-3084/96/$15.00 ,~ 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0009-3084(96)02586-8
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W. Pohle, C. Selle / Chemisto, and Physics oj Lipide' 82 (1996) 191 198
1. Introduction Phospholipids forming major constituents of biological membranes are distinguished by a very pronounced polymorphism. Its specification is realized, in each case, via the consideration of different, partly contradicting influences which arise from a number of intrinsic and external factors. Amongst the latter, a particular role is played by water, not only because of its omnipresence in living systems, but also since it delivers the physical-chemical background for asserting the amphiphilicity of lipids as the essential for their ability to self-organize into various supramolecular structures. The phase behaviour of lipids is mostly abstracted in the form of phase diagrams, which are usually established based on the results of physicochemical methods which provide data of a more or less integral character, such as differential scanning calorimetry and X-ray scattering. A valuable methodical supplement or extension for these methods is given by Fourier-transform infrared spectroscopy. This method can be likewise used to follow phase transitions in model membranes, but has further merits especially because of its capability to elucidate more detailed structural implications due to the different forms of lipid self-assembling. These as well as a number of further advantages have accounted for the fairly frequent application of FTIR spectroscopy in this field [1-5]. Nevertheless, one of the principal conclusions in the most recent of these reviews [4] is that hitherto the potentialities of FTIR spectroscopy have not been exhaustively utilized, This statement holds, for instance, for a certain lack of investigations by vibrational spectroscopy devoted to the gradual hydration of lipids; such measurements which are potentially appropriate to study the lyotropic polymorphism ofphospholipids were performed up to now only in several specific cases [6-8]. Therefore, we have been developing a technique based on FTIR spectroscopy to conduct these investigations in order to apply it to a whole series of phospholipids [9]. In this short communication, a first set of exciting data, focussing on DOPE, are reported and compared to those obtained for the related, common lipids DPPC and DPPE. DOPE was chosen since this molecule is of
considerable interest as being a paradigm for a lipid with a great preference to form non-bilayer aggregates, and, in particular, to adopt the inverse hexagonal (Ha) phase as the apparently most prominent non-lamellar phase [10] - - over a wide range of parameters including those vouching for physiological conditions [11-15]. DOPE is also able to form, under certain conditions, an inverted cubic phase [15,16]. Non-bilayer aggregations of lipids were first observed and defined by Luzzati and co-workers [17,18], and they are suspected to have also some implicit functional meaning, for instance in the process of cell fusion [19]. Actually, looking at the IR-spectroscopic behaviour of DOPE in the course of stepwise hydration/dehydration results in the observation of extraordinarily strong variations of many of the spectral parameters and may be, after all, suitable to shed new light on some peculiarities of the complex phase behaviour of this molecule.
2. Experimental procedures Samples from different lots of DOPE purchased from Sigma Co. (Munich) as pure chloroformic solutions (20 mg/ml)have been investigated. DPPC and DPPE were obtained from Bachem Biochemica (Heidelberg). All these compounds were used without further purification since uniformity was confirmed by thin-layer chromatography. Chloroform (UV-grade) from Baker (Deventer, Netherlands) was used as a solvent for DPPC and DPPE. Films of these lipids were prepared by casting the chloroformic solutions (10 mg/ml in the case of DPPC and DPPE) on IR-transparent ZnSe windows and completely evaporating the solvent whilst simultaneously stroking the solution unidirectionally by a spatula. Dehydration-rehydration cycles have been performed with these samples in situ; the water content of the films was varied changing the relative humidity between 98 and 0% in the cells by saturated solutions of different appropriate salts. Reaching equilibrium was assured by applying sufficiently long waiting times of at least 24 h (which were determined in pre-experiments). Reproducibility of the measurements was excellent, and hystereses along the dehydration-rehydration cycles could not be observed.
W. Pohle, C. Selle / Chemistry and Physics q/' Lilfids 82 (1996) 191 198
Infrared spectra were recorded, for each R H step adjustment, by means of an IFS-66 F T I R spectrometer from Bruker (Karlsruhe) equipped with a shuttle device under standard conditions and at a temperature of about 28°C. Thirty-two scans have been performed at a resolution of 2 cm t and using a zero-filling factor of 2. Data processing was done by the OPUS software package (Bruker). Wavenumbers of the peak maxima were determined by the OPUS peak-picking routine in the standard mode resulting in an accuracy of 0.1 cm ~. Fig. 4 was prepared by use of the Grams/386 programme (Galactic Industries Corp., Salem, N H , USA).
193
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In Fig. 1, comprehensive IR spectra of D O P E obtained at very low (A) and high (B) R H values are depicted. Already a glance reveals drastic differences between these t w o c a s e s with respect t o a set of spectral features. As a matter of course, the water content of D O P E indicated by the intensity of the O H stretching-vibration band near 3400 cm ~ is much higher in the wetted sample (spectrum B); the integral absorbance of this band, A o u , c a n be used to follow the consecutive sorption of water by the lipid film. Not so self-evidently and therefore worth mentioning is the finding of AOH = 0 at zero R H (spectrum B) implying that D O P E does not already contain ' ' , .......
,//
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Fig. 2. Wavenumber displacements of the bands due to the symmetric PO 3 stretching vibration (A) and the antisymmetric P OC stretching vibration (B) compared for DOPE (left ordinate axis, dots are circles) and DPPC (right ordinate axis, dots are triangles), respectively, in dependence on the relative humidity surrounding the tilms.
some residual, tightly bound water after drying the film under gentle conditions; the latter type of water is known to occur in DPPC [20], as could be confirmed also by us [9]. A second and even more striking aspect refers to the observation that there are several more or less dramatic wavenumber displacements of lipid bands arising in the course of D O P E hydration (for details, see below). This behaviour is remarkable in so far as it is not quite usual for cephalins: for instance, infrared spectra of DPPE as a saturated-chain phosphatidylethanolamine (which are not shown h r
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peak wavenumbers on hydration, but essentially rather an invariance of those wavenumbers [21]. Looking now' at the mode of frequency shifting in greater detail by monitoring the stepwise hydration process of D O P E unravels a unique characteristic of that particular lipid not observed in the other related substances studied so far: in the former case, the wavenumber shifts occur very often as pronounced 'jumps', i.e. relatively drastic
w. Pohle, C. Selle / Chemistry and Physics ~/ Lipids 82 (/996) 19/ 198
194
changes within very narrow hydration intervals are observed. This is illustrated by Fig. 2 where the peak-wavenumber displacements are exemplarily compared for DOPE and DPPC in terms of R H by two relevant vibrational modes, namely those represented by the symmetric PO) and antisymmetric P - O C stretching vibration bands, respectively. Although the sign of frequency shift is in both cases the same, the steepness of the typical D O P E 'jumpings' is in a striking contrast to the essentially smooth and continuous type of wavenumber shifting found for DPPC. An interesting approach, enabled by F T I R spectroscopy, is to study more intricate features of how a lipid responds to the influence of hydration on a submolecular level. Fig. 3 shows a respective comparative picture for DOPE involving the regions due to the apolar acyl chains, the polar/apolar interface, and the polar headgroup represented by the bands assigned to the symmetric CH 2 stretching vibration near 2850 cm ~ (A), to the
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carbonyl stretching vibration at ~ 1740 c m l ( B ) and to the symmetric CCN + stretching vibration around 915 cm 1 (C), respectively. The behaviour of the pertinent wavenumbers reveals a typical diversity and can be taken as to reflect the involvement of each of the lipid sub-regions into the molecular processes induced by hydration in a qualitative (is there a response or not) and in a quantitative (order of magnitude of the response, if present) respect. The invariance of the wavenumbers due to both the symmetric methylene stretch (Fig. 3A) and other acyl-chain bands (not shown) indicates that: (i) the apolar tail region is, according to rough expectations, not directly affected by water molecules; and (ii) and more importantly, there is no change in the state of ordering of the hydrocarbon chains. Enhanced acyl-chain fluidity as caused by, e.g. increasing temperatures, is evidenced via significant blue shifts of several of the C - H stretching vibration bands [1,2] ascribed to an enlargement of the fraction of gauche conformers along the chains. In a qualitatively variant manner, the frequencies of the bands arising from the functional groups of the polar region are more or less changed. There appears to be, however, a quantitative difference in the size of wavenumber shifts between the carbonyl band on the one hand and 'headgroup' bands on the other. In the RH region of drastic changes, the wavenumber of the vC = O band decreases only very slightly during DOPE hydration (by about 0.5 c m 1, see arrow in Fig. 3B), whilst as in the example in Fig. 3C the "headgroup' bands are much more strongly influenced. For the sake of obtaining a better survey about the headgroup region, in Fig. 4 the FTIR spectra of DOPE were plotted in the range of 1300 700 cm ' in a three-dimensional manner with hydration as the third axis. This kind of illustration is instructive in elucidating that many of the spectral alterations observed, or, in other terms, all the dramatic ones, are correlated with each other since they occur just at one and the same relative humidity, namely at about 50% RH (cf. Fig. 2. corresponding to AOH = 0.2, see Figs. 3 and 4). Marking in black in Fig. 4 is to label the edge which indicates, in this contour plot, that defined locus on the hydration scale (according t o
C
L_//
o "~ 912 , , o, , , e, , o, , , , ,~, , 0.0 0.5 1.0 1.5 %, Fig. 3. W a v e n u m b e r s of characteristic DOPE bands due to (A) the symmetric methylene stretch, (B), the carbonyl stretch, and (C), the symmetric C C N + stretch, depicted vs. AoH which is a measure of the water content of the films (cf. text) and RH; arrows indicate the abscissa where wavenumber j u m p s occur,
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.
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Chemistry and Physics ~/ Lipids 82 (1996) 191 198
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Fig. 4. 3D representatiou of the FT-IR spectra of DOPE in terms of its water content in the wavenumber range of 1300 700 cm ~: black marking is to feature the edge which indicares the occurrence of major structural reorientations in the lipid headgroup,
AoH = 0.2) at which major structural reorientations in the headgroup and, presumably to a lesser degree, also in the carbonyl regions (according to the data in Fig. 3B) of DOPE molecules seem to occur. In order to roughly estimate the stoichiometry of water-per-lipid molecules for this critical hydration level, nwt, one can simply correlate a series of Aon values obtained spectroscopitally for several lipids with the corresponding water-per-lipid ratios determined gravimetrically at the same RH. This estimation based on the data t¥om different gravimetric studies [22-24] yields n~t = 0.6 _+ 0.1. In view of both the multitude and magnitude of the spectral changes found, it is suggestive to assume that these rearrangements, supposed to occur for DOPE, do affect the mode of lipid assembling or that they are a consequence of an hydration-induced change of the latter. In other words, these spectral alterations appear to indicate, by virtue of their distinctness, the existence of a lyotropic phase transition between two supermolecular assemblies with sufficiently variant geometrical characteristics. Making such a far-reaching assumption is promoted by the fact that the spectral effects observed in this case are much more dramatic than even those which are known to accompany, e.g., the main transition of lipids [1,2]. Once having accepted the idea that the experimental findings presented here reveal a lyotropic
195
phase transition, interpretation in terms of its nature is, however, not quite so easy in view of the present state of knowledge. As already mentioned above, DOPE belongs to the particular class of lipids that have a great propensity to form non-bilayer phases [10 16]. According to the most extensive and recent phase diagrams, DOPE exists under conditions near room temperature as used in the present experiments in the inverse hexagonal phase over a very wide range of water contents [15]. Let us start imaginarily with hydrated DOPE as being in the HH phase, and then continue to stepwise diminish the w a t e r c o n t e n t of the sample. When reaching a certain critical threshold (at a water-to-lipid ratio of ~ 0.6, s e e above), the H H structure persisting so far collapses and is substituted, apparently in a cooperative process, by another one (as spectroscopic findings reported here suggest), which will be called the X phase in the following. What could be the nature of this second phase of DOPE? First, the likely idea of X being the lamellar L~ phase should be considered. Actually, the spectroscopic findings for the proposed phase transition of DOPE (Figs. 2-4) are markedly different from the respective pattern which was reported for the L ~ - H u phase transition [25~27] that consisted essentially of slight wavenumber increases of the v~CH2 band of the methylene groups due to the acyl chains by not more than 1 cm - ~, the vC = O band by approximately 3 cm ~, and, sometimes, also the v~PO2 band by 3 - 4 cm ~ (only in [27]). Nevertheless, we have also recorded temperaturedependent FTIR spectra of DOPE, and the relevant results for the L~ HH transition qualitatively reproduce the literature data mentioned above [25 27] instead of the data presented here for DOPE hydration (Figs. 2-4), In particular, the wavenumber of the v~CH, band due to the methylene groups of the acyl chain of DOPE in the films investigated is hydration-independently situated at about 2853.6 cm ~, i.e. at a value typical for the HH phase with a relatively large degree of chain disorder and, thus, significantly higher than for phospholipids in the L~ phase, where generally ~ 2852.5 cm- ~ are found ([1 4], own data, not shown). Furthermore, the poorly hydrated X phase of DOPE is maintained in a
196
W. Pohle, C. Selle / Chemistry and Physics o[ Lipids 82 (1996) 191 198
largely unvaried fashion down to zero water fraction. This represents, however, a state of sample, where the existence of L~ is highly improbable for any lipid and nearly to be excluded for cephalins, as can be proved by the inspection of relevant phase diagrams [17,18,28-30]. It is, moreover, very improbable from molecular-physical grounds to expect, under the experimental conditions as described above, a change from the HH to the L~ phase, since a decrease of the water content in the headgroup region of a molecule like DOPE with a cross-section area of its headgroup significantly smaller than for its apolar tails should rather deteriorate the prerequisites for a lamellar phase to exist, Therefore it becomes quite clear that the phase transition postulated here for D O P E cannot be identic with the L~-H~. transition. Nevertheless, the X phase must be suggested to be sufficiently different with respect to the Hli phase. Defining the former has to proceed from the experimentally ensured facts: this 'superstructure' contains very little or no water, has highly fluid hydrocarbon chains, and upon its formation from the H,. phase the lipid headgroups are strongly influenced, Taken together, these three or four specifics appear to be best realized by taking into account that X is given by another non-lamellar phase, Then two alternatives arise: (i) the cubic phase already descibed as to occur for DOPE [15,16] or (ii) the inverted form of the so-called ribbon phase denoted as Pn [10] which is thought to be constructed by strains of headgroups-centered lipid assemblies having an ellipse as the cross-section, This phase has been reported primarily to exist in a region of the phase diagrams of certain lecithins which is characterized by very low water content and higher temperatures [17,18,30] and, later on, similarly also for DPPE at somewhat lower ternperatures [29]. In either of these cases, the P phase under debate is specified by suffix ci representing a partly ordered state of lipids [17,18,29,30]. Our data demonstrate, however, a strong prevalence of largely disordered hydrocarbon tails in the assumed P phase with a fraction of gauche conformers as high as in the more strongly hydrated H H or H~ phase; therefore we are inclined to term the phase observed in dried fihns of DOPE as P~
differing from the P s phase defined on the basis of previous X-ray scattering data [17,18,29] by including a more fluid apolar part. It should be noted that, to the best of our knowledge, nonlamellar PI. phases have, up to now, not been discussed to occur in DOPE; likewise, we found no mention of a P~ phase in the relevant literature. Remarkably, the poorly hydrated X phase of DOPE is maintNned in a largely unvaried fashion down to zero water fraction (as already stated above). This fact is very much in favour of our idea to assign the X phase to the fluid ribbon phase: all the relevant phase diagrams of different lipids (lecithins [17,18,30], DPPE [29]) show that the P,~ phase occurs only at extremely low water contents of n~. < ~ 0.5, with the highest vertical extension of the related area at n,,, = 0 (along the temperature axis). These nw boundaries of the P,~ phase fit fairly well the existence region of the X phase found by us to be stable at nw < ~ 0.6. Thus, P~ phase is in a principal contrast to L~ phase generally not touching the y-axis of phase diagrams at all (cf. above). A direct consequence of the absence of water in the ribbon phase is that the opposite headgroups (which are more or less separated by solvent molecules in the H,~ phase) come there inevitably into an immediate contact with each other. This particular superstructural element that we would like to mark with the term 'headgroup interdigitation' can be suggested to account very well for the apparently high quality of geometrical rearrangements spectroscopically indicated as to proceed precisely in the headgroup region. Moreover, the HH-P~ transition seems to be not very expensive from a 'superstructural' point of view: upon gradually detaching water from the array of DOPE molecules being in the HH phase, a critical point will be reached (at a ratio of ~ 0.6 water per lipid, see above), where the radius of the central 'water cylinder' inside the H H tubes becomes too small to maintain the overall structure underlying this phase. Thus, the facing headgroups are induced to stick together, whereby a great deal of them is displace& from sterical demands, such that the cross-section (according to one headgroup layer of the strains formed) is now
W. Pohle, C. Selle / Chemistt3' and Physics of Lipids 82 (19961 191 198
given by a rod or ellipse, as shown in the pictures in Fig. 7 of ref. [18] or in Fig. 5b of ref. [10], instead of the circle for H , . F r o m these last lines of argumentation, we tend to strongly prefer an assignment of X to the P~ phase vs. the cubic phase, which, however, cannot be quite excluded to exist in water-depleted D O P E by spectroscopic means,
Eventually identifying the real nature of this phase transition will be facilitated by a more detailed analysis of the spectroscopic data arising from extended measurements as well as by the use of other structural methods, above all X-ray diffraction studies, which are underway.
4. C o n c l u s i o n s
The lyotropic phase behaviour of dioleoylphosphatidylethanolamine has been probed by F T I R spectroscopic investigations of the successive hydration and dehydration of cast lipid films. At room temperature and at a certain high lipid content, drastic and simultaneous spectral changes occur which are interpreted in terms of indicating the existence of a reversible lyotropic phase transition. The latter is tentatively ascribed to a Hu-X transformation (going from high to low water contents) with X being represented most probably by a highly fluid ribbon phase, denoted as P~, possibly also by one of the cubic phases, but by no means by the L~ phase,
Acknowledgements The authors are indebted to the Thuringian Ministry for Science ( T M W F K ) for a grant and to Prof. H. Fritzsche for a critical reading of the paper.
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