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An FT-IR study of water hydrating dipalmitoylphosphatidylcholine multibilayers and reversed micelles
Journal
of
MOLECUlAR STRUCTURE
ELSEVIER
Journal of Molecular Structure 322 (1994) 93- 103
An FT-IR study of water hydrating dipalmitoylphosphatidylcholine multibilayers and reversed micelles’ J. Grdadolnik,
J. KidriE, D. Hadii*
National Institute of Chemistry,
61115 Ljubljana. Slovenia
(First received 13 September 1993;in final form 13 January 1994)
Abstract Infrared spectra of HZ0 and HDO (5% D20 in HzO) in the stretching and bending regions are presented and discussed with respect to location and energy of bonding of the first water molecules hydrating the polar heads of the title systems. Spectral features of water in the higher hydrated bilayers are compared to those of ordinary water. The v,,~ and v2 bands of water at low hydration are only slightly sensitive to low temperatures. As an indication that these water molecules do not freeze, we take the absence of any major effects of cooling as the main evidence for the origin of the bandwidth. We propose that the v1,3 band broadening is of a statistical character, i.e. it is caused by various binding topologies of the water molecules, and not by homogeneous broadening mechanisms. With highly hydrated bilayers, the formation of amorphous ice is observed. Differences are noted between some spectral features of water inside the reverse micelles and water hydrating the bilayers, particularly at subzero temperatures.
1. Introduction The structure and properties of phospholipid assemblies, and their function in biological membranes, are inextricably linked to hydration. Its pervasive effects have been investigated, particularly in the example of bilayers, by a variety of physical methods, which were also applied to the elucidation of the organization of water sandwiched between the polar heads (for reviews, see Refs. l-3). Interpretations of the experimental results with ’ Presented at the Xth Workshop “Horizons in Hydrogen Bond Research”, Autrans, France, 12- 17 September 1993. * Corresponding author.
respect to water organization at progressive hydration lead to two views. At one extreme, water would form distinct layers or hydration shells, whereas at the other extreme, the measured properties are considered as smoothly evolving changes in the water organization. The views strongly depend on the methods of investigation. The concept of shells - up to four - emerged from calorimetric [4,5] and sorption measurements [6,7] and NMR studies [7,8], but results of dielectric [9,10] and chemical potential measurements [l l] were interpreted in terms of continuous changes. However, almost all methods demonstrate that the first hydrating water molecules (between six and ten) are more firmly bound to the polar heads of phospholipids and that with increasing hydration
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the mobility of the water molecules increases [7-lo]. This also seems to be true for the water molecules inside the reverse micelles [ 12,131. The increasing mobility of the water molecules at higher hydration levels is usually attributed to a weakening of the hydrogen bonding [8-lo]. The water-to-phosphate bonding, prevailing in the lowest hydrates, is stronger than water-to-water bonding. However, definite data on hydrogen bond energies are scarce or reflect, in fact, the activation energies of rotation. Raman spectra [ 141 in the region of the water stretching of gradually dehydrated bilayer samples of dipalmitoylphosphatidylcholine (DPPC) confirm the contention of more strongly bound water that is closer to the polar heads, but no detailed analysis has been undertaken. ‘H NMR spectra, particularly the chemical shifts of water protons on increasing stepwise the water inside DPPC reverse micelles in benzene [13], are not readily interpretable in terms of the variation of strength of hydrogen bonding. It is rather surprising that relatively little use has been made of infrared spectroscopy to characterize the organization of the water hydrating the phospholipids [ 15-171, although this is one of the principal tools for investigating hydrogen bonding in general. To fill this gap, we have investigated the infrared spectra of HZ0 and isotopically diluted HDO in the region 37001600cm-’ of progressively hydrated DPPC, and, partly, dimyristoylphosphatidylcholine (DMPC) multibilayers (abbreviated to films) in the temperature range from - 150 to 70°C. The appearance of a fairly broad HZ0 stretching band even at the lowest levels of hydration (n M 1-2, where n is the water to phospholipid molar ratio) prompted us to also investigate some crystalline samples for comparison. Although the smoothly increasing trend of the water stretching frequencies and intensities with increasing hydration yield no evidence of various classes of bound water, this may be seen in the trends of the bandwidth at half the maximum height and, more clearly, by the effects of subzero temperatures. Particular attention will be paid in the discussion to the first water of hydration because the frequency of the stretching vibration is used to estimate the strength of the water-to-
phosphate hydrogen bonding, and its bandshape is taken as evidence for disordered bonding at the polar heads. Low temperature spectra of water in multibilayers demonstrate the formation of amorphous ice without the tendency for a transition to any form of crystalline ice.
2. Experimental Phosphatidylcholines (dipalmitoyl +99%, dimyristoyl +99% and egg yolk lecithin 98%) of highest declared purity were purchased from Sigma. Solvents (CHCls, PA purity and CDCls, Stohler 99.8%) were dried over molecular sieves as described in Refs. 18 and 19. Dry multibilayer films were formed on CsI or CaF2 (higher hydrates) plates by evaporation of solution in CHCls. To remove traces of the solvent the films were warmed in vacuum to 40°C for 5 h. Hydrated films up to n = 10 were obtained by soaking the dried films in the appropriate amount of HZ0 or D20/H20 mixture (5% vol. of D20). To achieve proper hydration, the films were warmed above the main phase transition temperature T,,, cooled to 21°C before measurement. This was performed in the closed cell to prevent water losses. Higher hydrated films were prepared from centrifuged liposomes as described in Ref. 19. Water content was checked by (i) redissolving the films in dry CDC& and measuring the ‘HNMR signal integrals and (ii) by measuring the integrated absorbance of the ~i,~ band and comparing it with standards obtained by exposing films to air of controlled humidity. The hydration interval between n = 7 and n = 15 was examined with films prepared in both ways, i.e. hydrating the films cast from CHC13 and drying the liposome suspension. There was no difference in the water band parameters. Reversed micelles were prepared from dry CDC13 and dry phosphatidylcholines (60 mM). Appropriate amounts of water were added to solutions with a 1~1 Hamilton syringe. The solutions were sonicated in a bath sonicator for 10 min and incubated for 12 h before measuring. Hydrated crystals of the respective phosphatidylcholines were grown from chloroform-
J. Grdadolnik et al.lJ. Mol. Struct. 322 (1994) 93-103
diethylether solutions by slowly lowering the temperature. Some of the crystals were also generously donated by Professor H. Hauser (E.T.H., Zurich). Spectra were recorded on a Digilab FT 15-80 spectrometer. Nominal resolution was 2 cm-‘. Typically, 256 interferograms were averaged and apodized with a triangular function. The accuracy of the frequencies of the yl,3 bands is f2 cm-i and of the bandwidths is f4 cm-‘. Micellar solutions in CDC13 were measured in a Harrick liquid cell with CdTe windows and teflon spacers 0.012mm thick. The low- and hightemperature spectra were measured using a variable-temperature cell (Beckmann) with KBr windows. The temperature was measured with a platinum resistor (PtlOO) and iron-constantan thermocouple. The accuracy of the temperature reading was in the range between 100 and -50 f 2°C and at lower temperatures f5”C.
3. Results 3.1. Water spectra ofjilms and micelles above 20°C Some examples of spectra in the Hz0 (v,,~) region of gradually hydrated DPPC films and micelles are displayed in Fig. 1. Between the monohydrate and the maximally hydrated samples, the peak frequency smoothly increases from 3380 cm-’ (films) or 3376cm-i (micelles) to 3400cm-’ and 3395 cm-‘, respectively. The corresponding values of the decoupled OD stretching bands (vOD) are 2489 cm-’ and 2507cn-’ (films), 2488cm-’ and 2507 cm-’ (micelles). Note that the maximal hydration of micelles was n = 12 after which cloudiness appeared and no attempt at further hydration seemed sensible. The centres of intensity show a very similar trend as the peak frequencies in both types of samples (not shown). The integrated intensities increase linearly with n. Figure 2 shows the bandwidths at half the maximum height as a function of hydration. The decrease of slope after n > 7 is notable in all examples. However, there are minor differences in the trends of broadening between the films and micelles, particularly in the range between n = 3 and n = 7. The starting half-bandwidth (n = 1) is
95
3 12 cm-’ (film) and 334 cm-’ (micelles). The width of the band of the films reaches the value of 450cm-’ at maximal hydration. Thus the width of the vi,3 band of Hz0 of hydration exceeds by about 50 cm-’ the one observed with a thin film of pure water. The currently obtained value of 405cm-’ for the latter is in agreement with the literature data [20,21]. The initial width of the vOD band under isotopic dilution is 127cm-’ (150cm-‘, micelles) and reaches the maximum value of 170cm-‘, but the broadening does not show the same trend as in the example of Hz0 hydration. A more detailed examination of the bandshapes of films at small n (including n = 1) reveals a shoulder on the high frequency slope at about 3465 cm-’ (band fitting value). A very small peak appears at 3600 The depression at about 3280cm-’ corresponds to the one observed in the spectrum of ordinary liquid water [22], whereas the high frequency shoulder, which is also observed in the non-decoupled spectrum of D20 (50% D20 in H20) hydrates, merges into the main band. With increasing hydration the depression at the low frequency band slope remains and its counterpart also appears in the spectrum of D20 hydrated samples (not shown). The stretching bands of the micelles are similar except that the shoulder on the high frequency slope is less pronounced and the small band at 36OOcm-’ is missing. The y band at about 1650 cm-’ does not exhibit any peculiarities except for the increase of intensity with hydration.
cn-i.
3.2. Spectra of some hydrated phosphatidylcholine crystals.
The spectra of water in the examined crystal hydrates of lauroylpropanediolphosphatidylcholine (LPPC), DPPC and DMPC differ essentially (Fig. 3) from those of films and micelles in that sharper peaks protrude from a broad background. Only in the example of LPPC are the peaks like those of well-ordered crystal hydrates. In the other examples the protruding peaks assume various values between 3450 and 3180 cm-‘. Although the bands in the region below 1800cm-’ are sharp, with developed crystal field splittings, the stretching bands of hydrating water
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J. Grdadolnik et al.lJ. Mol. Struct. 322 (1994) 93-103
A
b S 0 r b a n C e
3800
3400
3600
3200
I
3000
3800
Wavenumbers
3000 Wavenumbers
Fig. 1. Examples of spectra in the q3 region of: (A) hydrated DPPC films (a, b, c, d; n = 1,4,10,30);(B) reversed micelles in CDCl, (a, b, c, d; n = 1,2,4,10) (chloroform is subtracted, absorbances normalized).
indicate only partial ordering. Differences in band frequencies and crystal field splittings in the region 1800-500 cm-’ are indicative of the polymorphism of the crystalline samples. Unfortunately, a better characterization of the crystals was not possible because of the small quantity of samples available. Nevertheless, the observed peaks demonstrate clearly that quite a large variety of bonding possibilities is available for water molecules in the crystals. 3.3. Effects of temperature
little on cooling to - 150°C; we show in Fig. 5 only the spectra at -40°C. Some intensity is lost at the high frequency side of the stretching band; the v2 band remains unaffected. The minimal narrowing of the v1,3 band strongly contrasts with the behaviour of the usual hydrogen-bonded systems 1251. 500 450
variation
The profound influence of hydration on thermotropic phase transitions has been extensively investigated [2,23]. We have recorded several spectra at temperatures below and above the main transition of variously hydrated DPPC, but little change was observed with low hydrated samples (Fig. 4). The slight frequency shifts and broadening of the ~i,~ band in the case of the more hydrated samples (not shown) correspond to those of ordinary water [20,24]. The effects of low temperatures upon the spectra differ strongly depending on the level of hydration. In the example of films, the limit between the two types of spectral response to cooling is n z 10. From n = 1 to n = 9 the water bands change very
400 &
350
n
Fig. 2. Plot of halfwidths of the tq3 and YOD bands (5% vol. D20 in H20) of stepwise hydrated DPPC films and reversed micelles in CDC& (60 mM) against n. (0) IQ Hz0 micelles; (0) v,,~ Hz0 films; (w) vOD micelles; (0) vOD films.
J. Grabdolnik et al/J. Mol. Struct. 3.22 (1994) 93-103
3aoo
3600
3400
3200
3000
Wavenumbers Fig. 3. Representative spectra of some phosphatidylcholine crystal hydrates in the ~1.3 region: (a) DMPC; (b) and (c) DPPC; (d) DMPC; (e) LPPC. (a, b, c: crystals grown from chloroform-diethylether solution (see Experimental; d, e: crystals donated by Prof. H. Hauser).
The effects of cooling the films with n M 10 to subzero temperatures (Fig. 6) are much more dramatic than in the example of the less hydrated ones; the changes, moreover, depend on the chemical composition of the lipid and the thermal
3800
3600
3400 Wavenumbers
3200
3000
Fig. 4. v,,s bands of a hydrated DPPC film (n % 6) below and above the temperature of the main phase transition (T, = 60”). The arrow indicates increasing temperatures (30-7O”C, 5°C step).
91
history. For instance, on the first cooling of the highly hydrated DPPC films the water spectrum undergoes no major changes down to -30°C; obviously water is undercooled. On further cooling a sudden frequency lowering to 3200cm-’ of the v1,3band centre is observed along with a change of the bandshape. This becomes quite like one of amorphous ice [26,27]. The decoupled vOD band becomes narrow (halfwidth = 50 cm-‘) whereas the y H20 band is strongly broadened (Fig. 6). Lowering the temperature and cycling at various intermediate temperatures did not provoke any notable changes in the bandshapes that might indicate an ice transformation. However, after warming up to room temperature and cooling the same sample for a second time, freezing occurred at 0°C. The same results were obtained with DMPC films but egg yolk lecithin exhibited a different behaviour in that freezing sets in at -20°C; this is not influenced by cycling. The spectrum of the film at n M 10 is peculiar in that its vi,3 Hz0 band moves to lower frequencies on cooling, but only for the 50 cm-’ centre of intensity with simultaneous replacement of the depression on the low frequency slope by a maximum. The decoupled (H)OD band also moves and remains broad. The u2 band is markedly flattened. Samples of reversed micelles hydrated to n = 4 and n = 9 were examined and the effects of cooling are illustrated by Figs. 5(C) and 5(D). In the example of n = 9, no major changes appeared either in Y~,~or decoupled vOD bands until a temperature of -30°C was reached; the cooling time was 50 min. After keeping the sample for 20min. at this temperature, the vOD band began to display an asymmetry and the CH2 scissoring band split. Obviously DPPC is no longer associated in the form of the reverse micelles under this condition. Nevertheless, cooling was continued and at -50°C the spectrum of amorphous ice appeared. The less hydrated sample (n = 4) was cooled only to -30°C; no splitting of the CH2 scissoring band occurred and we presume that the micellar structure persisted to this temperature. The vi,3 band gradually changed shape, in that the depression near 3280cm-’ was replaced by
98
absorption. more flat.
J.Grdadolniket al.lJ. Mol.Struct.322 (1994) 93-103
Simultaneously
the v2 band became
4. Discussion Discussion of the results will be centred around the following questions. What can be inferred from the spectra about the location and energy of binding of the first (up to n = 10) water molecules? How close are the spectroscopic characteristics of water at high hydration to those of ordinary bulk water?
Are there any major differences between water inside the reverse micelles and interlayer water? It is largely accepted by now that the first one to two water molecules are more tightly bound than the following ones and that this is due to direct binding of the first water molecules to the charged phosphate oxygens of the headgroups (see Ref. 19 and references cited therein). Considering the crystal structures of LPPC +H20 [28] and DMPC. 2H20 [29] it may be assumed that the monohydrate water in the less ordered aggregates also forms bridges between the phosphate groups
I-
A
,
\
i
’
,T=ZV
I I I I I I I I I I I I I 3600 3600 3400 3200 3000 2800 2600 2400 2200 2000 1600 1600 1400 Wavenumbers
0
I I I I I I I I I I 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800
I 1600
I 1400
Wavenumbers
Fig. 5. Spectra of hydrated DPPC films and DMPC reversed micelles at room and low temperature (RT spectra displaced film (n = 6); (B) film (n = 10); (C) reversed micelles (n= 4: only V,,J region); (D) reversed micelles (n = 9).
upwards).
(A)
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J. Grdadolnik et al/J. Mol. Struct. 322 (1994) 93-103 Ts-50'
A b S 0
A
_
b S 0 r b a n C e
-
6 a n C e
-
-
3800
I
I
I
I
3600
3400
3200
3000
Wavenumbers
3800
3600
3400
3200
3000
Wavenumbers
2800
2600
2400
Fig. 5. (Continued.)
of one layer, like those found in the crystal structures. The second water molecule, and perhaps some more, may be engaged in hydrogen bonding with one hydroxyl to a phosphate group and to one of the first water molecules with the other hydroxyl group. Molecular mechanics refinement of the crystal structure of DMPC - 2H20 has indeed revealed two possible and energetically similar binding modes for the second water molecule [30]. The width and shape of the v1,3 band of the monohydrated films and micelles are not easily reconciled with the view of a strongly privileged binding mode such as that offered by a regular chain of water-bridged phosphate groups. Such an arrangement would not be expected to result in a broad band with n = 1, as already observed. We contend that the bandwidth of monohydrated films and micelles originates from a distribution of the hydrogen bonds of variable strengths owing to a variety of binding sites. Each component band is, of course, broadened by any of the proposed mechanisms for homogeneous broadening [25]. However, that this homogeneous broadening cannot be at the origin of the overall bandwidth is clearly shown by the negligible effect on the latter of cooling to -150°C; this contrasts strongly with the behaviour of homogeneously broadened
stretching bands in most of the hydrogen-bonded systems [25]. This reasoning can also be extended to higher hydrates (up to n M 9). Further support for the assumption of the statistical character of the y1,3 bandwidth of the lower hydrates is offered by the spread of frequencies of the peaks in the spectra of the hydrated crystalline samples (Fig. 3). These peaks easily cover the widths of the water stretching bands of low hydrated films and micelles. However, this comparison is not meant to be an allusion to a crystalline mosaic structure of the films. Considering the details of the band contour, we note a shoulder at 3465cm-’ in the spectra of the monohydrates and the other low hydrated samples. It can be assigned to less strongly bonded hydroxyl groups. The small peak at 3600cn-’ may consequently be attributed to some nearly free OH groups. The inflection at about 3280cm-i is obviously created by Fermi resonance with 22~~as in the example of the y1,3 band of bulk water [22]. The view of disordered water binding, which also includes very weakly bonded OH groups, is in accord with a semiempirical MO calculation [31] on the model system consisting of a cluster of four phosphatidylcholine molecules (with curtailed hydrocarbon chains) with added water molecules, and with the results of the molecular
J.Grdadolnik etal./J. Mol.Struct. 322 (1994)93-103
3800 3600
3400 3200 3000 2800 2600 2400 2200 2000 Wavenumbers
1800 1600 1400
0.8A b ; 0.66 ; 0.4C e
I 3600
I
I
I
I
3200 2800 Wavenumbers
I
I 2400
Fig. 6. (A) Spectra of centrifuged DPPC liposomes at room temperature and -40°C in the regions between 3800 and 14OOcm-’ (RT spectrum displaced upwards). (B) v,,~ region of pure ice (water frozen to -40” between CaF2 plates).
mechanics refinement of the DMPC - 2H20 crystal structure [30]. The latter calculation revealed two possible, energetically similar, binding modes for the second water molecule; an extrapolation to more water molecules appears to be justified by the small differences in the bandwidths of the low hydrates. A rough estimate of the hydrogen bond strength of the first bound water molecules can be made by considering the frequency of the v],~ band. This is 20cm-’ lower than the one of bulk water
(3400cm-‘); the enthalpy of the most likely water-water hydrogen bonds amounts to about 4 kcalmol-’ [32,33]. Thus the majority of the binding sites in phosphatidylcholines would offer bonding energies for the first bound molecules in the range 4-5 kcalmoi-‘; the width of the v],~ band can accommodate both stronger and weaker hydrogen bonding. The estimate is valid within the limits of the general relation between the low frequency shifts of the X-H stretchings due to hydrogen bonding, and its strength [34]. The
J. Grdadolnik et al.lJ. Mol. Struct. 322 (1994) 93-103
estimate of 5 kcalmol-’ can be compared with the value for the DMPC - 2HzO crystal [30] which is markedly higher (7.8 kcal mol-‘). This somewhat exceeds the energy of the hydrogen bonds in ice (7.4 kcal mol-‘, ref. 35). Note, however, that the frequency of the central peak in the spectrum of ice is at 3200cn-’ which is considerably lower than the peak frequencies of the water in the low hydrates of phosphatidylcholines. The present estimate of the most likely bonding energy for water in the low hydrates is quite close to that deduced from dielectric relaxation studies [9]. At any rate, these hydrogen bond energies are much inferior to the theoretically calculated interaction energies of water with dimethyl phosphate, which is often taken as a model for the phosphate group in phospholipids. The theoretically computed interaction energies are in the range 20-30 kcal mol-’ , depending on the level of theory [36-381. However, these energies were obtained with optimized geometries, neglecting the effect of the neighbourhood of the positively charged trimethylammonium group of the choline chain and other possible medium effects. With increasing hydration the availability of various binding sites for water also increases, causing further band broadening. Changes in the slope of the plot relating the half-bandwidths with the level of hydration (Fig. 2) are probably making the transition from a more frequent water-tophosphate bonding to increasing water-to-water bonding. An intermediate state may be imagined in which water molecules bind to those in direct contact with phosphate groups. These water molecules are expected to be more polarized, thus warranting somewhat stronger hydrogen bonding to the next incoming molecules, but this effect decays as the thickness of the water layer increases. Beyond n = 10 the shape of the v1,3band closely parallels that of bulk water except for the width, which at the maximal hydration (n = 30) exceeds by approximately 50 cm-’ the width of the band of bulk water (405cm-‘). The bandshape, including the depression at 3280 cm-‘, can be interpreted by analogy with bulk water [22]. The excess bandwidth may signify a wider spread in the variation of hydrogen bonds resulting from long-range effects of the polar heads. The small spectral
101
changes noted at hydration levels beyond n = 10 evolve smoothly at room temperature and even above the main transition (65°C for DPPC at this level of hydration [4,5]). Even more dramatic differences between the water spectrum of the films hydrated below n = 10 and at n > 10 are observed on cooling to subzero temperatures. At the higher level of hydration, cooling elicits spectral changes that are indicative of freezing to amorphous ice. Formation of amorphous ice in DPPC multibilayers has been observed previously [17]; high pressure forms of crystalline ice were obtained by subjecting dispersions of mixed chain phosphatidylcholines to pressures exceeding 21 kbar [39]. The formation of amorphous ice at relatively high temperatures and the excess width of the ~r,~ band of water in the fully hydrated multibilayers are indicative of differences with respect to ordinary bulk water. The bandshapes of the 21r,~bands and the overall bandwidths of the micelles are slightly different from those of the films. The somewhat narrower Hz0 bands in the former may be taken as an indication of a lesser disorder in the organization of the headgroup-to-water interface than in the bilayers, which are expected to offer more binding possibilities for water molecules than the single spherical (or nearly so) interior surface of a reverse micelle. The most conspicuous difference between water in multibilayers and that in reverse micelles appears on cooling to subzero temperatures. The interpretation of the gradual disappearance with decreasing temperature of the depression in the ~1.3 band of micellar water and the concomitant broadening of the y band, which seem to be in a causal relation, is not possible at present because the mechanism of this broadening in pure water is not yet well understood. Further work on this and some other aspects of low temperature effects on water of hydration is ongoing; it will also include NMR spectroscopy. The presently investigated types of phospholipid assemblies are not the best models of biological membranes. However, the results demonstrate that interesting information about the organisation of water of hydration can be extracted from the infrared spectra. Obviously, there is ample scope for more detailed investigations, with inclusion of
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other spectroscopic techniques, and we are already pursuing several lines to complete the presented results.
5. Conclusions The width of the 13~ band of water hydrating phosphatidylcholine films and micelles up to n = 10 is explained in terms of a distribution of hydrogen bond strengths caused by multiple binding possibilities within the polar heads. The main support for this view is the lack of low temperature effects upon the band frequencies and shapes, which also demonstrate that at this level of hydration water does not freeze. Freezing phenomena in films in micelles are somewhat different. Minor differences between water in the two forms of aggregation appear in the trends of v1 s band broadening with increasing hydration, which also permit a differentiation between water molecules binding in the range from about Al= 1 to II = 6. However, no sharp limits in the sense of hydration shells are observed except for the freezing above n M 10 and its absence at lower hydration.
6. Acknowledgements This investigation was supported by the Ministry of Science and Technology of Slovenia. Thanks are due to Miss Silva Zagorc for assistance in the experimental work and to Mrs. Tatjana Karba for skilful typing.
7. References [1] G. Cevc, in E. Westhof (Ed.), Hydration of Biological Macromolecules, McMillan Press, New York, 1992, pp. 338-389. [2] H. Hauser and M.C. Phillips, Prog. Surf. Membr. Sci., 13 (1979) 297. [3] P. Walde, A.M. Giuliani, C.A. Boicelli and P.L. Luisi, Chem. Phys. Lipids, 53 (1990) 265. [4] M. Kodama, M. Kuwabara and S. Seki, Biochim. Biophys. Acta, 689 (1982) 567.
PI C. Gabrielle-Madelmont
and R. Perron, J. Colloid Interface Sci., 95 (1983) 483. WIP.H. Elworthy, J. Chem. Sot., (1961) 4897. [71 D.A. Wilkinson, H.J. Morowitz and J.H. Prestegard, Biophys. J., 20 (1970) 169. PI G. Finer and A. Darke, Chem. Phys. Lipids, 12 (1974) 1. [91 H. Enders and G. Nimtz, Ber. Bunsenges. Phys. Chem., 88 (1984) 512. DOI G. Nimtz, Physica Ser., T13 (1986) 172. Vll L.J. Lis, M. McAlister, N. Fuller, R.P. Rand and V.A. Parsegian, Biophys. J., 37 (1982) 657. WI K. Arnold, L. Pratsch and K. Gawrisch, Biochim. Biophys. Acta, 728 (1983) 121. [I31 G. Klose and F. Stelzner, Biochim. Biophys. Acta, 363 (1974) 1. 1141S. Kim, P.H. Wermer and J.R. Scherer, J. Phys. Chem., 96 (1992) 446. P51 D. Chapman, The Structure of Lipids, Methuen, London, 1965, pp. 53-132. E. Oamura, I. Umemura [I61 L. Ter-Minassian-Saraga, and T. Takenaka, Biochim. Biophys. Acta, 946 (1988) 417. 1171E. Okamura, J. Umemura and T. Takenaka, Vibr. Spectrosc., 2 (1991) 95. WI J. Grdadolnik and D. Hadii, Chem. Phys. Lipids, 63 (1993) 121. 1191J. Grdadolnik, J. KidriE and D. Hadii, Chem. Phys. Lipids, 59 (1991) 57. [201 M. Falk and T.A. Ford, Can. J. Chem., 44 (1966) 1699. PI J. Schiffer and D.F. Hornig, J. Chem. Phys., 49 (1968) 4150. P21 J.R. Scherer, in R.J.H. Clark and R.E. Hester (Eds.) Advances in IR and Raman Spectroscopy, Vol. 5, Heyden and Sons, 1978, pp. 149-216. v31 M.J. Janiak, D.M. Small and G.G. Shipley, J. Biol. Chem., 254 (1979) 6068. v41 M.G. Sceats and K. Belsley, Mol. Phys., 40 (1980) 1389. [25] D. Hadii and S. Bratos, in P. Schuster, G. Zundell and C. Sandorfy (Eds.), The Hydrogen Bond Vol. 2, NorthHolland, 1976, pp. 565-612. [26] E. Mayer, J. Phys. Chem., 89 (1985) 3474. [27] G. Nielson and S.A. Rice, J. Chem. Phys., 78 (1983) 4824. [28] H. Hauser, I. Pascher and S. Sundell, J. Mol. Biol., 137 (1980) 249. [29] R.H. Pearson and I. Pascher, Nature, 281 (1979) 499. [30] G. Vanderkooi, Biochemistry, 30 (1991) 10760. [31] J. Koller and D. Had&, in preparation. [32] D. Eisenberg and W. Kauzmann, The Structure and Properties of Water, Oxford University Press, 1969, pp. 177-179.
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MOLECUlAR STRUCTURE
ELSEVIER
Journal of Molecular Structure 322 (1994) 93- 103
An FT-IR study of water hydrating dipalmitoylphosphatidylcholine multibilayers and reversed micelles’ J. Grdadolnik,
J. KidriE, D. Hadii*
National Institute of Chemistry,
61115 Ljubljana. Slovenia
(First received 13 September 1993;in final form 13 January 1994)
Abstract Infrared spectra of HZ0 and HDO (5% D20 in HzO) in the stretching and bending regions are presented and discussed with respect to location and energy of bonding of the first water molecules hydrating the polar heads of the title systems. Spectral features of water in the higher hydrated bilayers are compared to those of ordinary water. The v,,~ and v2 bands of water at low hydration are only slightly sensitive to low temperatures. As an indication that these water molecules do not freeze, we take the absence of any major effects of cooling as the main evidence for the origin of the bandwidth. We propose that the v1,3 band broadening is of a statistical character, i.e. it is caused by various binding topologies of the water molecules, and not by homogeneous broadening mechanisms. With highly hydrated bilayers, the formation of amorphous ice is observed. Differences are noted between some spectral features of water inside the reverse micelles and water hydrating the bilayers, particularly at subzero temperatures.
1. Introduction The structure and properties of phospholipid assemblies, and their function in biological membranes, are inextricably linked to hydration. Its pervasive effects have been investigated, particularly in the example of bilayers, by a variety of physical methods, which were also applied to the elucidation of the organization of water sandwiched between the polar heads (for reviews, see Refs. l-3). Interpretations of the experimental results with ’ Presented at the Xth Workshop “Horizons in Hydrogen Bond Research”, Autrans, France, 12- 17 September 1993. * Corresponding author.
respect to water organization at progressive hydration lead to two views. At one extreme, water would form distinct layers or hydration shells, whereas at the other extreme, the measured properties are considered as smoothly evolving changes in the water organization. The views strongly depend on the methods of investigation. The concept of shells - up to four - emerged from calorimetric [4,5] and sorption measurements [6,7] and NMR studies [7,8], but results of dielectric [9,10] and chemical potential measurements [l l] were interpreted in terms of continuous changes. However, almost all methods demonstrate that the first hydrating water molecules (between six and ten) are more firmly bound to the polar heads of phospholipids and that with increasing hydration
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the mobility of the water molecules increases [7-lo]. This also seems to be true for the water molecules inside the reverse micelles [ 12,131. The increasing mobility of the water molecules at higher hydration levels is usually attributed to a weakening of the hydrogen bonding [8-lo]. The water-to-phosphate bonding, prevailing in the lowest hydrates, is stronger than water-to-water bonding. However, definite data on hydrogen bond energies are scarce or reflect, in fact, the activation energies of rotation. Raman spectra [ 141 in the region of the water stretching of gradually dehydrated bilayer samples of dipalmitoylphosphatidylcholine (DPPC) confirm the contention of more strongly bound water that is closer to the polar heads, but no detailed analysis has been undertaken. ‘H NMR spectra, particularly the chemical shifts of water protons on increasing stepwise the water inside DPPC reverse micelles in benzene [13], are not readily interpretable in terms of the variation of strength of hydrogen bonding. It is rather surprising that relatively little use has been made of infrared spectroscopy to characterize the organization of the water hydrating the phospholipids [ 15-171, although this is one of the principal tools for investigating hydrogen bonding in general. To fill this gap, we have investigated the infrared spectra of HZ0 and isotopically diluted HDO in the region 37001600cm-’ of progressively hydrated DPPC, and, partly, dimyristoylphosphatidylcholine (DMPC) multibilayers (abbreviated to films) in the temperature range from - 150 to 70°C. The appearance of a fairly broad HZ0 stretching band even at the lowest levels of hydration (n M 1-2, where n is the water to phospholipid molar ratio) prompted us to also investigate some crystalline samples for comparison. Although the smoothly increasing trend of the water stretching frequencies and intensities with increasing hydration yield no evidence of various classes of bound water, this may be seen in the trends of the bandwidth at half the maximum height and, more clearly, by the effects of subzero temperatures. Particular attention will be paid in the discussion to the first water of hydration because the frequency of the stretching vibration is used to estimate the strength of the water-to-
phosphate hydrogen bonding, and its bandshape is taken as evidence for disordered bonding at the polar heads. Low temperature spectra of water in multibilayers demonstrate the formation of amorphous ice without the tendency for a transition to any form of crystalline ice.
2. Experimental Phosphatidylcholines (dipalmitoyl +99%, dimyristoyl +99% and egg yolk lecithin 98%) of highest declared purity were purchased from Sigma. Solvents (CHCls, PA purity and CDCls, Stohler 99.8%) were dried over molecular sieves as described in Refs. 18 and 19. Dry multibilayer films were formed on CsI or CaF2 (higher hydrates) plates by evaporation of solution in CHCls. To remove traces of the solvent the films were warmed in vacuum to 40°C for 5 h. Hydrated films up to n = 10 were obtained by soaking the dried films in the appropriate amount of HZ0 or D20/H20 mixture (5% vol. of D20). To achieve proper hydration, the films were warmed above the main phase transition temperature T,,, cooled to 21°C before measurement. This was performed in the closed cell to prevent water losses. Higher hydrated films were prepared from centrifuged liposomes as described in Ref. 19. Water content was checked by (i) redissolving the films in dry CDC& and measuring the ‘HNMR signal integrals and (ii) by measuring the integrated absorbance of the ~i,~ band and comparing it with standards obtained by exposing films to air of controlled humidity. The hydration interval between n = 7 and n = 15 was examined with films prepared in both ways, i.e. hydrating the films cast from CHC13 and drying the liposome suspension. There was no difference in the water band parameters. Reversed micelles were prepared from dry CDC13 and dry phosphatidylcholines (60 mM). Appropriate amounts of water were added to solutions with a 1~1 Hamilton syringe. The solutions were sonicated in a bath sonicator for 10 min and incubated for 12 h before measuring. Hydrated crystals of the respective phosphatidylcholines were grown from chloroform-
J. Grdadolnik et al.lJ. Mol. Struct. 322 (1994) 93-103
diethylether solutions by slowly lowering the temperature. Some of the crystals were also generously donated by Professor H. Hauser (E.T.H., Zurich). Spectra were recorded on a Digilab FT 15-80 spectrometer. Nominal resolution was 2 cm-‘. Typically, 256 interferograms were averaged and apodized with a triangular function. The accuracy of the frequencies of the yl,3 bands is f2 cm-i and of the bandwidths is f4 cm-‘. Micellar solutions in CDC13 were measured in a Harrick liquid cell with CdTe windows and teflon spacers 0.012mm thick. The low- and hightemperature spectra were measured using a variable-temperature cell (Beckmann) with KBr windows. The temperature was measured with a platinum resistor (PtlOO) and iron-constantan thermocouple. The accuracy of the temperature reading was in the range between 100 and -50 f 2°C and at lower temperatures f5”C.
3. Results 3.1. Water spectra ofjilms and micelles above 20°C Some examples of spectra in the Hz0 (v,,~) region of gradually hydrated DPPC films and micelles are displayed in Fig. 1. Between the monohydrate and the maximally hydrated samples, the peak frequency smoothly increases from 3380 cm-’ (films) or 3376cm-i (micelles) to 3400cm-’ and 3395 cm-‘, respectively. The corresponding values of the decoupled OD stretching bands (vOD) are 2489 cm-’ and 2507cn-’ (films), 2488cm-’ and 2507 cm-’ (micelles). Note that the maximal hydration of micelles was n = 12 after which cloudiness appeared and no attempt at further hydration seemed sensible. The centres of intensity show a very similar trend as the peak frequencies in both types of samples (not shown). The integrated intensities increase linearly with n. Figure 2 shows the bandwidths at half the maximum height as a function of hydration. The decrease of slope after n > 7 is notable in all examples. However, there are minor differences in the trends of broadening between the films and micelles, particularly in the range between n = 3 and n = 7. The starting half-bandwidth (n = 1) is
95
3 12 cm-’ (film) and 334 cm-’ (micelles). The width of the band of the films reaches the value of 450cm-’ at maximal hydration. Thus the width of the vi,3 band of Hz0 of hydration exceeds by about 50 cm-’ the one observed with a thin film of pure water. The currently obtained value of 405cm-’ for the latter is in agreement with the literature data [20,21]. The initial width of the vOD band under isotopic dilution is 127cm-’ (150cm-‘, micelles) and reaches the maximum value of 170cm-‘, but the broadening does not show the same trend as in the example of Hz0 hydration. A more detailed examination of the bandshapes of films at small n (including n = 1) reveals a shoulder on the high frequency slope at about 3465 cm-’ (band fitting value). A very small peak appears at 3600 The depression at about 3280cm-’ corresponds to the one observed in the spectrum of ordinary liquid water [22], whereas the high frequency shoulder, which is also observed in the non-decoupled spectrum of D20 (50% D20 in H20) hydrates, merges into the main band. With increasing hydration the depression at the low frequency band slope remains and its counterpart also appears in the spectrum of D20 hydrated samples (not shown). The stretching bands of the micelles are similar except that the shoulder on the high frequency slope is less pronounced and the small band at 36OOcm-’ is missing. The y band at about 1650 cm-’ does not exhibit any peculiarities except for the increase of intensity with hydration.
cn-i.
3.2. Spectra of some hydrated phosphatidylcholine crystals.
The spectra of water in the examined crystal hydrates of lauroylpropanediolphosphatidylcholine (LPPC), DPPC and DMPC differ essentially (Fig. 3) from those of films and micelles in that sharper peaks protrude from a broad background. Only in the example of LPPC are the peaks like those of well-ordered crystal hydrates. In the other examples the protruding peaks assume various values between 3450 and 3180 cm-‘. Although the bands in the region below 1800cm-’ are sharp, with developed crystal field splittings, the stretching bands of hydrating water
96
J. Grdadolnik et al.lJ. Mol. Struct. 322 (1994) 93-103
A
b S 0 r b a n C e
3800
3400
3600
3200
I
3000
3800
Wavenumbers
3000 Wavenumbers
Fig. 1. Examples of spectra in the q3 region of: (A) hydrated DPPC films (a, b, c, d; n = 1,4,10,30);(B) reversed micelles in CDCl, (a, b, c, d; n = 1,2,4,10) (chloroform is subtracted, absorbances normalized).
indicate only partial ordering. Differences in band frequencies and crystal field splittings in the region 1800-500 cm-’ are indicative of the polymorphism of the crystalline samples. Unfortunately, a better characterization of the crystals was not possible because of the small quantity of samples available. Nevertheless, the observed peaks demonstrate clearly that quite a large variety of bonding possibilities is available for water molecules in the crystals. 3.3. Effects of temperature
little on cooling to - 150°C; we show in Fig. 5 only the spectra at -40°C. Some intensity is lost at the high frequency side of the stretching band; the v2 band remains unaffected. The minimal narrowing of the v1,3 band strongly contrasts with the behaviour of the usual hydrogen-bonded systems 1251. 500 450
variation
The profound influence of hydration on thermotropic phase transitions has been extensively investigated [2,23]. We have recorded several spectra at temperatures below and above the main transition of variously hydrated DPPC, but little change was observed with low hydrated samples (Fig. 4). The slight frequency shifts and broadening of the ~i,~ band in the case of the more hydrated samples (not shown) correspond to those of ordinary water [20,24]. The effects of low temperatures upon the spectra differ strongly depending on the level of hydration. In the example of films, the limit between the two types of spectral response to cooling is n z 10. From n = 1 to n = 9 the water bands change very
400 &
350
n
Fig. 2. Plot of halfwidths of the tq3 and YOD bands (5% vol. D20 in H20) of stepwise hydrated DPPC films and reversed micelles in CDC& (60 mM) against n. (0) IQ Hz0 micelles; (0) v,,~ Hz0 films; (w) vOD micelles; (0) vOD films.
J. Grabdolnik et al/J. Mol. Struct. 3.22 (1994) 93-103
3aoo
3600
3400
3200
3000
Wavenumbers Fig. 3. Representative spectra of some phosphatidylcholine crystal hydrates in the ~1.3 region: (a) DMPC; (b) and (c) DPPC; (d) DMPC; (e) LPPC. (a, b, c: crystals grown from chloroform-diethylether solution (see Experimental; d, e: crystals donated by Prof. H. Hauser).
The effects of cooling the films with n M 10 to subzero temperatures (Fig. 6) are much more dramatic than in the example of the less hydrated ones; the changes, moreover, depend on the chemical composition of the lipid and the thermal
3800
3600
3400 Wavenumbers
3200
3000
Fig. 4. v,,s bands of a hydrated DPPC film (n % 6) below and above the temperature of the main phase transition (T, = 60”). The arrow indicates increasing temperatures (30-7O”C, 5°C step).
91
history. For instance, on the first cooling of the highly hydrated DPPC films the water spectrum undergoes no major changes down to -30°C; obviously water is undercooled. On further cooling a sudden frequency lowering to 3200cm-’ of the v1,3band centre is observed along with a change of the bandshape. This becomes quite like one of amorphous ice [26,27]. The decoupled vOD band becomes narrow (halfwidth = 50 cm-‘) whereas the y H20 band is strongly broadened (Fig. 6). Lowering the temperature and cycling at various intermediate temperatures did not provoke any notable changes in the bandshapes that might indicate an ice transformation. However, after warming up to room temperature and cooling the same sample for a second time, freezing occurred at 0°C. The same results were obtained with DMPC films but egg yolk lecithin exhibited a different behaviour in that freezing sets in at -20°C; this is not influenced by cycling. The spectrum of the film at n M 10 is peculiar in that its vi,3 Hz0 band moves to lower frequencies on cooling, but only for the 50 cm-’ centre of intensity with simultaneous replacement of the depression on the low frequency slope by a maximum. The decoupled (H)OD band also moves and remains broad. The u2 band is markedly flattened. Samples of reversed micelles hydrated to n = 4 and n = 9 were examined and the effects of cooling are illustrated by Figs. 5(C) and 5(D). In the example of n = 9, no major changes appeared either in Y~,~or decoupled vOD bands until a temperature of -30°C was reached; the cooling time was 50 min. After keeping the sample for 20min. at this temperature, the vOD band began to display an asymmetry and the CH2 scissoring band split. Obviously DPPC is no longer associated in the form of the reverse micelles under this condition. Nevertheless, cooling was continued and at -50°C the spectrum of amorphous ice appeared. The less hydrated sample (n = 4) was cooled only to -30°C; no splitting of the CH2 scissoring band occurred and we presume that the micellar structure persisted to this temperature. The vi,3 band gradually changed shape, in that the depression near 3280cm-’ was replaced by
98
absorption. more flat.
J.Grdadolniket al.lJ. Mol.Struct.322 (1994) 93-103
Simultaneously
the v2 band became
4. Discussion Discussion of the results will be centred around the following questions. What can be inferred from the spectra about the location and energy of binding of the first (up to n = 10) water molecules? How close are the spectroscopic characteristics of water at high hydration to those of ordinary bulk water?
Are there any major differences between water inside the reverse micelles and interlayer water? It is largely accepted by now that the first one to two water molecules are more tightly bound than the following ones and that this is due to direct binding of the first water molecules to the charged phosphate oxygens of the headgroups (see Ref. 19 and references cited therein). Considering the crystal structures of LPPC +H20 [28] and DMPC. 2H20 [29] it may be assumed that the monohydrate water in the less ordered aggregates also forms bridges between the phosphate groups
I-
A
,
\
i
’
,T=ZV
I I I I I I I I I I I I I 3600 3600 3400 3200 3000 2800 2600 2400 2200 2000 1600 1600 1400 Wavenumbers
0
I I I I I I I I I I 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800
I 1600
I 1400
Wavenumbers
Fig. 5. Spectra of hydrated DPPC films and DMPC reversed micelles at room and low temperature (RT spectra displaced film (n = 6); (B) film (n = 10); (C) reversed micelles (n= 4: only V,,J region); (D) reversed micelles (n = 9).
upwards).
(A)
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A b S 0
A
_
b S 0 r b a n C e
-
6 a n C e
-
-
3800
I
I
I
I
3600
3400
3200
3000
Wavenumbers
3800
3600
3400
3200
3000
Wavenumbers
2800
2600
2400
Fig. 5. (Continued.)
of one layer, like those found in the crystal structures. The second water molecule, and perhaps some more, may be engaged in hydrogen bonding with one hydroxyl to a phosphate group and to one of the first water molecules with the other hydroxyl group. Molecular mechanics refinement of the crystal structure of DMPC - 2H20 has indeed revealed two possible and energetically similar binding modes for the second water molecule [30]. The width and shape of the v1,3 band of the monohydrated films and micelles are not easily reconciled with the view of a strongly privileged binding mode such as that offered by a regular chain of water-bridged phosphate groups. Such an arrangement would not be expected to result in a broad band with n = 1, as already observed. We contend that the bandwidth of monohydrated films and micelles originates from a distribution of the hydrogen bonds of variable strengths owing to a variety of binding sites. Each component band is, of course, broadened by any of the proposed mechanisms for homogeneous broadening [25]. However, that this homogeneous broadening cannot be at the origin of the overall bandwidth is clearly shown by the negligible effect on the latter of cooling to -150°C; this contrasts strongly with the behaviour of homogeneously broadened
stretching bands in most of the hydrogen-bonded systems [25]. This reasoning can also be extended to higher hydrates (up to n M 9). Further support for the assumption of the statistical character of the y1,3 bandwidth of the lower hydrates is offered by the spread of frequencies of the peaks in the spectra of the hydrated crystalline samples (Fig. 3). These peaks easily cover the widths of the water stretching bands of low hydrated films and micelles. However, this comparison is not meant to be an allusion to a crystalline mosaic structure of the films. Considering the details of the band contour, we note a shoulder at 3465cm-’ in the spectra of the monohydrates and the other low hydrated samples. It can be assigned to less strongly bonded hydroxyl groups. The small peak at 3600cn-’ may consequently be attributed to some nearly free OH groups. The inflection at about 3280cm-i is obviously created by Fermi resonance with 22~~as in the example of the y1,3 band of bulk water [22]. The view of disordered water binding, which also includes very weakly bonded OH groups, is in accord with a semiempirical MO calculation [31] on the model system consisting of a cluster of four phosphatidylcholine molecules (with curtailed hydrocarbon chains) with added water molecules, and with the results of the molecular
J.Grdadolnik etal./J. Mol.Struct. 322 (1994)93-103
3800 3600
3400 3200 3000 2800 2600 2400 2200 2000 Wavenumbers
1800 1600 1400
0.8A b ; 0.66 ; 0.4C e
I 3600
I
I
I
I
3200 2800 Wavenumbers
I
I 2400
Fig. 6. (A) Spectra of centrifuged DPPC liposomes at room temperature and -40°C in the regions between 3800 and 14OOcm-’ (RT spectrum displaced upwards). (B) v,,~ region of pure ice (water frozen to -40” between CaF2 plates).
mechanics refinement of the DMPC - 2H20 crystal structure [30]. The latter calculation revealed two possible, energetically similar, binding modes for the second water molecule; an extrapolation to more water molecules appears to be justified by the small differences in the bandwidths of the low hydrates. A rough estimate of the hydrogen bond strength of the first bound water molecules can be made by considering the frequency of the v],~ band. This is 20cm-’ lower than the one of bulk water
(3400cm-‘); the enthalpy of the most likely water-water hydrogen bonds amounts to about 4 kcalmol-’ [32,33]. Thus the majority of the binding sites in phosphatidylcholines would offer bonding energies for the first bound molecules in the range 4-5 kcalmoi-‘; the width of the v],~ band can accommodate both stronger and weaker hydrogen bonding. The estimate is valid within the limits of the general relation between the low frequency shifts of the X-H stretchings due to hydrogen bonding, and its strength [34]. The
J. Grdadolnik et al.lJ. Mol. Struct. 322 (1994) 93-103
estimate of 5 kcalmol-’ can be compared with the value for the DMPC - 2HzO crystal [30] which is markedly higher (7.8 kcal mol-‘). This somewhat exceeds the energy of the hydrogen bonds in ice (7.4 kcal mol-‘, ref. 35). Note, however, that the frequency of the central peak in the spectrum of ice is at 3200cn-’ which is considerably lower than the peak frequencies of the water in the low hydrates of phosphatidylcholines. The present estimate of the most likely bonding energy for water in the low hydrates is quite close to that deduced from dielectric relaxation studies [9]. At any rate, these hydrogen bond energies are much inferior to the theoretically calculated interaction energies of water with dimethyl phosphate, which is often taken as a model for the phosphate group in phospholipids. The theoretically computed interaction energies are in the range 20-30 kcal mol-’ , depending on the level of theory [36-381. However, these energies were obtained with optimized geometries, neglecting the effect of the neighbourhood of the positively charged trimethylammonium group of the choline chain and other possible medium effects. With increasing hydration the availability of various binding sites for water also increases, causing further band broadening. Changes in the slope of the plot relating the half-bandwidths with the level of hydration (Fig. 2) are probably making the transition from a more frequent water-tophosphate bonding to increasing water-to-water bonding. An intermediate state may be imagined in which water molecules bind to those in direct contact with phosphate groups. These water molecules are expected to be more polarized, thus warranting somewhat stronger hydrogen bonding to the next incoming molecules, but this effect decays as the thickness of the water layer increases. Beyond n = 10 the shape of the v1,3band closely parallels that of bulk water except for the width, which at the maximal hydration (n = 30) exceeds by approximately 50 cm-’ the width of the band of bulk water (405cm-‘). The bandshape, including the depression at 3280 cm-‘, can be interpreted by analogy with bulk water [22]. The excess bandwidth may signify a wider spread in the variation of hydrogen bonds resulting from long-range effects of the polar heads. The small spectral
101
changes noted at hydration levels beyond n = 10 evolve smoothly at room temperature and even above the main transition (65°C for DPPC at this level of hydration [4,5]). Even more dramatic differences between the water spectrum of the films hydrated below n = 10 and at n > 10 are observed on cooling to subzero temperatures. At the higher level of hydration, cooling elicits spectral changes that are indicative of freezing to amorphous ice. Formation of amorphous ice in DPPC multibilayers has been observed previously [17]; high pressure forms of crystalline ice were obtained by subjecting dispersions of mixed chain phosphatidylcholines to pressures exceeding 21 kbar [39]. The formation of amorphous ice at relatively high temperatures and the excess width of the ~r,~ band of water in the fully hydrated multibilayers are indicative of differences with respect to ordinary bulk water. The bandshapes of the 21r,~bands and the overall bandwidths of the micelles are slightly different from those of the films. The somewhat narrower Hz0 bands in the former may be taken as an indication of a lesser disorder in the organization of the headgroup-to-water interface than in the bilayers, which are expected to offer more binding possibilities for water molecules than the single spherical (or nearly so) interior surface of a reverse micelle. The most conspicuous difference between water in multibilayers and that in reverse micelles appears on cooling to subzero temperatures. The interpretation of the gradual disappearance with decreasing temperature of the depression in the ~1.3 band of micellar water and the concomitant broadening of the y band, which seem to be in a causal relation, is not possible at present because the mechanism of this broadening in pure water is not yet well understood. Further work on this and some other aspects of low temperature effects on water of hydration is ongoing; it will also include NMR spectroscopy. The presently investigated types of phospholipid assemblies are not the best models of biological membranes. However, the results demonstrate that interesting information about the organisation of water of hydration can be extracted from the infrared spectra. Obviously, there is ample scope for more detailed investigations, with inclusion of
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other spectroscopic techniques, and we are already pursuing several lines to complete the presented results.
5. Conclusions The width of the 13~ band of water hydrating phosphatidylcholine films and micelles up to n = 10 is explained in terms of a distribution of hydrogen bond strengths caused by multiple binding possibilities within the polar heads. The main support for this view is the lack of low temperature effects upon the band frequencies and shapes, which also demonstrate that at this level of hydration water does not freeze. Freezing phenomena in films in micelles are somewhat different. Minor differences between water in the two forms of aggregation appear in the trends of v1 s band broadening with increasing hydration, which also permit a differentiation between water molecules binding in the range from about Al= 1 to II = 6. However, no sharp limits in the sense of hydration shells are observed except for the freezing above n M 10 and its absence at lower hydration.
6. Acknowledgements This investigation was supported by the Ministry of Science and Technology of Slovenia. Thanks are due to Miss Silva Zagorc for assistance in the experimental work and to Mrs. Tatjana Karba for skilful typing.
7. References [1] G. Cevc, in E. Westhof (Ed.), Hydration of Biological Macromolecules, McMillan Press, New York, 1992, pp. 338-389. [2] H. Hauser and M.C. Phillips, Prog. Surf. Membr. Sci., 13 (1979) 297. [3] P. Walde, A.M. Giuliani, C.A. Boicelli and P.L. Luisi, Chem. Phys. Lipids, 53 (1990) 265. [4] M. Kodama, M. Kuwabara and S. Seki, Biochim. Biophys. Acta, 689 (1982) 567.
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and R. Perron, J. Colloid Interface Sci., 95 (1983) 483. WIP.H. Elworthy, J. Chem. Sot., (1961) 4897. [71 D.A. Wilkinson, H.J. Morowitz and J.H. Prestegard, Biophys. J., 20 (1970) 169. PI G. Finer and A. Darke, Chem. Phys. Lipids, 12 (1974) 1. [91 H. Enders and G. Nimtz, Ber. Bunsenges. Phys. Chem., 88 (1984) 512. DOI G. Nimtz, Physica Ser., T13 (1986) 172. Vll L.J. Lis, M. McAlister, N. Fuller, R.P. Rand and V.A. Parsegian, Biophys. J., 37 (1982) 657. WI K. Arnold, L. Pratsch and K. Gawrisch, Biochim. Biophys. Acta, 728 (1983) 121. [I31 G. Klose and F. Stelzner, Biochim. Biophys. Acta, 363 (1974) 1. 1141S. Kim, P.H. Wermer and J.R. Scherer, J. Phys. Chem., 96 (1992) 446. P51 D. Chapman, The Structure of Lipids, Methuen, London, 1965, pp. 53-132. E. Oamura, I. Umemura [I61 L. Ter-Minassian-Saraga, and T. Takenaka, Biochim. Biophys. Acta, 946 (1988) 417. 1171E. Okamura, J. Umemura and T. Takenaka, Vibr. Spectrosc., 2 (1991) 95. WI J. Grdadolnik and D. Hadii, Chem. Phys. Lipids, 63 (1993) 121. 1191J. Grdadolnik, J. KidriE and D. Hadii, Chem. Phys. Lipids, 59 (1991) 57. [201 M. Falk and T.A. Ford, Can. J. Chem., 44 (1966) 1699. PI J. Schiffer and D.F. Hornig, J. Chem. Phys., 49 (1968) 4150. P21 J.R. Scherer, in R.J.H. Clark and R.E. Hester (Eds.) Advances in IR and Raman Spectroscopy, Vol. 5, Heyden and Sons, 1978, pp. 149-216. v31 M.J. Janiak, D.M. Small and G.G. Shipley, J. Biol. Chem., 254 (1979) 6068. v41 M.G. Sceats and K. Belsley, Mol. Phys., 40 (1980) 1389. [25] D. Hadii and S. Bratos, in P. Schuster, G. Zundell and C. Sandorfy (Eds.), The Hydrogen Bond Vol. 2, NorthHolland, 1976, pp. 565-612. [26] E. Mayer, J. Phys. Chem., 89 (1985) 3474. [27] G. Nielson and S.A. Rice, J. Chem. Phys., 78 (1983) 4824. [28] H. Hauser, I. Pascher and S. Sundell, J. Mol. Biol., 137 (1980) 249. [29] R.H. Pearson and I. Pascher, Nature, 281 (1979) 499. [30] G. Vanderkooi, Biochemistry, 30 (1991) 10760. [31] J. Koller and D. Had&, in preparation. [32] D. Eisenberg and W. Kauzmann, The Structure and Properties of Water, Oxford University Press, 1969, pp. 177-179.