Interaction of uranyl ions with phospholipid and cholesterol monolayers

Interaction of Uranyl Ions with Phospholipid and Cholesterol Monolayers 1 D I N E S H O. S H A H 2

Exobiology Division, Ames Research Center, NASA, Moffett Field, California 94035 Received May 23, 1968 Surface pressures and potentials of egg and dipalmitoyl lecithins, dipalmitoyl phosphatidic acid, cholesterol, cholestanol, and eicosanyl trimethylammonium monolayers were measured on subsolutions of uranyl acetate and NaC1. Among these films only lecithin monolayers were influenced by the presence of uranyl acetate in the subsolution. It is evident from surface potential measurements that uranyl ions interact with phosphate groups in lecithin monolayers but not in phosphatidie acid monolayers. It is suggested that steric factors and ionic charges are involved in the interaction between phosphate groups and uranyl ions. The interaction of uranyl ions with the phosphate groups tends to solidify lecithin monolayers and to increase the surface potential. A comparison of surface pressure-area curves of dipalmitoyl lecithin and dipalmitoyl phosphatidic acid monolayers suggests that the trimethylammonium group causes a long-range repulsion up to a distance of 12 A in lecithin monolayers. Surface chemistry of various stains has been discussed in relation to electron microscopy of membranes. INTRODUCTION Uranyl acetate has been widely used as a stain in electron microscopy to preserve and to reveal the structure of biomembranes

(i, 2). It has been also known that uranyl ions significantly influence the electrophoretic mobility of cells (3) and phospholipid colloids (4). The monolayer approach was taken in our laboratory in order to understand the surface chemistry of staining for electron microscopy and the mode of action of h e a v y metal ions on membranes. Since phospholipids and cholesterol are the major lipid components in m a n y biomembranes, I have studied the interaction of uranyl acetate with monolayers of these compounds. Moreover, it has been reported previously t h a t the un1 P r e s e n t e d a t t h e 12th A n n u a l M e e t i n g of t h e

Biophysical Society, Pittsburgh, Pennsylvania, 1968. Present adress: Laboratory of Surface Chemistry, Lamont Geological Observatory, Columbia University, Palisades, New York 10964.

saturation of fatty acyl chains influences the following surface properties: ionic structure, binding with metal ions, effect of cholesterol, and enzymic hydrolysis of lecithin monolayers (5-7). Therefore, b o t h saturated and unsaturated lecithins have been used in the present studies. T h e effect of OsO4 on monolayers and bilayers of lecithin, cholesterol, and their mixtures has been reported previously (8, 9). MATERIALS AND METHODS Chromatographically pure L-a-dipalmitoyl lecithin was purchased from M a n n Research Laboratories, Inc. (New York, New York), and egg lecithin was supplied b y the Sylvana Chemical C o m p a n y (Orange, New Jersey). n-a-Dipalmitoyl phosphatidie acid was a gift from Dr. A. F. Rosenthal of the Long Island Jewish Hospital. These phospholipids gave single spots on a T L C plate with a chloroform-methanol-water 60: 35: 5 solvent system. High-purity cholesterol and cholestanol were supplied b y Applied Science Laboratories, Inc. (State

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URANYL

IONS

AND

PHOSPHOLIPID

AND

College, Pennsylvania), and Aldrich Chemical Co., Inc. (~iilwaukee, Wisconsin), respectively. Eieosanyl trimethylammonium was purchased from K & K Laboratories Inc. (Plain View, New York). All lipid solutions were prepared in spectroscopic grade ehloroform-methanol-hexane 1:1: 3. Inorganic chemicals of reagent grade and twice distilled water were used in all experiments. The fatty acid composition of the egg lecithin, which contains approximately equal amounts of saturated and unsaturated fatty acids, has been reported previously (10). The molecular weights of egg lecithin, dipalmitoyl lecithin, dipalmitoyl phosphatidie acid, cholesterol, eholestanol, and eieosanyl trimethylammonium used in the calculations were 796, 752, 649, 386.6, 388.6 and 340.5, respectively. The surface pressure was measured by a modified Wilhelmy plate method, and surface potential by a radioactive electrode as described previously (10). The surface measurements were taken on subsolutions of 1 X 10-3 M uranyl acetate, pH 4.8, and 2 X 10-s M NaC1, pH 6.0 at 25°C. It should be mentioned that a requirement for measuring surface tension by the Wilhelmy plate method is that the contact angle between the subsolution and platinum blade should be zero. This is usually accomplished by sandblasting the surface of the blade. However, it was found that after spreading positively charged monolayers, the contact angle changes presumably owing to ionic repulsion between the film and the positively charged surface of the platinum blade. This results in a tendency of the blade to jump out of the subsolution when the tensiometer pointer approaches the equilibrium mark. For this reason, it was difficult to measure surface pressures of eieosanyl trimethylammonium monolayers. Similar observations were also reported by Rogeness (11) for films of primary and secondary amines. The state of films was inferred from the mobility of sprinkled tale particles when a gentle stream of air was blown at the particles (12). RESULTS

Egg Lecithin and Cholesterol Monolayers. The surface pressure-area curves of egg

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FIG. 1. Surface pressure-area curves of egg lecithin and cholesterol monolayers on subsolutions of I X 10-3 M uranyl acetate, pH 4.8 (A), and 2 X 10.8 M NaC1, pH 6.0 (O), at 25°C. lecithin and cholesterol on subsolutions of uranyl acetate and NaC1 are shown in Fig. 1. Cholesterol monolayers are not influenced by uranyl acetate whereas egg lecithin monolayers exhibit small changes in the surface pressure-area curve. However, the effect of uranyl acetate on egg lecithin is shown more strikingly by surface potential-area curves (Fig. 2). The surface potential of egg lecithin monolayers at 65 A2/moleeule increases by 155 my in the presence of uranyl acetate, whereas the surface potential of cholesterol monolayers decreases by 20 my. This decrease is presumably due to physical adsorption of uranyl ions to cholesterol monolayers. Cholesterol monolayers are in the liquid state on subsolutions of NaC1 or uranyl acetate at all surface pressures. At high surface presures (>30 dynes/em) egg lecithin monolayers are in the liquid state on subsolutions of NaC1 but in the gel state in the presence of uranyl acetate.

Dipalmitoyl Lecithin and Phosphatidic Acid Monolayers. The surface pressure-area and surface potentiM-area curves of dipalmitoyl lecithin and dipalmitoyl phosphatidic acid monolayers are shown in Figs. 3 and 4. The changes in surface pressure-area curves

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212

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FIG. 2. Surface potential-area curves of egg lecithin and cholesterol monolayers on subsolutions of 1 X 10-3 M uranyl acetate, pH 4.8 (&), and 2 X 10-3 M NaC1, pH 6.0 (O), at 25°C. of dipalmitoyl lecithin b y the presence of uranyl acetate are similar to those occurring in egg lecithin monolayers. I n addition, the changes in surface potential indicate that uranyl ions interact with dipalmitoyl lecithin monolayers but not with phosphatidic acid monolayers (Fig. 4). Dipalmitoyl lecithin monolayers are in the gel state at high surface pressures (40 dynes/cm) on subsolutions of NaC1, but in the solid state above a surface pressure of 15 d y n e s / c m in the presence of uranyl acetate in the subsolution (arrows in Fig. 3 indicate solidification of films). Dipalmitoyl phosphatidic acid monolayers are in the solid state above a surface pressure of 0-2 dynes/ cm in the presence of NaC1 or uranyl acetate in the subsolution. I t should be emphasized that the presence of a trimethylammonium group considerably expands the surface pressure-area curve of lecithin as compared to that of phosphatidic acid (Fig. 3).

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FIG. 3. Surface pressure-area curves of dipalmitoyl lecithin and dipalmitoyl phosphatidie acid monolayers on subsolutions of 1 X 10-3 M uranyl acetate, pit 4.8 (A), and 2 X 10-3 M NaC1, pit 6.0 (©), at 25°C. Arrows at 45 and 54 A2/molecule indicate solidification of the films. 800

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Cholestanol and Eicosanyl Trimethylammonium Monolayers. Cholestanol mono-

FIG. 4. Surface potential-area curves of dipalmitoyl lecithin and dipalmitoyl phosphatidic acid monolayers on subsolutions of 1 X 10-3 M uranyl acetate, pH 4.8 (A), and 2 X 10-3 M NaC1, pit 6.0 (O), at 25°C.

layers were studied to determine the contribution of the double bond to the surface properties of cholesterol. The surface pressure-area curves of eholestanol on subsolutions of NaCI or uranyl acetate are

identical to those of cholesterol shown in Fig. 1. The surface potentials of cholestanol on subsolutions of NaC1 and uranyl acetate are shown in Fig. 5. The difference between

Journal of Colloid and Interface Science,

Vol. 29~No. 2, February 1969

URANYL IONS AND PHOSPHOLIPID AND CHOLESTEROL MONOLAYERS DISCUSSION

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Egg Lecithin and Cholesterol Monolayers. It has been shown (8) that Os04 increases the surface potential of cholesterol monolayers but not that of cholestanol monolayers, indicating that OsQ reacts with the double bond in cholesterol molecules. In contrast, uranyl acetate does not interact with cholesterol monolayers (Fig. 1, 2). A lowering of 15-20 my of the surface potential of cholesterol monolayers in the presence of uranyl acetate is presumably due to physical adsorption of uranyl ions. A similar decrease in the surface potential of cholestanol monolayers is in agreement with this explanation (Fig. 5). Previous studies from this laboratory (8) and the results reported here indicate that OsQ reacts with the double bond of the fatty aeyl chains and not with the polar group of egg lecithin, and thereby causes degradation of the monolayers. On the other hand, uranyl acetate reacts only with the phosphate group of lecithin and not with the fatty acy] chains. Moreover, nranyl acetate stabilizes lecithin monolayers by increasing the surface viscosity (or solidity) of the films. The changes in surface pressure-area curves of egg lecithin in the presence of uranyl acetate can be explained as follows. The binding (or interaction) of uranyl ions to phosphate groups in lecithin monolayers results in a net positive surface charge due to trimethy]ammoninm groups. This positively charged monolayer presumably changes the contact angle between the platinum blade and the subsolution resulting in an apparent shift in the surface tension, and hence in the surface pressure. An increase of 155 mv in the surface potential of egg lecithin monolayers at 65 A2/molecule in the presence of 10-~ M uranyl ions is considerably greater than the 75 my increase caused by 10-2 M AI+~+ ions (10). This indicates a strong interaction (or affinity) between uranyl ions and the phosphate groups in lecithin monolayers. This is also in agreement with the finding that at very low concentrations uranyl ions as compared to other divalent cations

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Fro. 5. Surface potential-area curves of cholestanol and eicosanyltrimethylammoniummonolayers on subsolutions of 1 X 10-~ M uranyl acetate, pit 4.8 (£~), and 2 X 10-3 M NaC1, pH 6.0 (O), at 25°C. the surface potentials of cholesterol and cholestanol is about 15 mv on subsolutions of N a G (Figs. 2 and 5). Since they both have the same molecular area, this difference indicates the contribution of the double bond to the surface potential of cholesterol. Both cholesterol and cholestanol monolayers are in the liquid state on subsolutions of ~xrac1 or uranyl acetate. A decrease of 10-15 my in the surface potentials of cholesterol and eholestanol by the presence of uranyl acetate is presumably due to physical adsorption of uranyl ions to the monolayers. In order to understand the effect of the trimethylammoninm group of lecithin, the monolayers of eieosanyl trimethylammonium were studied. As has been mentioned, it was not possible to measure surface pressurearea curves of eicosanyl trimethylammonium monolayers. It was inferred from the mobility of tale particles that these monolayers were in the liquid state on subsolutions of NaC1 or uranyl aeetate. The surface potentials of these monolayers on subsolutions of uranyl acetate or NaCI are shown in Fig. 5, which indicates that uranyl acetate does not interact with trimethylammonium groups in the monolayers.

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SHAH

cause a charge reversal of phospholipid micelles in aqueous dispersions (4).

Dipalmitoyl Lecithin and Phosphatidic Acid MonoIayers. On subsohitions of NaC1, dipalmitoyl phosphatidic acid forms solid condensed monolayers, whereas dipalmitoyl lecithin forms liquid expanded monolayers. Therefore, it is evident that in the phosphatidie acid monolayers cohesive forces between hydrocarbon chains are greater than the ionic repulsion between the phosphate groups. On the other hand, the presence of trimethylammonium groups considerably expands the lecithin monolayers because of ionic repulsion between the polar groups. If one assumes that the increase of surface pressure at 105 A2/molecule (Fig. 3) is due to a long-range ionic repulsion between the polar groups, then the intermolecular spacing at which the repulsive forces act in lecithin monolayers can be calculated as follows. If we consider the area/molecule as the area of a circle with a radius r, then the intermolecular spacing is 2r. By taking 105 A2/molecule as the area of the circle, the intermolecular spacing is found to be 11.6 A. This suggests that the trimethylammonium groups in lecithin monolayers would repel one another if their charge centers are closer than 12 A. Parsegian (13) has reported a distance of up to 25 A for the repulsion between the leaflets of egg lecithin in the liquid-crystalline state. The fact that uranyl acetate tends to solidify lecithin monolayers (Fig. 3) suggests that it may be able to form a two-dimensional polymeric lattice with phosphate groups in lecithin monolayers. Similar polymeric lattices of carboxyl groups and divalent metal ions in fatty acid monolayers have been reported by Deamer and Cornwell (14). The changes in surface pressure-area curve of lecithin in the presence of uranyl acetate are presumably due to changes in the contact angle and in the structure of monolayers. Figure 4 suggests that uranyl ions interact with the phosphate groups in lecithin monolayers but not in phosphatidic acid monolayers. This peculiar property of uranyl ions is not exhibited by other

divalent metal ions such as Ca ++ or Mg++, which increase the surface potential of phosphatidic acid monolayers by 100 my (6). Figure 6 represents schematically the influence of uranyl ions on dipalmitoyl lecithin and phosphatidic acid monolayers. At pH 4.8, the phosphate group of lecithin has one ionic oxygen whereas that of phosphatidic acid has two. It is presumably the sterie factors that prevent the interaction of uranyl ions with the divalent phosphate groups in phosphatidie acid monolayers.

Cholestanol and Eicosanyl Trimethylammonium Monolayers. A difference of 20 my between surface potentials of cholesterol and cholestanol monolayers on subsohitions of NaC1 (Figs. 2, 5) can be attributed to the presence of a double bond in cholesterol. Ig has been known that a double bond situated immediately above a polar group contributes as an induced dipole to the surface potential (10, 12, 15). Since the double bond in cholesterol molecules is not immediately adjacent to the polar group (--OH), its contribution to the surface potential is rather small. It is evident from Fig. 5 that uranyl acetate does not influence the surface potential of trimethylammonium groups. Therefore, the change in the surface potential of lecithin monolayers is due to an interaction of uranyl ions with the phosphate groups.

Surface Chemistry of Staining for Electron Microscopy. The results reported above indicate that the monolayer approach is useful in elucidating the mode of action of heavy metal ions on cell surfaces. Stoeckenius, Sehulman, and Prince (16) showed that uranyl and osmium ions associate with LECITHIN

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® Fro. 6. A schematic representation of the ionic structure of lecithin and phosphatidie acid monolayers and their interaction with uranyl ions. Uranyl ions (hatched circles) i n t e r a c t with leeithir~ but not with phosphatidic acid monolayers.

Journal of Colloid and Interface Science, Vol. 29, No. 2, February 1969

URANYL IONS AND PHOSPHOLIPID AND CHOLESTEROL MONOLAYERS the polar groups of phospholipids. Therefore, the dark bands in electron micrographs of myelin figures correspond to polar groups of phospholipids. A l t h o u g h OsO4 and K M n 0 4 react with double bonds in f a t t y acyl chains of phospholipids, the local concentration of b o u n d metal ions is p r e s u m a b l y too small to give contrasts in electron micrographs (17). A decrease in X - r a y diffraction spacing of phospholipids after I(l~InO4 or OsO4 treatment (18, 19) is presumably due to degradation of hydrocarbon chains. Among the stains we studied uranyl acetate seems to be the only stain which does not cause degradation of phospholipids and which stabilizes phospholipid films by increasing the surface viscosity of the films. A detailed discussion on the effect of various stains on the thickness of cell membrane has been given in an excellent review article by Elbers (20). ACKNOWLEDGMENT I wish to express my sincere gratitude to the late Professor Jack I-I. Schulman for his advice and encouragement in this investigation. This work was carried out at Columbia University, New York, and was supported by the National Science Foundation (Grant No. GB-5273). REFERENCES i. STOECK~;NIUS, W., In G. E. W. Wolstenholme and M. O'Cormor, eds., Ciba Foundation Symposium, "Principles of Bimolecular Organization," p. 418. Little, Boston, 1966. 2. K~tY, D. H., In "Techniques for Electron Microscopy," 2nd ed., p. 262. F. A. Davis Co., Philadelphia., Pennsylvania, 1965.

215

3. RIDEAL, E. K., AND ADAMS, D. IV[., Surface Phenomena Chem. Biol. 1958, 309. 4. TEUNISSEN, VON I-I. P., AND DE JONG, I~. G. BUNGENBERG,KoUoid Beih. 48, 33 (1938). 5. SHAH, D. 0., AND SCHUSMAN,J. H., J. Lipid Res. 8, 215 (1967). 6. SHAH, D. O., AND SCHULMAN,J. It., Y. Lipid Res. 8, 227 (1967). 7. SHAH, D. O., AND SCHULMAN,J. H., J. Colloid and Interface Sci. 26,107 (1967). 8. DREHER, K. D., SCHULMAN,J. H., ANDERSON, O. R., AN/) ROEI~S,O. A., J. Ultrastruct. Res. 19, 586 (1967). 9. ANDERSON,O. R., ROELS, O. A., DREHER, K. D., ANDSCHCLMAN,j . t-I., J. Ultrastruct. Res. 19, 600 (1967). 10. SHAH, D. O., AND SCHUL1VI&N,J. I~l., J. Lipid Res. 6, 341 (1965). 11. ROG~N~SS, G. A., J. Colloid and Interface Sci. 26, 131 (1968). 12. SHAH, D. O., AND SCHU~AN, J. H., Lipids 2, 21 (1967). 13. PARSEGIAN, V. A., J. Theoret. Biol. 16, 70 (1967). 14. DE~ER, D. W., AND CORNWELL,D. G., Biochim. Biophys. Acta 116, 555 (1966). 15. HUGHES, A., AND RIDEAL, E. K., Proc. Roy. Soc. (London) A140, 253 (1933). 16. STOECKENIVS, W., SCHVLMAN, J. tI., AND PRINCE, L. M., Kolloid-Z. 169, 170 (1960). 17. STOECKENIUS,W., In A. L. Houwink and B. J. Spit, eds., "The Proc. European Conf. Electron Microscopy," Vol. II, p. 716. 1960. 18. ROBERTSON,J. D., I n G. E. W. Wolstenholme and M. O'Connor, eds., Ciba Foundation Symposium, "Principles of Bimolecular Organization," p. 357. Little, Boston, 1966. 19. STOECKENIUS, W., J. Cellular Biol. 12, 221 (1962). 20. ELBE~S, P. F., Recent Progr. Surface Sci., p. 443 (1964).

Journal of Colloidand Inter]aveScience,Vol. 29, No. 2, l~ebruary1969