Phase transitions of phospholipids in monolayers and surface viscosity

Chemistry and Physics of Lipids 15 (1975) 209-215 © North-Holland Publishing Company

PHASE TRANSITIONS OF PHOSPHOLIPIDS IN MONOLAYERS AND SURFACE VISCOSITY Makoto HAYASHI *, Toshio MURAMATSU **, Ichiro HARA ** and Tsutomu SEIMIYA ***

* Laboratory of Chemistry, College ofArtsand Sciences, Chiba University, Yayoicho, Chiba. ** Laboratory of Chemistry, The Department o f General Education, Tokyo Medical and Dental University, Ichikawa. *** The Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Setagaya-ku, Tokyo, Japan.

Received May 12, 1975,

accepted August 5, 1975

The surface pressures and the surface viscosities of lecithin, cephalin and its analogs were measured at the air-water and the oil-water interfaces. It was found that the surface viscosity of the phospholipids used in this study was very high, and was comparable to those of some polymer films at the oil-water interface as well as at the air-water interface under the conditions where the monolayers were condensed. The plateaus indicating the phase transitions in monolayers were clearly observed on the pressure-area curves at the oil-water interface in all of the specimens studied. It was found that the phase transitions exactly corresponded to the abrupt increases in surface viscosity. From the results thus obtained, an intermolecular ionic linkage between neighboring molecules in the monolayers is discussed.

1. Introduction Phospholipids with choline or ethanolamine as polar groups form a typical zwitter ionic structure, which enables us to take a specific configuration in membrane state into account. For this purpose, synthesized phospholipids consisting of various polar groups are conveniently available. Dipalmitoyl phosphatidyl ethanolamine forms a stable and condensed monolayer on water surface at room temperature, and it gives a film of an extremely rigid nature, which is observable from its high surface viscosity. On the contrary, dipalmitoyl phosphatidyl choline gives an expanded mobile film under the same condition, and a low surface viscosity is observed at the same surface pressures [1,2]. From this viewpoint, it is likely that these two groups of phospholipids are basically different from each other in the monolayer properties at this temperature. However, phosphatidyl choline behaves in an almost identical way to phosphatidyl ethanolamine in the monolayer state, when it is brought to a sufficiently low temperature [2] or when the acyl chains are elongated. For 209

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M. Itayashi et al., Phase transitions in monolayers

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instance, distearoyl phosphatidyl choline gives a condensed monolayer on water surface even at room temperature [3], and its surface viscosity is comparable to that of a dipalmitoyl phosphatidyl ethanolamine film as seen in fig. 1. Most of the phospholipids clearly show the phase change at their respective transition temperatures found not only in the aqueous dispersion systems [4], but also in the monolayer state [2]. It is emphasized by some workers that attention must be paid to this kind of phase change in any discussion of membrane properties [5, 6]. The phase transitions which appear on the surface pressure-area curves have been simply considered to be a change from expanded to condensed monolayers in the same manner as in the cases of simple lipids as fatty acids. It is, however, clearly seen in the surface viscosity measurements that the condensed monolayer of dipalmitoyl phosphatidyl ethanolamine, for example, reveals a characteristic behavior similar to polymer films, from which we must take a special conformation into account for this condensed film. Therefore, the surface viscosities of the monolayers were measured with several synthetic phospholipids at the air-water and the dodecane-water interfaces as well as the phase changes in the monolayers. II.

Experimental

A circular glass dish 14 cm in diameter, which was treated with dimethyldichlorosilane for repelling water was used as a trough for spreading monolayers at the a i r -

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water interface. The surface pressure was measured by the hanging plate method with a glass plate which was roughened by carborundum for maintaining zero contact angle. For the dodecane-water interface, a platinum plate coated with carbon black was used for the surface pressure measurements, and a circular glass frame, the top face of which was treated with dimethyldichlorosilane was placed in a circular glass dish 13.5 cm in diameter for spreading the monolayers. The monolayers were spread at the dodecane-water interface which was formed at the top level of the inside frame in the dish. In both cases, the monolayers were made by intermittently spreading from a micro syringe one or two/al of spreading solutions; the surface pressures and the surface viscosities were recorded at each step, from which the relationships were plotted between these parameters. The surface viscosities were measured by the oscillation damping method making use of a cylindrical teflon pendulum 3 cm in diameter and 7 mm in height. The oscillation processes were electrically recorded by a special device, details of which will be reported elswhere. The whole apparatus was placed in a thermally insulated chamber, temperature of which was controlled as desired within -+ 0.1°C. The synthetic phospholipids used in this study are dipahnitoyl D, L-phosphatidyl ethanolamine, dipalmitoyl D,L-phosphatidyl n-propanolamine, dipalmitoyl D,L-phosphatidyl i-propanolamine and distearoyl D,L-phosphatidyl choline, the preparation of which was reported elsewhere [7]. Each of them was chromatographically purified before use and a single spot was confirmed on TLC plate for each specimen. Water was twice distilled over alkaline permanganate, and other inorganic reagents were passed through active charcoal columns for removing surface active impurities. Dodecane was distilled after washing with alkaline solutions and water, to remove acidic impurities.

III. Results Fig. 1 shows the surface viscosities measured at the air-water interface as a function of surface pressure. As seen in this figure, all of the phospholipids used show the extremely high surface viscosity after arriving at the close-packed areas, which in creases with increasing surface pressure. It is also seen in this figure that distearoyl phosphatidyl choline is distinguishable from the other two lipids contaning primary amine end groups. The relationships between surface pressure and area, and those between surface viscosity and surface pressure were separately measured at the dodecane-water interface for the same phospholipids as those used at the air-water interface; the results are shown in figs. 2 - 5 . It is commonly seen in figs. 2 - 4 that all of the curves clearly give the plateaus at the definite surface pressures, which show the phase changes from the expanded to the condensed states. Fig. 5 shows the surface viscosities as a function of surface pressure at the dodecane-water interface. At the surface pres-

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M. Hayashi et al., Phase transitions in monolayers

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sures below the respective plateaus, all of the monolayers exhibit the low surface viscosity in the order of 10 - 3 ~ 10 - 4 g/sec, though they cannot be quantitatively measured by the present device. However, when the monolayers are compressed up to the respective plateaus and beyond them, abrupt increases in surface viscosity occur, and they attain the extremely high values in the order of g/sec with further increase in pressure. This trend is commonly observed in all systems studied.

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Fig. 3. Pressure-area curves of dipalmitoyl D,L-phosphatidyl n-propanolamine at the dodecanewater interface. Subphase, see fig. 1. (1) 28.6°C and (2) 24.2 ° C.

M. Hayashi et aL, Phase transitions in monolayers

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M. Hayashi et al., Phase transitions in monolayers

IV. Discussion

It may be seen in fig. 1 that the distearoyl phosphatidyl choline monolayer behaves somewhat differently from the other two lipids, which suggests an enhanced effect of the polar groups on the rheological nature of the monolayers. At the oilwater interface, it has been inferred that the cohesive forces between hydrocarbon chains of amphipathic molecules should be mostly eliminated [8-10]. As predicted from this argument, the monolayers are expanded at the dodecane-water interface, and the phase transitions clearly appear between the expanded and the condensed monolayers in all cases studied; the surface viscosities are as low as those of fatty acid monolayers in the expanded and the transition regions. The surface viscosity of a close-packed monolayer of stearic acid on water surface is reported to be less than 10 -3 g/sec even at more than 20 dynes/cm (11). The monolayer of calcium stearate has been recognized to form a rigid film, and a polymeric lattice structure has been predicted [ 11 ]. Despite of that, the surface viscosity of it is in the order of 10 . 2 g/sec, which was also confirmed in our laboratory. Taking these facts into account, the surface viscosity obtained in the dipalmitoyl phosphatidyl ethanolamine monolayer, for example, must be considered to be enormously high, which leads to the conclusion that the monolayers in this study have a polymeric structure at the closely packed area at either interface. For this monolayer structure, it may be reasonably postulated that an intermolecular ionic linkage is formed between phosphate and amine, which is in agreement also with the molecular configuration in dipalmitoyl phosphatidyl ethanolamine monotayer deduced by Standish and Pethica in their surface potential measurements [12]. It is seen in figs. 2 and 3 that in the case of dipalmitoyl phosphatidyl n-propanolamine higher pressures are required before this structure is present than was found for dipalmitoyl phosphatidylethanolamine. This fact would support the above postulation, since it indicates a remarkable effect of the chain length of the polar moiety on the phase transition process. Incidentally, it is also worth noting that the lifting-off pressures in fig. 5 exactly correspond to the respective plateaus in the pressure-area curves (figs. 2 - 4 ) . From these facts, it may be further inferred that the monolayers do not form this structure in the expanded fihns, but it starts to be formed at the areas where the phase transitions occur. We conclude, therefore, that the plateaus on the pressure-area curves indicate the respective transitional processes from the monomer state to the polymeric lattice structure in the monolayers. The distearoyl phosphatidyl choline also shows the high surface viscosity at the close-packed areas at both interfaces, but the transition pressure at which the high surface viscosity appears is higher than in the dipalmitoyl phosphatidyl ethanolamine. This indicates that the former phospholipid requires the higher pressure for making the structure. This is also clear from the dipalmitoyl phosphatidyl choline monolayers shown in the same figure. Weconsider, therefore,that the choline group renders the formation of this intermolecular linkage more difficult, probably

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due to the stronger hydration of the choline groups as compared with the ethanolamine moiety.

Acknowledgements The authors wish to thank Dr. Y. Nagai of Tokyo Metropolitan Institute of Geron tology for supplying distearoyl lecithin synthesized in his laboratory, and also to Mr. N. Ito for his assistance. This work has been financially supported in part by a Grant No. 834031 of the Ministry of Education for Scientific Research of Japan.

References [1] [2] [3] [4] [5]

F. Vilallonga, Biochim. Biophys. Acta 163 (1968) 290 M. Hayashi, T. Muramatsu and I. ltara, Biochim. Biophys. Acta 291 (1973) 335 M.C. Phillips and D. Chapman, Biochim. Biophys. Acta 163 (1968) 301 B.D. Ladbrooke and D. Chapman, Chem. Phys. Lipids 3 (1969) 304 D. Chapman, N.F. Owens, M.C. Phillips and D.A. Walker, Biochim. Biophys. Acta 183 (1969) 458 I6] M.C. Phillips, Progress in Surface and Membrane Science, Vol. 5 ed. by D.A. Cadenhead, J.F. Danielli and M.D. Rosenberg, Academic Press, New York (1972) p. 139 [7] T. Muramatsu and 1. Hara, Bull. Soc. Chim. Biol. Fr. (1971) 3335 [8] J.T. Davies, Biochim. Biophys. Acta 11 (1953) 165 [9] J.T. Davies and E.K. Rideal, Interfacial Phenomena, Academic Press, New York (1963) pp. 158,228 [10] R.A. Demel and P. Joos, Chem. Phys. Lipids 2 (1968) 35 [111 D.W. Deamer and D.G. Cornwell, Biochim. Biophys. Acta 116 (1966) 555 [12] M.M. Standish and B.A. Pethica, Trans. f.'araday Soc. 64 (1968) 1113