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Hydration of phosphatidylcholine reverse micelles multilayers an infrared spectroscopic study and. J. Grdadolnik a, J. Kidri6 a and D. Had~i b. aBoris Kidri?
Chemistry and Physics of Lipids, 59 (1991) 57-68 Elsevier Scientific Publishers Ireland Ltd.

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Hydration of phosphatidylcholine reverse micelles multilayers an infrared spectroscopic study

and

J. G r d a d o l n i k a, J. K i d r i 6 a a n d D . H a d ~ i b aBoris Kidri? Institute o f Chemistry, P.O. Box 30, 61115 Ljubljana and hLek - - Pharmaceutical and Chemical Works, Ljubljana (Yugoslavia)

(Received January 25th, 1991; revision received May 13th, 1991; accepted May 16th, 1991) Effects of incremental hydration from 0 to 10 water moleculesper lipid were observed on the indicating C=O and asOPO stretching bands in the spectra of chloroformdispersions and cast films of DMPC, DPPC and DSPC. Besidesthese usual indicators we introduced a new one, antisymmetric CH3 stretching of the quaternary ammonium, which yields very useful information. The results indicate that the first water molecules diminish the interaction between the phosphate and quaternary ammonium groups by binding to the phosphate oxygens. Concomitant conformational change of the choline chain is revealed by changes in C--N frequencies in the 904--930 cm-l absorption. No evidence is found for early water binding to carbonyl groups. In discussing these results use is made of ab-initio calculations of model systems and of NMR hydration studies of micelles. Keywords: phospholipids; hydration; FTIR spectra; indicator groups; polar head; conformation.

Introduction Many physiologically important processes evolve at the external membrane surface and most of them include at a certain stage the interaction of active molecules with groups constituting the polar heads of phospholipids. Water molecules covering the membrane surface become necessarily involved in such processes. For instance, adsorbing molecules are expected to either displace the bound water or become bonded through bridging water molecules. In either case the water molecules which are in intimate contact with the membrane surface are the most important ones and therefore a description at the molecular level of the interactions between polar head constituents and water molecules is the prerequisite for understanding other, even more complex interactions at the membrane surface. It may be expected that the most intimately bound water molecules are those which Correspondence to: D. Had~i, Boris Kidri6 Institute of Chemistry, P.O. Box 30, 61115 Ljubljana, Yugoslavia.

become first bonded on hydration of a dry phospholipid, hence the interest in the early stages of phospholipid hydration. By contrast with the thoroughly investigated structure and dynamics of the highly hydrated phases [1--3], there are some obscure spots and, indeed, controversies in the literature about functional groups to which the first waters may become bonded ([4 71 and references therein) and about the intimately connected question of consequences regarding the conformation of the polar head, the acylester interface and, eventually, the packing of phospholipid molecules. Whereas Levine and coworkers [4,5] interpret their Raman spectroscopic observation of the antisymmetric stretchings of the non-ester PO oxygens (asOPO-, for short) in solid DPPC near 1250 cm -1 on dehydration and heating cycles in terms of structural changes with exclusion of direct water bonding to the phosphate, Wong and Mantsch [6] argue on hand of pressure effects on the analogous infrared band that water molecules do bind first to the phosphate. Similarly, the behaviour of the com-

0009-3084/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

58 ponents of the complex carbonyl stretching band (sCO) between 1745 and 1700 cm -I are alternatively attributed to steric [7,8] and hydrogen bonding effects [6,9,10], or to conformational and packing effects arising from water binding elsewhere [7,8]. Obviously the clarification is needed because groups used as indicators for both the environmental and steric effects are the same ones that have to be used in investigating the interactions of other interesting molecules with the polar head constituents. Sources of controversies have to be sought in the sensitivity of the frequencies of the PO2- and the carbonyl groups to both stereoelectronic and environmental effects as well as in the problems of sample preparation [11--14]. An additional hurdle with the phosphate group arises from the lack of reference for the frequency of the unperturbed group. Ionic phosphate esters are either bound to a counterion or richly solvated, in phosphatidylcholines or ethanolamines, for instance, the phosphate lays close to quaternary ammonium groups [15--18] which lower the asOPOfrequency with respect to the (virtual) free one. The latter is accessible only in theoretical calculations, but the precision is sufficient only for relative comparisons (M. Hodo~ek and D. Had~i, unpublished). With hydration, the effect of the ammonium group may be either supplemented or (partially) replaced by water bonding. The way out of these quandaries requires the implementation of both vibrational and NMR spectroscopies to a reasonably wide choice of phospholipid samples, supplemented by studies of model systems. Theoretical calculations on model systems should be helpful in framing the experimental findings by calculated interaction energies. Particularly precious would be the adoption of additional indicator groups. We therefore endeavour to examine critically the spectroscopic properties of groups already used for monitoring environmental effects and to exploit so far unused groups, in order to construct a whole representation of the hydration process which will be in accord with NMR measurements as well as with the theoretical predictions and will also serve in studies of interactions between active molecules and models of cell membranes.

In this paper we report mainly on the infrared spectra of stepwise hydrated dispersions of DPPC in CDCI3 and planar multibilayers cast on CsJ plates. Less systematically, other phosphatidylcholines (DSPC, DMPC, DHPC) were investigated. Crystalline phases were also investigated, but will be only partially reported here. In terms of experimental model systems, we followed the hydration effects on tetramethylammonium dicetylphosphate (TMA. DCP). The ab-initio calculations on interacting model systems consisting of tetramethylammonium with water or dimethylphosphate (DMP) will be described elsewhere, but we shall make use of the main results in the discussions. Perhaps the most useful novelty is the use of the antisymmetric stretching frequencies of ammonium methyl groups (asNCH) as indicators of environmental changes in hydration. We shall not be dealing in this paper with the H20 stretching bands. This complex absorption certainly contains additional information on hydration and we are currently engaged in extracting this information. Materials and Methods

Dipalmitoyl L-cc-phosphatidylcholine (DPPC), dimyristoyl L-ot-phosphatidylcholine (DMPC) and distearoyl L-ot-phosphatidylcholine (DSPC) were commercial samples (Sigma) of +99% purity (TCL) and were used without additional purification. Phosphatidylcholines were dissolved in dry benzene (1% solutions) which was slowly distilled off at 80°C. Residues of the solvent were removed under vacuum (P 10-2 torr). Dried phosphatidylcholines did not show any water bands in the IR spectra nor IH signals of water protons (CDCI3 solution). Scepticism about the complete removal of water from phosphatidylcholines is often expressed in the literature; "dry" or "anhydrous" when used in this paper means that water has been removed to the extent of being imperceptible in infrared or NMR spectra. This is definitely < w o = 0.5. Solvent CDCl 3 used in preparing inverted micelles was dried over molecular sieves (4 A). Phosphatidylcholine solutions (60 mM) and cast films were prepared in a glove-box flushed with dry nitrogen (rel. humidity < 4%).

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measurement. The extent of hydration was checked by redissolving the films in dry CDCI3 and measuring the I H - N M R signal integrals. Hydration of micelles and films was eventually cross checked by the areas of the complex H 2 0 stretching absorption in the IR spectra. The preparation and recording of IR spectra of D P P C were done in triplicate, Moreover, the hydration of the other phosphatidylcholines was included in the N M R and IR checkings so that the error in w o is estimated to be < 15%. IR spectra were recorded on a Digilab FT 15-80

Water was added to micellar solutions in appropriate amounts with a 1-/zl Hamilton syringe. The solutions were sonicated in a bath sonicator for 10 min and incubated for 12 h before measuring. Layers were formed on CsJ plates by evaporation of solvent (CHCI3). Films were treated in vacuum to 40°C for 5 h to remove traces of chloroform. Hydration was achieved by exposing the layers to air of controlled humidity (bubbling through H20/H2SO 4 solutions of appropriate ratios). To achieve proper hydration, films were heated above Tc and cooled to 21°C before e

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