22 Electron Microscope Studies of Monolayers of Lecithin H. E. RIES, JR., M. MATSUMOTO, N. UYEDA, and E. SUITO Downloaded via YORK UNIV on December 2, 2018 at 16:51:39 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
1
Institute for Chemical Research, Kyoto University, Kyoto, Japan
Electron micrographs of the monolayers of a synthetic lecithin,β,γ-dipalmitoyl-DL-α-glycerylphosphorylcholine,are markedly different from those of fatty acids and choles terol. However, the overall thinfilmproperties of these materials, as indicated by pressure-area isotherms, are quite similar. At low surface pressures, the island structures of lecithinfilmsare far less regular in contour than those of fatty acid or cholesterolfilms.The lecithinfilmsalso have an unusual microporosity. This perforated structure persists at intermediate and high pressures. After monolayer col lapse, the long,flat,ribbonlike structures formed by lecithin are less regular than the well defined collapse structures of fatty acids and cholesterol. Such similarities and differences are related to molecular geometry and the location and strength of polar groups. T e c i t h i n , one of the principal lipids in cell membranes, controls many important biological processes. Nevertheless, little is known about the structure of its films (1, 2, 3, 4). Electron micrographs now show remarkable properties for the thin film or monolayer of dipalmitoyl leci thin transferred quantitatively from a water surface. In many respects the water corresponds to the aqueous phases that bound cell membranes. Experimental Pressure-area isotherms were determined for synthetic lecithin and compared with those for related compounds. Electron micrographs were obtained for samples transferred from representative films before and after collapse. Basic apparatus and techniques have been described (1,5,6,7). Present address: Whitman Laboratory, Department of Biology, University of Chicago, Chicago, Ill. 60637. 1
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Materials. A l l materials had the highest purity available. The synthetic lecithin, yS,y-dipalmitoyl-DL-û:-glycerylpnosphorylcholine, was obtained from the Sigma Chemical Co. Chloroform, the principal volatile solvent used for spreading, was obtained from the Bojin Pharmaceutical Chemical Laboratories. Twice-distilled benzene was used for some of the experiments with related materials. Film-balance measurements with the solvents alone showed that remaining impurities were negligible. The water used as a substrate was twice distilled and had a p H slightly below 7.0 and a specific conductivity of about 0.5 /xmho. Because the p H was always below 7.0, its variation had a negligible effect on the lecithin isotherm. Thorough testing of the water surface on the film balance ensured freedom from significant capillary-active contamination. Surface-Pressure Measurements. Both the Wilhelmy and the L a n g muir-Adam-Harkins techniques have been used for pressure—area measurements ( 1 ). For most of the experiments a long, shallow trough coated with Teflon contained the twice-distilled water on which the monolayer was spread (6). The apparatus was housed i n a glass-walled cabinet that could be kept closed during pressure and area adjustments and during the transfer of samples to electron microscope screens. The entire apparatus was mounted on a concrete base that was essentially free of vibration as indicated by a long optical lever reflected from the water surface. Glass weighing pipets were used to spread a few drops of the dilute solutions of film-forming materials in volatile solvents. To minimize contamination, all parts of the system were thoroughly cleaned before each experiment; the water surface was swept many times with small Teflon coated barriers before the film was spread. During pressure-area experiments, the film area was reduced in small decrements and pressures were measured at 2-min intervals. The compression continued until collapse; this was indicated by a constant or falling pressure. Temperatures rarely varied more than 0.1 °C during individual experiments. Electron Microscopy. A modified Langmuir-Blodgett method was used to transfer monolayer samples to electron microscope screens that were sandwiched between Formvar and a glass plate (5, 6). A motor drive raised the plate slowly through the water-air interface, and a variable-speed motor drive moved the compressing barrier at a rate that maintained constant surface pressure during the transfer. Many samples were transferred at low surface pressures because the lipids in membranes are undoubtedly subjected to relatively small "horizontal" or surface pressures. Following transfer, film samples were placed in a high vacuum and were shadowcast with germanium at an angle of approximately 10°. The samples were then examined at a direct magnification of at least 5000 times in a modified J E M - T y p e 7 electron microscope. There are many difficulties in transferring monolayer samples for electron microscope studies—the evaporation or removal of the interposed water film between the monolayer and the Formvar, vibration, mechanical problems, and various other strains. Therefore, we do not claim a one-to-one correspondence between the state of a film on the water surface and the structures observed in the micrographs. Nevertheless, it is of interest to compare the sequence of changes that occur during
Goddard; Monolayers Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
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compression on the water surface with the in the microscope.
sequence
of changes observed
Results and Discussion Pressure-Area Isotherms. In spite of the apparently bulky structure of the lecithin molecule (Figure 1), the isotherms presented in Figure 2 indicate strong and well-behaved monolayers. The isotherm at 20.0°C has an initial pressure rise near 70 A / m o l e c u l e , a pronounced inflection at 50 A and 4 dynes/cm, and a steep high-pressure portion that extrapo2
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Goddard; Monolayers Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
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ίο Figure
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Lecithin
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40 50 60 70 80 AREA, SQUARE ANGSTROMS PER MOLECULE
30
Pressure-area
isotherms for a synthetic temperatures (°C)
lecithin
at several
lates to 39.5 A /molecule. The collapse pressure is relatively high, close to 40 dynes/cm, and the compressibility, 0.0060, is relatively low for such a complex structure. These data support the approximate orientation shown in Figure 1 although details are not yet established (2, 3, 4). The effects of increasing temperature are demonstrated in Figure 2 and Table I. The extrapolated area increases linearly with the tempera ture. Moreover, the inflection-point pressure at 50 A also increases linearly with increasing temperature. A marked reorganization or repack2
2
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MONOLAYERS
ing of the molecules, intra- and/or intermolecular, evidently takes place at 50 A . Staggered packing that involves a vertical shift of the lecithin molecules might account for such a phase change. This vertical shift could greatly reduce the effect of repulsive forces between the large polar groups. As expected, the phase change or inflection-point plateau is less pronounced at higher temperatures. Also, as might be anticipated if collapse is a mechanical phenomenon, the collapse pressures are essentially the same at the four temperatures studied. Compressibility values for the films in the upper pressure region are also remarkably similar. The isotherm for cholesterol ( I ) is surprisingly similar to that for lecithin ( Table II ) ; both are important components of many cell mem branes. The extrapolated area of 39.0 A /molecule approximates that of a double-chain system, and the cohesive and adhesive forces give a collapse pressure of 43 dynes/cm, close to the 40 dynes/cm for lecithin. In Table II cholesterol is also compared with stearic acid, the classic compound in monolayer research. Collapse pressures and compressibili ties are similar. The rigid packing of the complex cholesterol molecules is clearly demonstrated by its monolayer compressibility—0.0012, the smallest value in these studies. 2
2
Table I. Temperature, °C
20.0 24.6 26.9 27.6
Monolayer Properties of a Synthetic Lecithin Inflection Point, dynes/cm
Area, A /molecule 2
0
b
39.5 45.0 47.5 48.5
4.0 12.5 16.5 17.5
° Extrapolated area at zero pressure. Effectively at 50 A /molecule. Compressibility is (α -α1)/α /1, where a and ai is a smaller area at pressure f\. b
Collapse Pressure, dynes/cm
(39) (40) (42) (41)
Cornpressibility, cm/dyne c
0.0060 0.0064 0.0061 0.0058
2
c
0
Table II.
Q
0
is the extrapolated area at zero pressure
Comparison of Monolayer Properties for Stearic A c i d , Cholesterol and Lecithin at 2 5 ° C Collapse Pressure, dynes/cm
Area, A /molecule 2
Stearic acid Cholesterol Lecithin
0
(41) (43) (40)
20.6 39.0 45.0
Extrapolated area at zero pressure. Compressibility is (α -α1)/α /1, where a and di is a smaller area at pressure fi.
Compressibility, cm/dyne b
0.0015 0.0012 0.0064
α
6
0
0
Q
is the extrapolated area at zero pressure
Goddard; Monolayers Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
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Electron Micrographs. Representative electron micrographs are shown i n Figure 3. Arrows indicate the direction of shadowcasting, and the magnification is shown by the 1μ scale. The blank sample of Figure 3A was obtained by raising the Formvarcovered screen through a clean water-air interface before the film was spread. Such micrographs establish both the flat smooth surface of the Formvar as well as the fine texture of the vapor-deposited germanium. A typical island or cluster structure for the lecithin film transferred
Figure 3. Electron micrographs of lecithin films transferred at various surface pressures. The scale shows 1μ, and the arrows indicate the direc tion of shadowcasting.
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at a low surface pressure (2 dynes/cm) is shown in Figure 3B. Monolayer thickness (ca. 40 A ) as observed in the electron microscope suggests an elongated molecular structure with extended polar groups ( Figure 1 ). Lecithin islands contain more small holes and have edge contours that are considerably more irregular than those for n-hexatriacontanoic acid (C-36) (5, 6) and cholesterol. A t 5 dynes/cm (Figure 3 C ) , the micrographs show large areas of a two-dimensional continuous phase with discontinuous so-called uncovered areas. Micro-islands and individual molecules beyond the limit of resolution of the microscope may be present in the so-called uncovered area. A t 15 dynes/cm ( 3 D ) , further compression increases the ratio of the covered to the uncovered area, but the small holes persist. A t 25 dynes/cm ( 3 E ) , much of the so-called uncovered area evidently disappears, and large areas are covered homogeneously. Following film collapse ( 3 F ) , long, flat, ribbonlike structures, apparently two molecules thick, appear. This collapsed material is less regular in structure than that formed in the collapse of C-36 acid films. There are three principal differences between the film structures observed for lecithin and those for the C-36 acid (as well as for cholesterol): (a) lecithin islands have many more holes and less regular edges; ( b ) the small holes in lecithin films persist at much higher surface pressures; (c) the collapse fragments formed by lecithin are less structured. Such differences are, of course, related to molecular geometry and polarity. The hydrocarbon chains of lecithin are much shorter than those of the C-36 acid; the forces of cohesion are thus smaller. The polar extremity of lecithin is larger and stronger (high dipole moment) than the carboxy group of the acid (or the hydroxy group of cholesterol), and may, therefore, interfere with close packing. Conclusion Combined film balance and electron microscope studies reveal some remarkable properties of lecithin monolayers. A microporosity of the film, which is observed at low surface pressures and persists into the intermediate and high pressure regions, might, if present in membrane structures, be related to the penetration of proteins and other materials into cell membranes. Films of related materials and their mixtures warrant detailed study. Acknowledgment W e thank Tohru Takenaka for helpful suggestions and the use of some of his film balance apparatus and Yoshio Saito and Tsunoru Yoshida for valuable assistance in the experimental work.
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Literature Cited 1. Gaines, G. L., Jr., "Insoluble Monolayers at Liquid-Gas Interfaces," Interscience Publishers, New York, 1966. 2. Cadenhead, D. Α., "Recent Progress in Surface Science," J. F. Danielli, A. C. Riddiford, M. D. Rosenberg, Eds., Vol. 3, p. 169, Academic Press, New York, 1970. 3. Shah, D. O., Schulman, J. H., J. Lipid Res. (1967) 8, 227. 4. Standish, M. M., Pethica, Β. Α., Trans. Faraday Soc. (1968) 64, 1113. 5. Ries, Η. Ε., Jr., Kimball, W. Α., "Proceedings of the Second International Congress on Surface Activity," Vol. 1, p. 75, Butterworths Scientific Publications, London, 1957. 6. Ries, Η. Ε., Jr., Walker, D. C., J. Colloid Sci. (1961) 16, 361. 7. Adamson, A. W., "Physical Chemistry of Surfaces," 2nd ed., Interscience, New York, 1967. RECEIVED September 23, 1974.
Goddard; Monolayers Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
