Langmuir 1990,6, 222-230
222
Interactions of Large Ions with Phospholipid hlonolayers D. Gorwyn and G. T. Barnes* Department of Chemistry, University of Queensland, Brisbane, Australia Received February 3, 1989. I n Final Form: July 13, 1989 Surface pressure-area isotherms of dimyristoylphosphatidylcholine,dipalmitoylphosphatidylcholine, dimyristoylphosphatidylethanolamine,and dipalmitoylphosphatidylethanolaminemonolayers have been measured at 25 O C on buffer solutions from pH 1.5 to 12.5, and the effects of phosphotungstate ions and of uranyl ions in the subphase have been determined. Phosphotungstate causes expansion of the monolayers at low pH values, indicating some penetration into the monolayer region and an associated increase in the bulk phase pH for protonation of the phosphate. Uranyl ions cause contraction of expanded monolayers at low pH, which is attributed to adsorption of the UOZ2+drawing neighboring phospholipid molecules together. Hydrolysis of the uranyl ions at higher pH values increases their size and reduces their charge, leading to decreased effects on the monolayers. It is concluded that the use of phosphotungstic acid or uranyl acetate as a “stain” for electron microscopy could lead to significant distortions in membrane or vesicle structure.
Introduction The electron-dense compounds uranyl acetate (UAc) and phosphotungstic acid (PTA) are used as “stains” in the preparation of biological samples and phospholipid vesicles’’2 for viewing in the electron microscope. In such situations, there is always the possibility that the stain might alter the sample and thus introduce artifacts into the image in the electron m i c r o ~ c o p e . ~This - ~ possibility has led us t o study the interactions between these ions and phospholipid monolayers, since the phospholipids are major components of animal cell membranes6-* and are frequently used in vesicles and since monolayers provide an experimentally convenient membrane mode1.’-12 The present study involves the measurement of SUJface pressure (n) as a function of monolayer area (A) and subphase pH for monolayers of four phospholipids: dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylethanolamine (DPPE), and dimyristoylphosphatidylethanolamine (DMPE). DPPC and DPPE were selected because of their biological importance: they are present to some extent in most animal cell membranes. The other two compounds, DMPC and DMPE, were included so that the effects of the lengths of the acyl chains could be observed.
Experimental Section Materials. The monolayer materials were dipalmitoyl-DLa-phosphatidylcholine (DPPC), from Sigma (purity 99%); di(1) Falls, A. H.; Davis, H. T.; Scriven, L. E.; Talmon, Y. Biochim. Biophys. Acta 1982,693,364. (2) Lukac, S.; Perovic, A. J . Colloid Interface Sci. 1985,103,586. (3) Dreher, K. D.; Schulman, J. H.; Anderson, 0. R.: Roels, 0. A. J. Ultrastructure Res. 1967,19,586. (4) Talmon. Y.J. Colloid Interface Sci. 1983.93. 366. ( 5 ) Kilpatrick, P. K.; Miller, W. G.; Talmon; Y.’J. Colloid Interface Sci. 1985,107,146. (6) Bretcher, M. Nature, new Biol. 1972,236,11. (7) Castleden, J. A. J.Pharm. Sci. 1969,58, 149. (8) van Deenen, L.L. M.; Houtsmuller, U. M. T.; de Haas, G. H.; Mulder, E. J. Pharm. Pharmacol. 1962,14,429. (9) Fendler, J. H. Membrane Mimetic Chemistry; Wiley: New York, 1982;Chapter 9. (10) Cadenhead, D. A. In Structure of Biological Membranes; Abrahamsson, S., Pascher, I., Eds.; Plenum: New York, 1976;pp 63-83. (11) Pethica, B.A.; Mingins, J.; Taylor, J. A. G. J. Colloid Interface Sci. 1976,55,2. (12) Phillips, M. C.; Chapman, D. Biochim. Biophys. Acta 1968, 163,301.
0743-7463/9Q/24Q6-0222$02.50/0
palmitoyl-DL-a-phosphatidylethanolamine (DPPE), from Sigma (purity 98%);L-&y-dimyristoyl-a-cephalin(DMPE),from Fluka (puriss. grade); and P,y-dimyristoyl-L-a-lecithin (DMPC),from Calbiochem (A grade). They were used without further purifi-
cation. For spreading, the lecithins (DPPC and DMPC) were dissolved in a mixture of n-hexane (90%) and ethanol and the cephalins (DPPE and DMPE) in a mixture of benzene (70%) and methanol (30%). The solventa were all from Merck (Uvasolspectroscopicgrade) and were used without further purification. Subphase solutions were all prepared with triply distilled rain water. Solutes were sodium chloride from BDH (Analar, 99.9%, roasted for 6 h at 500 “C); uranyl acetate (UAc) from Ajax Chemicals (AR grade); phosphotungstic acid, sodium salt (PTA)from Sigma;tris(hydroxymethy1)aminomethane ( G R Tris buffer) from Merck; hydrochloric acid 1 mol dm-3 standard solutions from May and Baker; and sodium hydroxide from Ajax Chemicals (AR grade, recrystallized from distilled water). The subphase solutions were all prepared to contain 0.1 mol dm-3 NaCl and 0.01 mol dm-3 Tris buffer, with additions of HC1 or NaOH to yield the required pH. One series of solutions also contained mol dm-3 PTA, and another series contained mol dm-3 UAc. Apparatus and Procedures. The surface film balance consisted of a PTFE trough mounted on a thermostated aluminium base and fitted with PTFE barriers arranged for symmetrical compression about the Wilhelmy plate. Surface pressures were measured with a Wilhelmy plate of roughened mica suspended from a strain gauge (Shinkoh Communication Co.). Monolayers were spread from an Agla micrometer syringe. Compressions were commenced about 3 min after spreading and proceeded in a stepwise manner with the surface pressure being allowed to come to a steady value between each step. The surface pressure-area isotherms presented here are each the average of three experimental runs. The major source of error lies in the areas, and from the reproducibility of the areas from the three different spreadings of each substance on each subphase we estimate that the average areas are accurate to iO.001 nm2 molecule-’. The temperature of the subphase was maintained at 298.0 f 0.5 K throughout.
Results Phospholipid Monolayers on Buffer. The surface pressure-area (II-a)isotherms of the four phospholip(13) Munden, J. W.;Swarbrick, J. J . Colloid Interface Sci. 1973,
42, 657.
(14) Cadenhead, D. A.; Kellner, B. M. J. J . Colloid Interface Sci. 1974,49,143.
0 1990 American Chemical Society
Langmuir, Vol. 6, No. 1, 1990 223
Ion Interactions with Phospholipid Monolayers DMPC I buffer
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ids on buffer with sodium chloride are shown in Figure
, 1.
There are numerous reports on the II-A isotherms of phospholipids a t the a i r / ~ a t e r ' * ' ~ -and ~ ~ oil/water (15) Cadenhead, D. A,; Demchak,
2.Polym. 1967, 220, 59.
R. J.; Phillips, M. C. Kolloid 2.
(16) Cadenhead, D. A.; Phillips, M. C. In Molecular Association in
Biological and Related S v s t e m : Goddard. E. D.. Ed.: Advances in Chemistry84, American Chemical Society: W&hin&on, DC, 1968; p 131. (17) Paltauf, F.; Hauser, H.; Phillips, M. C. Biochim. Biophys. Acta 1971,249,539. (18) Phillips, M. C.; Ladbrooke, B. D.; Chapman, D. Biochim. Biophys. Acta 1970, 196,35. (19) Green, J. P.; Phillips, M. C.; Shipley, G. G. Biochim. Biophys. Acta 1973,330, 243. (20) McGreeor. M. A,: Barnes. G. T. J. Pharm. Sci. 1978. 67. 364. (21) Bell, GT hi.; Combs, L. L.'; Dunne, L. J. Chem. Reu. 1981, 81, 15. (22) Anderson, P. J.; Pethica, B. A. Proc. 2nd Internat. Cong. on Biochemical Problems of Lipids; Ghent, 1955; p 24. (23) Standish, M. M.; Pethica, B. A. Trans. Faraday SOC.1968,64, 1113. (24) Pethica, B. A. In Structural and Functional Aspects oflipopro-
teins in Liuing Systems; Tria, E., Scanu, A. M., Eds.; Academic Press: London, 1969; Chapter A2. (25) Hayashi, M.; Muramatsu, T.; Hara, I. Biochim. Biophys. Acta 1972, 255, 98. (26) Vilallonga, F. Biochim. Biophys. Acta 1968, 163, 290. (27) Shah, D. 0.; Schulman, J. H. J. Lipid Res. 1965,6, 341.
interface^.'',^^-^^ In earlier work, there were some difficulties due to impurities and to spreading problem^,'^"^^^^ but most recent work is consistent and in reasonable accord with the data presented here. Most published isotherms of DPPC and DMPE'49'5*25'26 show the condensed-to-expanded phase transition as a curve similar to those of b and c of Figure 1. However, there have been recent indications from the work of Kim and Canne113' and Middleton and P e t h i ~ a , performed ~ ~ ' ~ ~ with extraordinary attention to purity and technique, that this transition occurs at constant surface pressure and is therefore a simple first-order phase transition. These workers attribute the rising (28) Shah, D. 0.; Schulman, J. H. J.Lipid Res. 1967,8, 215. (29) Yue, B. Y.; Jackson, C. M.; Taylor, J. A. G.; Mingins, J.; Pethica, B. A. J.Chem. SOC., Faraday Trans. I 1976, 72,2685. (30) Bell, G. M.; Mingins, J.; Taylor, J. A. G. J. Chem. Soc., Faraday Trans. 2 1978, 74, 223. (31) Mingins, J.; Taylor, J. A. G.; Pethica, B. A.; Jackson, C. M.; Yue, B. Y. T. J. Chem. SOC.,Faraday Trans. I 1982, 78,323. (32) Kim, M. W.; Cannell, D. S. Phys. Reu. A 1976, 13, 411; 14, 1299. (33) Middleton, S. R.; Pethica, B. A. Faraday Symp. Chem. SOC. 1981, 16, 109. (34) Middleton, S. R.; Pethica, B. A. R o c . R. SOC.London A 1984, 396, 143.
224 Langmuir, Vol. 6, No. 1, 1990 Table I. Surface Pressures (mN m-') of the Le monolayer
subphase
1.5
3.0
DMPC
B B + PTA B + UAc B B + PTA B + UAc B B + PTA B + UAc B B + PTA B + UAc
>50 >50 14.3 7.6 31.9 50 14.3 10.8 34.9
DPPC DMPE DPPE
(I
