Lysolecithin-Cholesterol Interaction. A Spinaesonanee and Electron-Micrographic Study1 Can. J. Biochem. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/09/14 For personal use only.
A. D. PURDON, J. C. HSIA,L. PINTERIC, AND D, 0. TINKER" Depcsrtmertts oj' Br'c~chswistryarad Phsrrrnacology, Urtr'ver~ityof Toronto, T Q ~ Q Orttario ~ ~ o , M5S I A 8 AND
R. P. RAND Depar.tme/af cf Biological Scierlces, Brock University, St. Ccrtkarii~es,darttario L2S 3AI Received July 8, 1974 Purdon, A. D., Hsia, J. C., Pinteric, L., Tinker, D. 0.& Rand, R. P. (197%)LysolecithinCholesterol Interaction. A Spin-Resonance and Electron-Micrographic Study. Cam 9.Biochem. 53,196-206 Another publication (Rand, R. P., Pangborn, W., Purdon, A. D., and Tinker, D. 8,(1945) Can. 9. Biochem. 53, 189-195) has established that lysolecithia~and cholesterol interact to form an equirnolar complex. We have investigated this compIex using the techniques of electron spin resonance (e.s.r.1 and electron microscopy. By varying the cholesterol concentration with iysslecithin in both thin films and dispersions studied by these techniques, we have observed the interaction between lysolecithia~and equimolar complex below 50 rnol yGcholesterol, and between crystalline cholesterol and equirnolar complex above 50 rnol 4% cholesterol. We have observed an interesting alteration in morphology by electron n~icroscopy,and an isotropic to anisotropic spectral change using 3-doxylcholestane and 12-doxylskaric acid spin-labelled probes when the cholesterol concentration is increased from 20 to 33 mol $2. The equimolar complex is stable in the presence of crystalline cholesterol, and exhibits n s phase changes in the physiological temperature range. Implications for membrane structure are discussed. Purdon, A. D., Hsia, J. C., Pinteric, L., Tinker, D. 0.& Rand, R. P. (1975) LysolecithinCholesterol Interaction. A Spin-Resonance and Electron-Micrographac Study. Can. 9. Biochemr. 53, 196-206 Bans une autre publication (Wand, R. P., Pangborn, W., Purdon, A. B. et Tinker, 13. 8. (1975) Gra.9. Biochem. 53, 189-1951, nous avons dkmontrC que la 1ysolCcitkineet le cholest6rol rkagissent pour former un cornpielre Cquimolaire. Nous avom CtudiC ce complexe ii l'aide des techniques de rCsonance paran~agnCtiyueet de microscspie Clectronique. Era faisant varier la concentration du cholestkrol avec la lysolkcithine dans les films minces et les dispersions CtudiCs avec ces techniques, nous avons observC I'interaction entre la lysol&ithine et le cornplexe Cquimolaire au-dessous de 50 mol b& de choiestCro1 et entre le cholest6rol cristallin et ie comp l e x Cquimolaire au-dessus de 50 rnol L;; de cholestkrol. La microscopic Clectronique a perrnis &observer un changement intdressant dams la morghologie et, utilisant le 3-doxylcholestane et I'acide 1Zdoxylst6arique comme marqueurs de spin, nous avons constat6 un changement de spectre qui d'isotrope devient anisotrope quand la concentration du cholestCrol est augmentCe de 20 ii 33 mol 94. Le cornplexe Cquimolaire est stable en prCsence de cholestCroI cristallin et il we montre aucun changement de phase ii des tempCrature physiologiques. Nous discutons des implieations de ces d~nnkespour la structure rnembranaire. [Traduit par le journal]
water-soluble than lecithin, and has a measurable Introduction Lysolecithin ( s n - ~ - ~ ~ a c y ~ g ~ y c e ~ o ~ -critical 3 - ~ - pmicelle ~ O s S concentration of approximately phorylcholine) is a product of the hydrolysis of 0.001%' in water (1, 2)- A phase diagram for the lecithin (sn-l , ~ - ~ ~ ~ ~ ~ a c y ~ g l y c s r o ~ 1 system ~ 3 ~ 8 lysolecithin-water PhOSP~o~ has been published (3) rylcholine) catalyzed by the enzyme phospho- indicating the presence of concentration-dependlipase A?. Lysolecithin is considerably more ent hexagonal liquid crystalline (HI) and micellar phases above 30°C, while below this ISupported by the Medical Research Council of temperature a lamellar gel (microcrystalline) ~ a n a d athrough grants No. MA-4129, MA-2355, and phase is formed. Light-scattering studies have MT-23789 to J-C.H.9Lap., and D.Q.T.5 respectively. and shown that the molecular weight lysolecithin by a grant from the National Wesearcln Council of micelles in water is 93 008 (4). Electron-microCanada to R.P.R. seopic investigations have also confirmed the 2To whom correspondence should be addressed.
PURHPBN ET AL.: LYSBLECITHIN-CHOLESTEROL INTERACTION
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TABLE 1. Gas-liquid chromatographic analysis of existence of hexagonal and lamellar lysolecithinsubstrate and reaction products of phospholipase As water phases (5). hydrolysis of egg lecithin The capacity of lysolecithin to lyse cell membranes such as the erythrocyte membrane is Fatty-acid composition* presumably related to its water solubility, alOriginal though it has been shown that the lytic potential lecithin a'-Acids1 &Acids$ depends on more complex factors than water Fatty acid? solubility alone. Indeed, the highly water-soluble decanoyl lysoleci thin is not lytic to erythrocytes whereas lecithins with side chains of 8-12 carbon atoms are lytic (6). Theoretical considerations have suggested that lysolecithin may disrupt lipid bilayers by a wedge mechanism (7), and other studies have indicated that significant inter*Results given in mole percentage. actions occur between lecithin and lysolecithin tFatty acid nomenclature: first number, number of carbons; second number. number of double bonds. in vvitro (8). Proton magnztic-resonance studies of ref so lecithin fatty acids. lysolecithin interaction with erythrocyte ghosts 8Fatty acids refeased by phospholiparre A?. have shown a broadening of the (CM2), signal, but not the N(CH3)3signal from the membrane ing spin-labelled probes coupled with electron lipids (9); Collier and Chen (18) have suggested spin-resonance (e.s.r.) spectroscopy, and electhat lysolecithin-cholesterol interaction is an tron-micrographic techniques. important event in hernolysis s f erythrocytes. Materials and Methods Lucy and co-workers (11, 12) have suggested that lysolecithin is responsible for phase transiEgg-yolk lecithin was prepared by a method previously tions in membranes which play a role in cell described (18). Lysolecithin was obtained by the action of fusion, and recent direct evidence for lysolecithin- pure phospholipase A2 from Croralus arrox venom on an solution of egg lecithin (19). The lysolecithin was induced cell fusion has been presented (13, 14). ether purified by silicic-acid chromatography, the lysolecithin Thus lysolecithin interaction with phospholipids, being eluted with chloroform-methanol 1:9 (v/v). This cholesterol, and possibly membrane proteins material was dried and then twice dissolved and crystal( 1 9 , must all be considered when evaluating the lized from a minimal amount of warm ethanol. The material gave a single spot on thin-layer chromatography phenomena of membrane lysis and fusion. (Silica Gel G, using the solvent chloroform-methanolDervichian (16) has suggested that such inter- water 95:35:4 v/v). The fatty-acid composition of the actions are general phenomena, dependent on reaction products s f phospholipase A2 hydrolysis was the solubility parameters of the molecules in- analyzed by gas-liquid chromatography (28). In Table 1 are recorded the data from the gas-liquid volved; utilizing the hierarchy of increasing chromatographic analysis of the egg lecithin and its solubility in water of cholesterol, lecithin, and hydrolysis products. The data indicate that the lysolecithin Bysolecithin, he reasoned that an equimolar mix- was predominantly palmitoyllysolecithin with a significant ture of cholesterol and lysolecithin ought to have amount of the stearoyl species as well. The results are in a solubility intermediate between those of the keeping with the known positional specificity of the two pure components, and pointed out that such enzyme (19). Cholesterol was obtained from British Drug Houses a mixture has similar properties to Becithin in and recrystallized three times from methanol. The spin water. However, in the paper by Rand et a/. (17), labelled probe 3-(2'-N-oxyl-4,4'-dimethyloxazolidime> evidence has been given for the formation of a cholestane (hereafter referred to as 3-doxylcholestane) specific equimolar complex between lysolecithin was prepared by the method of Keana er a]. (21) while 12-(2'-N-oxyl-4,4'-dimethyloxazolidine jsearic acid spinand cho1esterol which forms a separate liquid labelled probe (hereafter referred to as 12-doxylstearis crystalline lamellar phase. It is this specificity of acid) was prepared by the method of Waggoner et al. (22). interaction, in contrast to the general consideraElectron spin-resonance (e.s.r.) spectra were recorded tions cited by Dervichian (I$), that we wish at 21 + 1 "C (unless otherwise noted) on a Varian E-6 X-band spectrometer. The magnetic field was calibrated to emphasize. with Fremys salt (a, 13.091) (23). Planar lipid films were In this study, the properties of the equimolar prepared by a method previously described (24). The complex and its interaction with excess lyso- spin label to lysolecithin molar ratio was always kept at lecithin and choBestero1 were investigated, utiliz- 1 :150. Spectra sf the films were recorded in three states of
198
CAW. J. BIOCHEM. V8L. 53. 8975
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TABLE 2. Hyperfine splitting constants for 3-doxylchoiestane in various media
Method
Matrix
ar I(G)
Single crystal (30) Multibilayer film (dry) (27) Multibilayer film (dry) (this study)
Cholesteryl chloride Equimolar dipalmitoyllecitkin-cholestersi Equimolar 1ysolecit.hin-cholesterol
5.8i-0.5 6.8k0.5 6.2L-8.5
~L(G>
36.8f 8.5 32.89 1.8 31,4_90.5
FIG.1. (a) 3-Doxylcholestane. T,, is essentially parallel to the molecular long axis. (b) 1ZBoxylstearic acid in the all tmns configuration. T,, is parallel to the molecular axis.
hydraQioan: (a), anhydrous (under vacuum); (6) exposed to water-saturated air (ZOOYO relative humidity); and (c), fully hydrated (bathed with 0.15 M NaC1 in 0.01 M phosphate buffer, pH 7.4). Methods for the electron-microscopic examination of lipid samples by the negatia~e-stainingtechnique have been described in previous publications from this laboratory (25, 26). The negative-staining reagent used was sodium phosphotungstate. Atzdysis of E.S.R. Spectra The terminology used is that of Boggs and Hsia (27). The a x i s systems for the two spin-labelled probes used have been defined by McConnell and co-workers (28,29) and are shown in Fig. 1. The hyperfine splitting tensor elements qj for the nitroxide radical have been deduced from the e.s.r. spectra of oriented single crystals of cholesteryl chloride, containing 3-doxylcholestane as T,,, 6 G, impurity (30), and have the values T,, TEz"-- 32 6 (1 G = 10-* V s The quantities all and al refer to the observed hyperfine splitting when the normal to the plane of a spin-labelled lipid film is parallel and perpendicular, respectively, to the laboratory magnetic field. Figure 2 shows the e.s.r. spectra of equirnolar lysolecithin-cholesterol films containing 3-doxylcholestane in the anhydrous and fully hydrated states. In the anhydrous state, a l lis 6.2 k 0.5 G, or essentially equal to T,,. In the perpendicular orientation, the spectrum is characteristic of the label randomly distributed in the plane defined by the a,, and T,, axes (24), with al = 31.4 i 0.5 G . These values are essentially the same as those found for cholesteryl chloride crystals, and equimolar dipalmitoyllecithin-ckolestero1 films (Table 2). Bt can be concluded that the 3-doxylcholestane probe Is uniformly oriented with the long axis perpendicular to the film, which therefore most probably consists sf an oriented multibilayer array. When the film is fullyhydrated, all is unchanged but a l
"
"
k2a,,+
FIG.2. E.S.W. spectra of equimolar lysolecithincholesterol films containing 3-doxylcholestane probe, recorded with the normal to the plane of the film respectively perpendicular (-) and parallel (- - -) t s the laboratory magnetic field. (a) The film is under vacuum (dry). (b) The film is hydrated with buffered saline, and there is a decrease in the a1 value from that observed with the dry film (32-20 G). Values of a1 1 were the same in both cases, that is, 6 G .
decreases to approximately 19 G, indicating that although the long axes of the probe m o l ~ u l e sremain perpendicular to the film, they are undergoing rapid (frequency > 73 MHz) rotation about the long axes (24). Motion of the 3-doxylcholestane probe, in addition to rotation about the long axis, was analyzed using the order parameter Sgof Seelig (31) which is defined by
If rotation about the long axis is rapid, and e2 is the angle between the y axis and the normal to the plane sf the bilayer, then
where the fences indicate statistical averaging. In addition to the order parameter, the peak-height ratios (ratios sf
PURDBN ET AL.: LYSOLECITWIN-CWOLBSTEROL INTERACTION
TABLE 3. 92.S.R.-spectral parameters for 3-doxylcholestane and 12-doxylstearic acid incorporated into lysolwithin-cholesterol films
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LysoBecithin :cholesterol ratio
Hydration state*
a11
QL
S8.r
8.r PC1
3-BoxylchoBestane 1:I
Dry 1 h . Water
6.60 6 -63 6.70
Dry 100% r.h. Water
6.41 6.95 7.21
Dry 1 h . Water Water
24.20 22.73 19.33 23.50
B ZDoxylstearic acid
1 :I
2:1
*Dry, film under vacuum; 100% relative humidity (r.A.), film equilibrated with water-saturated air; water, fiSm bathed in phosphate-saline buffer. ?These parameters are defined in the text. 13 is calculated from S; and represents an 'averagevindiation of an axis sf the doxy1 probe (the y-axis of doxylcholestane, the z-axis of doxytstearic acid) to the normal to the plane of the lipid film.
heights of M+,, M,, and ,M-, peaks in parallel spectrum, Fig. 2B) were also measured for the 3-doxylcholestane probe. These parameters have been found to depend on the fluidity, distribution sf orientations, and homogeneity of the ordered bilayer system (23). The order parameter S3 was also used to ehracterize the orientation of 12-doxylsteai-ic-acid probes in the bilayers, being related in this case to the angle o3 between the z-axis and the normal to the plane of the bilayer by
Motion ccnd Oriekzialion epf Spi~-L&r&el!ed Probes in Lipid Films 3-Dsxy%cholestanecannot be incorporated into pure dry lysolecithin films. Spectra obtained indicate spin-spin interaction, suggesting that the probe precipitates as a separate phase when the organic solvent is removed irt P~~UCIPO. Hydration of lysolecithin-3-doxylcholestanefilms, either by adding water-saturated air or buffer, resulted in an isotropic spectrum (all .v l a 1 2 16 6).On the other hand, inclusion of as little as 10 mol 76 cholesterol in a dry lysolecithin film containing 3-doxyllcholestane probe resulted in anisotropic spectra identical to that shown in Fig. 2a. Anisotropy was retained on hydration of films containing 33 moH ?{, cholesterol, either with buffer or with water-saturated air, indicating that in these films, both in the dry and hydrated states, 3-debxyleholestane probe was selectively
/17\
:I
FIG.3. E.S.R. spectra of equimolar lysslecithincholesterol films containing 12-doxylsteanc-acid probe and hydrated with buffered saline. Spectra were recorded with the normal to the plane of the film respectively parallel (-) and perpendicular (- - -) to the laboratory magnetic field.
incorporated into the lamellar phase formed by the equimolar complex of lysolecithin and cholesterol, and that the latter was oriented as a multibilayer system in the lipid films. 12-Boxylstearic acid was incorporated into lysolecithin films and gave isotropic spectra, regardless of the degree of hydration. On the other hand, in films containing as little as 10 mol yb cholesterol (dry), 12-doxylstearic acid gave anisotropic spectra. Hydrated films containing 33 mol U/, cholesterol or more also yielded anisotropic spectra with this probe. Figure 3 shows the spectra of 12-doxylstearic acid incorporated into hydrated equimolar lysolecithin-cholesterol films. In Table 3 are recorded representative e.s.r.-
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CAN. J.
Mole
W Chalesteml
BIOCHEM. VQL. 53.
8975
Mole % Cholesterol
Fro. 4, 3-Doxylcholestane spectral hyperfine splitting FIG,5. Peak-height ratios obtained from spectra of data from lysolecithin-chslesterol films of varying hydra- 3-doxylchslestane in hydrated lysslecithin films containindicates films were ing various amounts sf cholesterol. The decrease in both tion and cholesterol content. 'HsBV hydrated with buffered saline. Anisstropy occurs regard- ratios with increasing cholesterol concentration after less of degree of hydration a t 33 moll (i;; cholesterol, and 33 mol 5; cholesterol probably indicates a decrease in the above this concentration of cholesterol data from X20'-fluidity sf the bilayer. hydrated and 100% relative humidity-hydrated films are coincident. Films containing 20 mol 7; cholesterol exhibit anisotropic spectra when hydrated at 180% relative humidity, but isotropic spectra when further hydrated with buffered saline. If the buffered saline is again drained off such a film, an anisotropic spectrum recurs.
spectral parameters for the two spin-labelled probes incorporated into lysoIecithin~hoBestero1 films containing 33 and 50 moI 7 6 cholesterol. As previously mentioned, the cholestane is almost perfectly oriented perpendicular to the dry films, and immobilized (on the e.s.r. time scale). On hydration with water-saturated air, the Mole % Choiesterel probes acquire rotational freedom about the FIG. 6. 12-Doxylstearic acid spectral hyperfine splitting long axes with no change in orientation, while data from Bysolecithin-choleskrol films hydrated with on hydration with liquid buffer, a significant 100% relative humidity and with buffered saline. The change in the order parameter SQ indicates an data indicate that anisotropy in the hydrocarbon phase is increase in the freedom of motion of the long achieved regardless of hydration at 33 mol 7, cholesterol. axes about the perpendicular direction. Similar Films containing 20 mol 72,cholesterol exhibit anisoobservations aCplJ to the 12-doxylstearic-acid tropic spectra when hydrated at 100% relative humidity but isotropic spectra when further hydrated with buffered spectra. . the buffered saline is saline (points marked 'H20')If In Fig. 4 we have plotted the hyperfine splitting again drained off suck a film, an anisotropic spectrum constant all, (which is linearly related to S3) for R X U h S . 3-doxy%cholestaneprobe incorporated into films of varying cholesterol content. A value of 16 6 were recorded for films containing 20 mol yo indicates complete lack of orientation of the cholesterol. Indeed, suck films reversibly gave probe molecules. In Fig. 6, values of ail and 61.1 isotropic and anisotropic spectra when the e.s.r. for 12-doxylstearic-acid probe, incorporated into cell was alternately filled with and emptied of similar films, are receded. ~ e ~ a r d l e sofs the buffer. Films recorded at 1007c relative humiddegree of hydration, anisotropy of the hydro- ity showed that anisotropy was present in the carbon phase and ordering of the lamellar hydrocarbon phase when 10 mol yo cholesterol structure have occurred at 33 mol yocholesterol. was present (1%-doxylstearic-acid probe) alIn films hydrated with buffer, isotropic spectra though the spectra of 3-doxylcholestane probe
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PURDON ET AL.: LYSOLEC~~HIN-CHOLESTEROLINTERACTION
FIG.7. Electron micrographs of lysolecitkin-cholesterol dispersions in water. The mole percentage sf cholesterol in the dispersion is as indicated below. Markers indicate a length sf 5808 A (500 nm). A, 8896; B, 337,; C , 50%;; D, 80%.
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282
CAN. 5. BHOCHEM. VOL. 53, 1975
was isotropic. In Fig. 5, peak ratios have been plotted for 3-doxylchoiestaneprobe incsrporated into fully hydrated films of differing chslesterol content. This figure gives similar but more detailed information on the fluidity and orientation of the bilayers, as in the case of Fig. 4. While the increased ordering of the films between 20 and 33 moH y;, cholesterol seen in Fig. 4 is also evident in Fig. 5, in addition, a maximum in the Mole % Cholesterol peak ratios near 33 mol yoand a sharp decrease FIG. 8. Electron microscopy repeat spacings obtained abob7e80 msl 7, cholesterol reflect properties of from measuring lamellar spacing in projected microthe films which are not evident when ail or S3 graphs taken a t different cholesterol concentrations. alone are considered. Vertical bars indicate standard deviations. In order t s find whether any temperaturedependent phase transitions of the lysolecithinchslesterol complex occur in the accessible range, spectra were recorded for films containing 50 m01 yo cholesterol, hydrated with bufler, and containing 3-doxytcholestane or 12-doxylstearicacid probe. Values of call and a i from such spectra were measured as a function of temperature. There was a slight decrease in the anisotropy for both probes over the range 10-60 "C, but no pronounced phase transition was evident. Ultrastrt~ctureof Lipid Dispersions Mixtures of lysolecithin and cholesterol were prepared by evaporation of chloroform solutions of known concentration in YVC~CUO,with water added to give a lipid concentration of 1% (w/v), and dispersed by agitation on a mechanical mixer. After staining with phosghotungstate, the morphology was examined in the electron microscope. The existence of a lamellar phase was verified in mixtures containing 10-80 rnol yi cholesterol, but there was a definite change in morphology as the cholesterol concentration was varied. Figure '7 (A-D) demonstrates this convincingly. In pure lysolecithin dispersions (not shorn), the predominant structures observed were small spherical particles with apparently amorphous inn teriors. Lysotecitfain-skolesterol dispersions eontaining 10-20 mol% cholesterol exhibited similar structures, and in addition, structures resembling the rouleaux formed by erythrocytes, that is, linear stacks of flattened, hollow spheres forming lamellar structures (Fig. 7 4 . When 33 msl yo cholesterol was present, essentially the whole field consisted either of these rouleaux or what appeared to be large extensions of laterally fused rsu%aux,which gave the appearance of ran-
200 Mole % Cholesterol
FIG.9. Size variation of lipid particles in lysolecithincholesterol dispersions determined by taking the average diameter of a large number of particles at each cholestero1 concentration indicated. Vertical bars indicate standard
deviations.
domly oriented lamellar phase (Fig. 7B). When 5 0 mol% cholesterol was present, a few rouleaux were still evident but the predominant structures were spheres of varying sizes (Fig. 7C). In the presence of excess chofesterol(66-90 mol%) the only structures observed were large spheres showing lamellar fine structure at the edges, which indicates they are rnultilamellar liposomes whose structure resembles that of an onion (25). A large number of measurements were made on projected images sf the electron micrographs in order to obtain values of the repeat spacing in the lamellar phase at different chslesterol concentrations, Measurements were not performed on digpenions containing less than 33 mol cholesterol, as very little lamellar phase was evident below this concentration. The results are given in Fig. 8 ;the repeat spacing decreases from 72 to 60 A (6 mm) as the cholesterol content is increased from 33 to 66 mol &jl,. Figure 9 shows
PURDON ET AL. : LYSQLECITHIN-6:HOLESTEROL INTERACTION
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Discussion The results presented in this paper are to be discussed in the light of the finding, established by X-ray observations (171, that lysolecithin and cholesterol form an equimolar complex which exists as a stable, lamellar, liquid crystalline phase in the presence of excess lysolecithin or cholesterol. An exceedingly conclusion which - - significant can be drawn from the e.s.r.-spectroscopic studies presented here is that the spin-labelled probes partitition strongly into the lysolecithincholesterol lamellar phase in the presence of excess lysolecithin or cholesterol. Both probes give quite different spectra in the later two phases than in the first. For dry films, or for hydrated films containing more than 33 mol yc cholesterol, a single anisotropic spectrum was recorded with no evidence for two superimposed spectra as would be obtained if the probes were partitioned between two phases. Thus the e.s.r. spectra give information on only the properties of one of the phases in the system. This was an advantage in this study, but may be a complicating factor in studies with spin-labelled probes. For example, the spectra obtained in this study, for hydrated films containing 33-80 mol yG cholesterol, were characteristic of the spectrum of a spin label in an oriented, liquid crystalline phase, and without previous knowledge of the phase diagram could have been interpreted in terms of a single, liquid crystalline phzse of variable cholesterol content. It is possible that in hydrated films of low cholesterol content, the probes partition between the lysolecithin-cholesterol and the pure lysolecithin phases, since isotropic spectra were obtained. However, it is most probable that the effects of varying the cholesterol content of the film on the e.s.r. spectra seen in Figs. 4 4 arise from the interaction of the lamellar complex with its environment. As had been evident in X-ray diffraction studies, equimolar mixtures of lysolecithin and cholesterol have been found to form a lamellar phase, which also spontaneously yields an oriented rnultibilayer film when deposited onto the flat quartz surface of the e.s.r. cell in the dry or
FIG.10. Computer-simulated model of thin lipid film having SQ%of its area occupied by lysolecithirn-cholesterol lamellar complex (shaded areas). The model is a square-tikd floor (30 tiles by 30 tiks) randomly covered with shaded and white tiles of equal numbers (see Appendix).
hydrated state. The exceedingly regular orientation of the 3-doxylcholestane probe indicates that the cholesterol ring nucleus and the hydrocarbon region of the lamellar phase adjacent to the polar head groups are highly ordered and aligned perpendicularly to the plane of the bilayers. However, the spectrum of 12-doxylstearicacid probe indicates that the centre of the hydrocarbon region is much more disordered. Such a fluidity gradient is characteristic of liquid crystalline phases (32). In the presence of excess lysolecithin, hydrated films exhibit lesser or no orientation compared to the pure complex. This has been rationalized in terms of a 'surface' effect. For films containing 50 rnol 0/, cholesterol, the entire film area is composed of lamellar complex, whereas for films containing, say, 33 rnol yo ch~lesterol,half or less of the film is occupied by the complex. However, the complex must be considered to be randomly dispersed on the film as 'islands' in a lysolecithin 'sea'. The area:perirneter ratio of these islands varies with the total percentage area of the film that is covered with lamellar complex. In order to assess this variation, a simple model of the two-dimensional structure of the lipid films, as described in the appendix, was devised. Figure 10 shows the model of a lipid film in which half the area is covered in a random fashion with lamellar complex. In Fig. 11, the area:perimeter ratio is plotted for such random structures having various percentage coverage with lamellar complex. Above about 80% cover-
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VBL. 53,
1975
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large effect on the long-range microstructure, although the number of phases is not altered. The striking variation in lamellar repeat spacing seen in Fig. 8 does not confirm the X-ray studies. However, as has been pointed out previously (25, 33), the repeat spacings observed in the electron microscope are characteristic of the degree of hydration of the sample on the electron-microscope grid, and can be radically different from those existing in the sample from which the negatively stained specimen was pre0 3 pared. We believe that samples containing a high Per Cent Coverage of Film with Complex Phase proportion of lysolecithin are more hygroscopic, FIG.18. Area:perirneter ratios of shaded areas for and retain more water when placed in vacuum 'tile floors9 similar to that shown in Fig. 10 but with in the electron microscope than do samples with various percentage coverages with shaded 'tiles' reprelow lysolecithin content, hence the former exhibit senting lysolsithin-cholesterol lamelfar complex. larger repeat spacings in the lamellar phase.
age, a large increase in this ratio occurs, while below 88% coverage, this ratio is small, approaching a limiting value of 0.25 at low coverage values. In fully hydrated films of such low coverage, the edges of the lamellar areas are in contact with a surface-active, presumably partly micellar, lysolecithin phase. The complex may thus be continually dissolving and reforming at the edges, and the perpendicular orientation of the molecules in the lamellar phase may be perturbed. This effect could easily account for the dramatic difference between fully hydrated films containing 20 and 33 mol yo cholesterol. For films hydrated with water-saturated air, a higher proportion of lysolecithin phase is required to perturb the orientation of the lamellar complex; this may be due to a difference in the properties of the lysolecithin phase formed under these conditions. The same principle may be applied in modified form to the consideration of the morphology of lysofecithin-cholesterol dispersions observed with the electron microscope. At high concentrations of lysolecithin, the ordered multilamellar structure of the lysolecithin-cholesterol complex is disrupted by the surface-active lysolecithin solution. The bilayer structure of the complex, under these conditions, exists as small, single-bilayer, walled, hollow spherules, which aggregate to form the rouleau structures observed. These rouleaux exhibit lamellar-phase order on a short-range scale, as opposed to the long-range order found in the absence of lysslecithin. Thus variation of the composition of the system has a
Conclusions In this section, some biological implications of the observations, presented in this and another paper (17), are discussed. We have shown that lysolecithin and cholesterol form an equimolar complex which forms a stable lamellar phase over a wide temperature range, in the presence of a considerable excess sf pure lysolecithin phases or crystalline cholesterol. The structure and properties of the lysolecithincholesterol bilayers have been found, using spinlabelled probes, to be closely similar to those of lecithin-cholesterol bilayers (27, 34) Moreover, we have obtained data, to be presented elsewhere, that equimolar mixtures of lysolecithin, cholesterol, and free fatty acids form stable bilayers with identical structure to those formed by lysolecitbin and cholesterol, and preliminary evidence that lysophosphatidylethanolamine and cholesterol also form a lamellar phase. Thus, contrary to common belief, the formation of lysophosphatides by local or more extensive hydrolysis of phosphatides in a bilayer membrane is not disruptive of the bilayer structure, providing adequate cholesterol is present. This may be a hitherto unrecognized role of the large amounts of cholesterol present in certain biological membranes, that is, to provide a stabilizing factor against membrane lysis by local formation of lysolecithin. Cell fusion and cell lysis are two phenomena in which lysolecithin is thought to be involved. The two processes have been linked in two papers
PURDON ET AL.: LYSQLECITHIN-CHQL~TERQLINTERACTION
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by Lucy and co-workers (11, 12) in which they showed that the addition of lysolecithin to the medium led to fusion of red blood cells and heterokaryon formation from LS fibroblasts and hen erythrocytes. If, as prop~sedin these papers, local micellization by lysolecithin is involved in these processes, then the small areas of the membrane involved must have a low molar amount of cholesterol in order to allow the bilayer-micelle transition to occur. Kellaway and Saunders (35) have shown that the lytic activity of lysolecithin on erythrocytes is reduced by both progesterone and more effectively by cholesterol, and Collier and Chen (10) demonstrated that induced hypercholesterolemia results in an increased lysolecithin-hemolysis time for whole blood. The physical basis of these observations seems to have been clarified by the present work. A converse implication of this study is the ability of lysolecithin to prevent crystallization of any local accumulation of cholesterol in a membrane. Lysolecithin has been found to occur in significant amounts in tissues and subcellular organelles that are rich in cholesterol, for example, in bovine adrenal chromaffin granules (36) and in atherosclerotic plaques (37). These could be rationalized as homeostatic responses to cholesterol accumulation based on the ability of lysolecithin to maintain cholesterol in a liquid crystalline phase. Thus we feel that knowledge of lysolecithincholesterol interactions offers certain important refinements to present theories of membrane structure. The capable assistance of Mr. Michael Paul1 in preparing the figures is gratefully acknowledged. Facilities and equipment for electron microscopy were provided by Medical Science Building Services,University of Toronto, Toronto, Ont. 1. Robinson, N. & Saunders, L. (1958) J. Phurm. Pharmacol. 10,755-761 2. Hamori, E. & Michaels, A. M. (1971) Biochim. Biophys. Acta 231, 496-504 3. Reiss-Husson, F. (1967) J. Mol. Biol. 25, 363-382 4. Robinson, N. & Saunders, L. (1959) J. Pharm. Plmrmucol. Sirppl. I1, 115T- 119T 5. Junger, E., Hahn, M. H. & Reinauer, H. (1970) Biochim. Biophys. Actu 211, 381-388 6. Reman, F. C., Demel, R. A. & Be Gier, J. (1969) Cklcm. Phys. Lipids 3, 221-233
205
7. Haydon, D. A. & Taylor, J. (1963) J. Tlreor. Biol. 4, 281-296 8. Van Zutphen, H. & Van Deenen, L. L. M. (1976) Chem. Phys. Lipids 1, 389-391 9. Chapman, D., Kamat, V. B., De Gier, J. & Penkett, S. A. (1968) J. Mol. Biol. 31, 101-614 10. Collier, H. B. & Cken, H. L. (1950) Cast. J. Res. Sect. E 28, 289-297 11. Poole, A. R., Howell, J. 1. & Lucy, J. A. (1970) Nature 227, 810-814 12. Lucy, J. A. (1970) Naare 227, 815-817 13. Croce, C. M., Sawicki, W., Kritchevsky, D. & Koprowski, H. (1971) Exp. Cell Res. 67, 427-435 14. Gledhill, B. L., Sawicki, W., Croce, C. M. & Koprowski, H. (1972) Exp. Cell Res. 73, 33-40 15. Deamer, D. W. (1973) J. Biol. Chem. 248, 5477-5486 16. Dervichian, D. G. (1969) Mol. Cryst. 2, 55-62 17. Rand, R. P., Pangborn, W., Purdon, A. D. & Tinker, D. 0. (1975) Can. J. Biockem. 53, 189-195 18. Tinker, D. 0. & Saunders, L. (1968) Clrem. Plys. Lipids 2, 316-329 19. Wu, T.-W. & Tinker, D. 0. (1969) Biochemistry 8, 1558-1 568 20. Kuksis, A. (1966) Chromatogr. Rev. 8, 172-207 21. Keana, J. F. W., Keana, S. B. & Beatkam, D. (1967) J. Am. Cltem. Soc. 89, 3055-3056 22. Waggoner, A. S., Kingzett, T. S., Rottsckaefer, S., Griffith, 0.H. & Keith, A. D. (1969) Chem. Plrys. Lipids 3, 245-253 23. Faber, R. J. & Fraekel, G. K. (1967) J. Clrem. Phys. 47,2462-2476 24. Hsia, J. C., Sckneider, H. & Smith, I. C. P. (1971) Can. J. Biocltem. 49, 614-622 25. Tinker, D. 0. & Pinteric, L. (1971) Biochemistry 10, 860-865 26. Pinteric, L., Tinker, D. 0. & Wei, J. (1973) Biochim. Biophys. Actu 293, 630-638 27. Boggs, J. M. & Hsia, J. C. (1972) Biochim. Biophys. Acfa 290, 32-42 28. Hamilton, C. L. & McConnell, H. M. (1968) in Str~ictwalChemistry aitd Molecular Biology (Rich, A. & Davidson, N., eds), pp. 115-149, W. H. Freeman & Co., San Francisco, Calif. 29. McConnell, H. M. & McFarland, B. G. (1970) Q. Rev. Biophy~.3, 91-136 30. Libertini, L. J. & Griffith,0.H. (1970) J. Chem. Phys. 53, 1359-1367 31. Seelig, J. (1970) J. Am. Chem. Soc. 92, 3881-3887 32. McConnell, H. M. & McFarland, B. G. (1972) Ann. N. Y . Acad. Sci. 195, 207-217 33. Rand, R. P., Tinker, B. 0.& Fast, P. 6 . (1971) Chem. Phys. Lipids 6, 333-342 34. Lapper, R. D., Paterson, S. J. & Smith, I. C. P. (1972) Can. J. Biockem. 50, 969-981 35. Kellaway, I. W. & Saunders, L. (1969) J. Pharm. Pharmacol. 26, 189s-194s 36. Blasckko, H., Firemark, H., Smith, A. D. & Winkler, H. (1967) Bioclrem. J. 104, 545-549 37. Portman, 0.W. & Alexander, M. (1969) J. Lipid Res. 10, 158-165
206
CAN. 3. BIOCHEM. MOL. 53, 1975
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Appendix bTibe-FI~sr9 Model sf the $truelure of Thie Lipid Films The minimum stable area of lysolecithincholesterol lamellar phase is represented by a square 'tile9 of unit area, and the lipid film is considered as a 'floor' of area N which is to be covered with tiles of two kinds, namely, black, representing lysolecithin-cholesterol lamellar complex, and white, representing pure lysolecithin phase (if lysolecithin is in excess) or cholesterol (if cholesterol is in excess). The floor is first ruled off into M contiguous squares of unit area, and these squares are covered at
random with tiles randomly drawn from a pile containing N13 black tiles and Nw white tiles (MB Mw = N ) initially.
+
This process was simulated by digital computation wing a Monte Carlo technique. Figure 10 shows a drawing of a typical random matrix containing 900 elements (30 tile by 30 tile 'floor'), half of which are black. The perimeter to area ratio of the black areas (not including perimeters at the edges of the 'floor') was computed for various percentage coverages with black tiles, corresponding to various proportions of thin lipid films occupied by lysolecithin-cholesterol lamellar phase. These ratios are shown in Fig. 11.
A. D. PURDON, J. C. HSIA,L. PINTERIC, AND D, 0. TINKER" Depcsrtmertts oj' Br'c~chswistryarad Phsrrrnacology, Urtr'ver~ityof Toronto, T Q ~ Q Orttario ~ ~ o , M5S I A 8 AND
R. P. RAND Depar.tme/af cf Biological Scierlces, Brock University, St. Ccrtkarii~es,darttario L2S 3AI Received July 8, 1974 Purdon, A. D., Hsia, J. C., Pinteric, L., Tinker, D. 0.& Rand, R. P. (197%)LysolecithinCholesterol Interaction. A Spin-Resonance and Electron-Micrographic Study. Cam 9.Biochem. 53,196-206 Another publication (Rand, R. P., Pangborn, W., Purdon, A. D., and Tinker, D. 8,(1945) Can. 9. Biochem. 53, 189-195) has established that lysolecithia~and cholesterol interact to form an equirnolar complex. We have investigated this compIex using the techniques of electron spin resonance (e.s.r.1 and electron microscopy. By varying the cholesterol concentration with iysslecithin in both thin films and dispersions studied by these techniques, we have observed the interaction between lysolecithia~and equimolar complex below 50 rnol yGcholesterol, and between crystalline cholesterol and equirnolar complex above 50 rnol 4% cholesterol. We have observed an interesting alteration in morphology by electron n~icroscopy,and an isotropic to anisotropic spectral change using 3-doxylcholestane and 12-doxylskaric acid spin-labelled probes when the cholesterol concentration is increased from 20 to 33 mol $2. The equimolar complex is stable in the presence of crystalline cholesterol, and exhibits n s phase changes in the physiological temperature range. Implications for membrane structure are discussed. Purdon, A. D., Hsia, J. C., Pinteric, L., Tinker, D. 0.& Rand, R. P. (1975) LysolecithinCholesterol Interaction. A Spin-Resonance and Electron-Micrographac Study. Can. 9. Biochemr. 53, 196-206 Bans une autre publication (Wand, R. P., Pangborn, W., Purdon, A. B. et Tinker, 13. 8. (1975) Gra.9. Biochem. 53, 189-1951, nous avons dkmontrC que la 1ysolCcitkineet le cholest6rol rkagissent pour former un cornpielre Cquimolaire. Nous avom CtudiC ce complexe ii l'aide des techniques de rCsonance paran~agnCtiyueet de microscspie Clectronique. Era faisant varier la concentration du cholestkrol avec la lysolkcithine dans les films minces et les dispersions CtudiCs avec ces techniques, nous avons observC I'interaction entre la lysol&ithine et le cornplexe Cquimolaire au-dessous de 50 mol b& de choiestCro1 et entre le cholest6rol cristallin et ie comp l e x Cquimolaire au-dessus de 50 rnol L;; de cholestkrol. La microscopic Clectronique a perrnis &observer un changement intdressant dams la morghologie et, utilisant le 3-doxylcholestane et I'acide 1Zdoxylst6arique comme marqueurs de spin, nous avons constat6 un changement de spectre qui d'isotrope devient anisotrope quand la concentration du cholestCrol est augmentCe de 20 ii 33 mol 94. Le cornplexe Cquimolaire est stable en prCsence de cholestCroI cristallin et il we montre aucun changement de phase ii des tempCrature physiologiques. Nous discutons des implieations de ces d~nnkespour la structure rnembranaire. [Traduit par le journal]
water-soluble than lecithin, and has a measurable Introduction Lysolecithin ( s n - ~ - ~ ~ a c y ~ g ~ y c e ~ o ~ -critical 3 - ~ - pmicelle ~ O s S concentration of approximately phorylcholine) is a product of the hydrolysis of 0.001%' in water (1, 2)- A phase diagram for the lecithin (sn-l , ~ - ~ ~ ~ ~ ~ a c y ~ g l y c s r o ~ 1 system ~ 3 ~ 8 lysolecithin-water PhOSP~o~ has been published (3) rylcholine) catalyzed by the enzyme phospho- indicating the presence of concentration-dependlipase A?. Lysolecithin is considerably more ent hexagonal liquid crystalline (HI) and micellar phases above 30°C, while below this ISupported by the Medical Research Council of temperature a lamellar gel (microcrystalline) ~ a n a d athrough grants No. MA-4129, MA-2355, and phase is formed. Light-scattering studies have MT-23789 to J-C.H.9Lap., and D.Q.T.5 respectively. and shown that the molecular weight lysolecithin by a grant from the National Wesearcln Council of micelles in water is 93 008 (4). Electron-microCanada to R.P.R. seopic investigations have also confirmed the 2To whom correspondence should be addressed.
PURHPBN ET AL.: LYSBLECITHIN-CHOLESTEROL INTERACTION
197
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TABLE 1. Gas-liquid chromatographic analysis of existence of hexagonal and lamellar lysolecithinsubstrate and reaction products of phospholipase As water phases (5). hydrolysis of egg lecithin The capacity of lysolecithin to lyse cell membranes such as the erythrocyte membrane is Fatty-acid composition* presumably related to its water solubility, alOriginal though it has been shown that the lytic potential lecithin a'-Acids1 &Acids$ depends on more complex factors than water Fatty acid? solubility alone. Indeed, the highly water-soluble decanoyl lysoleci thin is not lytic to erythrocytes whereas lecithins with side chains of 8-12 carbon atoms are lytic (6). Theoretical considerations have suggested that lysolecithin may disrupt lipid bilayers by a wedge mechanism (7), and other studies have indicated that significant inter*Results given in mole percentage. actions occur between lecithin and lysolecithin tFatty acid nomenclature: first number, number of carbons; second number. number of double bonds. in vvitro (8). Proton magnztic-resonance studies of ref so lecithin fatty acids. lysolecithin interaction with erythrocyte ghosts 8Fatty acids refeased by phospholiparre A?. have shown a broadening of the (CM2), signal, but not the N(CH3)3signal from the membrane ing spin-labelled probes coupled with electron lipids (9); Collier and Chen (18) have suggested spin-resonance (e.s.r.) spectroscopy, and electhat lysolecithin-cholesterol interaction is an tron-micrographic techniques. important event in hernolysis s f erythrocytes. Materials and Methods Lucy and co-workers (11, 12) have suggested that lysolecithin is responsible for phase transiEgg-yolk lecithin was prepared by a method previously tions in membranes which play a role in cell described (18). Lysolecithin was obtained by the action of fusion, and recent direct evidence for lysolecithin- pure phospholipase A2 from Croralus arrox venom on an solution of egg lecithin (19). The lysolecithin was induced cell fusion has been presented (13, 14). ether purified by silicic-acid chromatography, the lysolecithin Thus lysolecithin interaction with phospholipids, being eluted with chloroform-methanol 1:9 (v/v). This cholesterol, and possibly membrane proteins material was dried and then twice dissolved and crystal( 1 9 , must all be considered when evaluating the lized from a minimal amount of warm ethanol. The material gave a single spot on thin-layer chromatography phenomena of membrane lysis and fusion. (Silica Gel G, using the solvent chloroform-methanolDervichian (16) has suggested that such inter- water 95:35:4 v/v). The fatty-acid composition of the actions are general phenomena, dependent on reaction products s f phospholipase A2 hydrolysis was the solubility parameters of the molecules in- analyzed by gas-liquid chromatography (28). In Table 1 are recorded the data from the gas-liquid volved; utilizing the hierarchy of increasing chromatographic analysis of the egg lecithin and its solubility in water of cholesterol, lecithin, and hydrolysis products. The data indicate that the lysolecithin Bysolecithin, he reasoned that an equimolar mix- was predominantly palmitoyllysolecithin with a significant ture of cholesterol and lysolecithin ought to have amount of the stearoyl species as well. The results are in a solubility intermediate between those of the keeping with the known positional specificity of the two pure components, and pointed out that such enzyme (19). Cholesterol was obtained from British Drug Houses a mixture has similar properties to Becithin in and recrystallized three times from methanol. The spin water. However, in the paper by Rand et a/. (17), labelled probe 3-(2'-N-oxyl-4,4'-dimethyloxazolidime> evidence has been given for the formation of a cholestane (hereafter referred to as 3-doxylcholestane) specific equimolar complex between lysolecithin was prepared by the method of Keana er a]. (21) while 12-(2'-N-oxyl-4,4'-dimethyloxazolidine jsearic acid spinand cho1esterol which forms a separate liquid labelled probe (hereafter referred to as 12-doxylstearis crystalline lamellar phase. It is this specificity of acid) was prepared by the method of Waggoner et al. (22). interaction, in contrast to the general consideraElectron spin-resonance (e.s.r.) spectra were recorded tions cited by Dervichian (I$), that we wish at 21 + 1 "C (unless otherwise noted) on a Varian E-6 X-band spectrometer. The magnetic field was calibrated to emphasize. with Fremys salt (a, 13.091) (23). Planar lipid films were In this study, the properties of the equimolar prepared by a method previously described (24). The complex and its interaction with excess lyso- spin label to lysolecithin molar ratio was always kept at lecithin and choBestero1 were investigated, utiliz- 1 :150. Spectra sf the films were recorded in three states of
198
CAW. J. BIOCHEM. V8L. 53. 8975
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TABLE 2. Hyperfine splitting constants for 3-doxylchoiestane in various media
Method
Matrix
ar I(G)
Single crystal (30) Multibilayer film (dry) (27) Multibilayer film (dry) (this study)
Cholesteryl chloride Equimolar dipalmitoyllecitkin-cholestersi Equimolar 1ysolecit.hin-cholesterol
5.8i-0.5 6.8k0.5 6.2L-8.5
~L(G>
36.8f 8.5 32.89 1.8 31,4_90.5
FIG.1. (a) 3-Doxylcholestane. T,, is essentially parallel to the molecular long axis. (b) 1ZBoxylstearic acid in the all tmns configuration. T,, is parallel to the molecular axis.
hydraQioan: (a), anhydrous (under vacuum); (6) exposed to water-saturated air (ZOOYO relative humidity); and (c), fully hydrated (bathed with 0.15 M NaC1 in 0.01 M phosphate buffer, pH 7.4). Methods for the electron-microscopic examination of lipid samples by the negatia~e-stainingtechnique have been described in previous publications from this laboratory (25, 26). The negative-staining reagent used was sodium phosphotungstate. Atzdysis of E.S.R. Spectra The terminology used is that of Boggs and Hsia (27). The a x i s systems for the two spin-labelled probes used have been defined by McConnell and co-workers (28,29) and are shown in Fig. 1. The hyperfine splitting tensor elements qj for the nitroxide radical have been deduced from the e.s.r. spectra of oriented single crystals of cholesteryl chloride, containing 3-doxylcholestane as T,,, 6 G, impurity (30), and have the values T,, TEz"-- 32 6 (1 G = 10-* V s The quantities all and al refer to the observed hyperfine splitting when the normal to the plane of a spin-labelled lipid film is parallel and perpendicular, respectively, to the laboratory magnetic field. Figure 2 shows the e.s.r. spectra of equirnolar lysolecithin-cholesterol films containing 3-doxylcholestane in the anhydrous and fully hydrated states. In the anhydrous state, a l lis 6.2 k 0.5 G, or essentially equal to T,,. In the perpendicular orientation, the spectrum is characteristic of the label randomly distributed in the plane defined by the a,, and T,, axes (24), with al = 31.4 i 0.5 G . These values are essentially the same as those found for cholesteryl chloride crystals, and equimolar dipalmitoyllecithin-ckolestero1 films (Table 2). Bt can be concluded that the 3-doxylcholestane probe Is uniformly oriented with the long axis perpendicular to the film, which therefore most probably consists sf an oriented multibilayer array. When the film is fullyhydrated, all is unchanged but a l
"
"
k2a,,+
FIG.2. E.S.W. spectra of equimolar lysolecithincholesterol films containing 3-doxylcholestane probe, recorded with the normal to the plane of the film respectively perpendicular (-) and parallel (- - -) t s the laboratory magnetic field. (a) The film is under vacuum (dry). (b) The film is hydrated with buffered saline, and there is a decrease in the a1 value from that observed with the dry film (32-20 G). Values of a1 1 were the same in both cases, that is, 6 G .
decreases to approximately 19 G, indicating that although the long axes of the probe m o l ~ u l e sremain perpendicular to the film, they are undergoing rapid (frequency > 73 MHz) rotation about the long axes (24). Motion of the 3-doxylcholestane probe, in addition to rotation about the long axis, was analyzed using the order parameter Sgof Seelig (31) which is defined by
If rotation about the long axis is rapid, and e2 is the angle between the y axis and the normal to the plane sf the bilayer, then
where the fences indicate statistical averaging. In addition to the order parameter, the peak-height ratios (ratios sf
PURDBN ET AL.: LYSOLECITWIN-CWOLBSTEROL INTERACTION
TABLE 3. 92.S.R.-spectral parameters for 3-doxylcholestane and 12-doxylstearic acid incorporated into lysolwithin-cholesterol films
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LysoBecithin :cholesterol ratio
Hydration state*
a11
QL
S8.r
8.r PC1
3-BoxylchoBestane 1:I
Dry 1 h . Water
6.60 6 -63 6.70
Dry 100% r.h. Water
6.41 6.95 7.21
Dry 1 h . Water Water
24.20 22.73 19.33 23.50
B ZDoxylstearic acid
1 :I
2:1
*Dry, film under vacuum; 100% relative humidity (r.A.), film equilibrated with water-saturated air; water, fiSm bathed in phosphate-saline buffer. ?These parameters are defined in the text. 13 is calculated from S; and represents an 'averagevindiation of an axis sf the doxy1 probe (the y-axis of doxylcholestane, the z-axis of doxytstearic acid) to the normal to the plane of the lipid film.
heights of M+,, M,, and ,M-, peaks in parallel spectrum, Fig. 2B) were also measured for the 3-doxylcholestane probe. These parameters have been found to depend on the fluidity, distribution sf orientations, and homogeneity of the ordered bilayer system (23). The order parameter S3 was also used to ehracterize the orientation of 12-doxylsteai-ic-acid probes in the bilayers, being related in this case to the angle o3 between the z-axis and the normal to the plane of the bilayer by
Motion ccnd Oriekzialion epf Spi~-L&r&el!ed Probes in Lipid Films 3-Dsxy%cholestanecannot be incorporated into pure dry lysolecithin films. Spectra obtained indicate spin-spin interaction, suggesting that the probe precipitates as a separate phase when the organic solvent is removed irt P~~UCIPO. Hydration of lysolecithin-3-doxylcholestanefilms, either by adding water-saturated air or buffer, resulted in an isotropic spectrum (all .v l a 1 2 16 6).On the other hand, inclusion of as little as 10 mol 76 cholesterol in a dry lysolecithin film containing 3-doxyllcholestane probe resulted in anisotropic spectra identical to that shown in Fig. 2a. Anisotropy was retained on hydration of films containing 33 moH ?{, cholesterol, either with buffer or with water-saturated air, indicating that in these films, both in the dry and hydrated states, 3-debxyleholestane probe was selectively
/17\
:I
FIG.3. E.S.R. spectra of equimolar lysslecithincholesterol films containing 12-doxylsteanc-acid probe and hydrated with buffered saline. Spectra were recorded with the normal to the plane of the film respectively parallel (-) and perpendicular (- - -) to the laboratory magnetic field.
incorporated into the lamellar phase formed by the equimolar complex of lysolecithin and cholesterol, and that the latter was oriented as a multibilayer system in the lipid films. 12-Boxylstearic acid was incorporated into lysolecithin films and gave isotropic spectra, regardless of the degree of hydration. On the other hand, in films containing as little as 10 mol yb cholesterol (dry), 12-doxylstearic acid gave anisotropic spectra. Hydrated films containing 33 mol U/, cholesterol or more also yielded anisotropic spectra with this probe. Figure 3 shows the spectra of 12-doxylstearic acid incorporated into hydrated equimolar lysolecithin-cholesterol films. In Table 3 are recorded representative e.s.r.-
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CAN. J.
Mole
W Chalesteml
BIOCHEM. VQL. 53.
8975
Mole % Cholesterol
Fro. 4, 3-Doxylcholestane spectral hyperfine splitting FIG,5. Peak-height ratios obtained from spectra of data from lysolecithin-chslesterol films of varying hydra- 3-doxylchslestane in hydrated lysslecithin films containindicates films were ing various amounts sf cholesterol. The decrease in both tion and cholesterol content. 'HsBV hydrated with buffered saline. Anisstropy occurs regard- ratios with increasing cholesterol concentration after less of degree of hydration a t 33 moll (i;; cholesterol, and 33 mol 5; cholesterol probably indicates a decrease in the above this concentration of cholesterol data from X20'-fluidity sf the bilayer. hydrated and 100% relative humidity-hydrated films are coincident. Films containing 20 mol 7; cholesterol exhibit anisotropic spectra when hydrated at 180% relative humidity, but isotropic spectra when further hydrated with buffered saline. If the buffered saline is again drained off such a film, an anisotropic spectrum recurs.
spectral parameters for the two spin-labelled probes incorporated into lysoIecithin~hoBestero1 films containing 33 and 50 moI 7 6 cholesterol. As previously mentioned, the cholestane is almost perfectly oriented perpendicular to the dry films, and immobilized (on the e.s.r. time scale). On hydration with water-saturated air, the Mole % Choiesterel probes acquire rotational freedom about the FIG. 6. 12-Doxylstearic acid spectral hyperfine splitting long axes with no change in orientation, while data from Bysolecithin-choleskrol films hydrated with on hydration with liquid buffer, a significant 100% relative humidity and with buffered saline. The change in the order parameter SQ indicates an data indicate that anisotropy in the hydrocarbon phase is increase in the freedom of motion of the long achieved regardless of hydration at 33 mol 7, cholesterol. axes about the perpendicular direction. Similar Films containing 20 mol 72,cholesterol exhibit anisoobservations aCplJ to the 12-doxylstearic-acid tropic spectra when hydrated at 100% relative humidity but isotropic spectra when further hydrated with buffered spectra. . the buffered saline is saline (points marked 'H20')If In Fig. 4 we have plotted the hyperfine splitting again drained off suck a film, an anisotropic spectrum constant all, (which is linearly related to S3) for R X U h S . 3-doxy%cholestaneprobe incorporated into films of varying cholesterol content. A value of 16 6 were recorded for films containing 20 mol yo indicates complete lack of orientation of the cholesterol. Indeed, suck films reversibly gave probe molecules. In Fig. 6, values of ail and 61.1 isotropic and anisotropic spectra when the e.s.r. for 12-doxylstearic-acid probe, incorporated into cell was alternately filled with and emptied of similar films, are receded. ~ e ~ a r d l e sofs the buffer. Films recorded at 1007c relative humiddegree of hydration, anisotropy of the hydro- ity showed that anisotropy was present in the carbon phase and ordering of the lamellar hydrocarbon phase when 10 mol yo cholesterol structure have occurred at 33 mol yocholesterol. was present (1%-doxylstearic-acid probe) alIn films hydrated with buffer, isotropic spectra though the spectra of 3-doxylcholestane probe
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PURDON ET AL.: LYSOLEC~~HIN-CHOLESTEROLINTERACTION
FIG.7. Electron micrographs of lysolecitkin-cholesterol dispersions in water. The mole percentage sf cholesterol in the dispersion is as indicated below. Markers indicate a length sf 5808 A (500 nm). A, 8896; B, 337,; C , 50%;; D, 80%.
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was isotropic. In Fig. 5, peak ratios have been plotted for 3-doxylchoiestaneprobe incsrporated into fully hydrated films of differing chslesterol content. This figure gives similar but more detailed information on the fluidity and orientation of the bilayers, as in the case of Fig. 4. While the increased ordering of the films between 20 and 33 moH y;, cholesterol seen in Fig. 4 is also evident in Fig. 5, in addition, a maximum in the Mole % Cholesterol peak ratios near 33 mol yoand a sharp decrease FIG. 8. Electron microscopy repeat spacings obtained abob7e80 msl 7, cholesterol reflect properties of from measuring lamellar spacing in projected microthe films which are not evident when ail or S3 graphs taken a t different cholesterol concentrations. alone are considered. Vertical bars indicate standard deviations. In order t s find whether any temperaturedependent phase transitions of the lysolecithinchslesterol complex occur in the accessible range, spectra were recorded for films containing 50 m01 yo cholesterol, hydrated with bufler, and containing 3-doxytcholestane or 12-doxylstearicacid probe. Values of call and a i from such spectra were measured as a function of temperature. There was a slight decrease in the anisotropy for both probes over the range 10-60 "C, but no pronounced phase transition was evident. Ultrastrt~ctureof Lipid Dispersions Mixtures of lysolecithin and cholesterol were prepared by evaporation of chloroform solutions of known concentration in YVC~CUO,with water added to give a lipid concentration of 1% (w/v), and dispersed by agitation on a mechanical mixer. After staining with phosghotungstate, the morphology was examined in the electron microscope. The existence of a lamellar phase was verified in mixtures containing 10-80 rnol yi cholesterol, but there was a definite change in morphology as the cholesterol concentration was varied. Figure '7 (A-D) demonstrates this convincingly. In pure lysolecithin dispersions (not shorn), the predominant structures observed were small spherical particles with apparently amorphous inn teriors. Lysotecitfain-skolesterol dispersions eontaining 10-20 mol% cholesterol exhibited similar structures, and in addition, structures resembling the rouleaux formed by erythrocytes, that is, linear stacks of flattened, hollow spheres forming lamellar structures (Fig. 7 4 . When 33 msl yo cholesterol was present, essentially the whole field consisted either of these rouleaux or what appeared to be large extensions of laterally fused rsu%aux,which gave the appearance of ran-
200 Mole % Cholesterol
FIG.9. Size variation of lipid particles in lysolecithincholesterol dispersions determined by taking the average diameter of a large number of particles at each cholestero1 concentration indicated. Vertical bars indicate standard
deviations.
domly oriented lamellar phase (Fig. 7B). When 5 0 mol% cholesterol was present, a few rouleaux were still evident but the predominant structures were spheres of varying sizes (Fig. 7C). In the presence of excess chofesterol(66-90 mol%) the only structures observed were large spheres showing lamellar fine structure at the edges, which indicates they are rnultilamellar liposomes whose structure resembles that of an onion (25). A large number of measurements were made on projected images sf the electron micrographs in order to obtain values of the repeat spacing in the lamellar phase at different chslesterol concentrations, Measurements were not performed on digpenions containing less than 33 mol cholesterol, as very little lamellar phase was evident below this concentration. The results are given in Fig. 8 ;the repeat spacing decreases from 72 to 60 A (6 mm) as the cholesterol content is increased from 33 to 66 mol &jl,. Figure 9 shows
PURDON ET AL. : LYSQLECITHIN-6:HOLESTEROL INTERACTION
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Discussion The results presented in this paper are to be discussed in the light of the finding, established by X-ray observations (171, that lysolecithin and cholesterol form an equimolar complex which exists as a stable, lamellar, liquid crystalline phase in the presence of excess lysolecithin or cholesterol. An exceedingly conclusion which - - significant can be drawn from the e.s.r.-spectroscopic studies presented here is that the spin-labelled probes partitition strongly into the lysolecithincholesterol lamellar phase in the presence of excess lysolecithin or cholesterol. Both probes give quite different spectra in the later two phases than in the first. For dry films, or for hydrated films containing more than 33 mol yc cholesterol, a single anisotropic spectrum was recorded with no evidence for two superimposed spectra as would be obtained if the probes were partitioned between two phases. Thus the e.s.r. spectra give information on only the properties of one of the phases in the system. This was an advantage in this study, but may be a complicating factor in studies with spin-labelled probes. For example, the spectra obtained in this study, for hydrated films containing 33-80 mol yG cholesterol, were characteristic of the spectrum of a spin label in an oriented, liquid crystalline phase, and without previous knowledge of the phase diagram could have been interpreted in terms of a single, liquid crystalline phzse of variable cholesterol content. It is possible that in hydrated films of low cholesterol content, the probes partition between the lysolecithin-cholesterol and the pure lysolecithin phases, since isotropic spectra were obtained. However, it is most probable that the effects of varying the cholesterol content of the film on the e.s.r. spectra seen in Figs. 4 4 arise from the interaction of the lamellar complex with its environment. As had been evident in X-ray diffraction studies, equimolar mixtures of lysolecithin and cholesterol have been found to form a lamellar phase, which also spontaneously yields an oriented rnultibilayer film when deposited onto the flat quartz surface of the e.s.r. cell in the dry or
FIG.10. Computer-simulated model of thin lipid film having SQ%of its area occupied by lysolecithirn-cholesterol lamellar complex (shaded areas). The model is a square-tikd floor (30 tiles by 30 tiks) randomly covered with shaded and white tiles of equal numbers (see Appendix).
hydrated state. The exceedingly regular orientation of the 3-doxylcholestane probe indicates that the cholesterol ring nucleus and the hydrocarbon region of the lamellar phase adjacent to the polar head groups are highly ordered and aligned perpendicularly to the plane of the bilayers. However, the spectrum of 12-doxylstearicacid probe indicates that the centre of the hydrocarbon region is much more disordered. Such a fluidity gradient is characteristic of liquid crystalline phases (32). In the presence of excess lysolecithin, hydrated films exhibit lesser or no orientation compared to the pure complex. This has been rationalized in terms of a 'surface' effect. For films containing 50 rnol 0/, cholesterol, the entire film area is composed of lamellar complex, whereas for films containing, say, 33 rnol yo ch~lesterol,half or less of the film is occupied by the complex. However, the complex must be considered to be randomly dispersed on the film as 'islands' in a lysolecithin 'sea'. The area:perirneter ratio of these islands varies with the total percentage area of the film that is covered with lamellar complex. In order to assess this variation, a simple model of the two-dimensional structure of the lipid films, as described in the appendix, was devised. Figure 10 shows the model of a lipid film in which half the area is covered in a random fashion with lamellar complex. In Fig. 11, the area:perimeter ratio is plotted for such random structures having various percentage coverage with lamellar complex. Above about 80% cover-
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large effect on the long-range microstructure, although the number of phases is not altered. The striking variation in lamellar repeat spacing seen in Fig. 8 does not confirm the X-ray studies. However, as has been pointed out previously (25, 33), the repeat spacings observed in the electron microscope are characteristic of the degree of hydration of the sample on the electron-microscope grid, and can be radically different from those existing in the sample from which the negatively stained specimen was pre0 3 pared. We believe that samples containing a high Per Cent Coverage of Film with Complex Phase proportion of lysolecithin are more hygroscopic, FIG.18. Area:perirneter ratios of shaded areas for and retain more water when placed in vacuum 'tile floors9 similar to that shown in Fig. 10 but with in the electron microscope than do samples with various percentage coverages with shaded 'tiles' reprelow lysolecithin content, hence the former exhibit senting lysolsithin-cholesterol lamelfar complex. larger repeat spacings in the lamellar phase.
age, a large increase in this ratio occurs, while below 88% coverage, this ratio is small, approaching a limiting value of 0.25 at low coverage values. In fully hydrated films of such low coverage, the edges of the lamellar areas are in contact with a surface-active, presumably partly micellar, lysolecithin phase. The complex may thus be continually dissolving and reforming at the edges, and the perpendicular orientation of the molecules in the lamellar phase may be perturbed. This effect could easily account for the dramatic difference between fully hydrated films containing 20 and 33 mol yo cholesterol. For films hydrated with water-saturated air, a higher proportion of lysolecithin phase is required to perturb the orientation of the lamellar complex; this may be due to a difference in the properties of the lysolecithin phase formed under these conditions. The same principle may be applied in modified form to the consideration of the morphology of lysofecithin-cholesterol dispersions observed with the electron microscope. At high concentrations of lysolecithin, the ordered multilamellar structure of the lysolecithin-cholesterol complex is disrupted by the surface-active lysolecithin solution. The bilayer structure of the complex, under these conditions, exists as small, single-bilayer, walled, hollow spherules, which aggregate to form the rouleau structures observed. These rouleaux exhibit lamellar-phase order on a short-range scale, as opposed to the long-range order found in the absence of lysslecithin. Thus variation of the composition of the system has a
Conclusions In this section, some biological implications of the observations, presented in this and another paper (17), are discussed. We have shown that lysolecithin and cholesterol form an equimolar complex which forms a stable lamellar phase over a wide temperature range, in the presence of a considerable excess sf pure lysolecithin phases or crystalline cholesterol. The structure and properties of the lysolecithincholesterol bilayers have been found, using spinlabelled probes, to be closely similar to those of lecithin-cholesterol bilayers (27, 34) Moreover, we have obtained data, to be presented elsewhere, that equimolar mixtures of lysolecithin, cholesterol, and free fatty acids form stable bilayers with identical structure to those formed by lysolecitbin and cholesterol, and preliminary evidence that lysophosphatidylethanolamine and cholesterol also form a lamellar phase. Thus, contrary to common belief, the formation of lysophosphatides by local or more extensive hydrolysis of phosphatides in a bilayer membrane is not disruptive of the bilayer structure, providing adequate cholesterol is present. This may be a hitherto unrecognized role of the large amounts of cholesterol present in certain biological membranes, that is, to provide a stabilizing factor against membrane lysis by local formation of lysolecithin. Cell fusion and cell lysis are two phenomena in which lysolecithin is thought to be involved. The two processes have been linked in two papers
PURDON ET AL.: LYSQLECITHIN-CHQL~TERQLINTERACTION
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by Lucy and co-workers (11, 12) in which they showed that the addition of lysolecithin to the medium led to fusion of red blood cells and heterokaryon formation from LS fibroblasts and hen erythrocytes. If, as prop~sedin these papers, local micellization by lysolecithin is involved in these processes, then the small areas of the membrane involved must have a low molar amount of cholesterol in order to allow the bilayer-micelle transition to occur. Kellaway and Saunders (35) have shown that the lytic activity of lysolecithin on erythrocytes is reduced by both progesterone and more effectively by cholesterol, and Collier and Chen (10) demonstrated that induced hypercholesterolemia results in an increased lysolecithin-hemolysis time for whole blood. The physical basis of these observations seems to have been clarified by the present work. A converse implication of this study is the ability of lysolecithin to prevent crystallization of any local accumulation of cholesterol in a membrane. Lysolecithin has been found to occur in significant amounts in tissues and subcellular organelles that are rich in cholesterol, for example, in bovine adrenal chromaffin granules (36) and in atherosclerotic plaques (37). These could be rationalized as homeostatic responses to cholesterol accumulation based on the ability of lysolecithin to maintain cholesterol in a liquid crystalline phase. Thus we feel that knowledge of lysolecithincholesterol interactions offers certain important refinements to present theories of membrane structure. The capable assistance of Mr. Michael Paul1 in preparing the figures is gratefully acknowledged. Facilities and equipment for electron microscopy were provided by Medical Science Building Services,University of Toronto, Toronto, Ont. 1. Robinson, N. & Saunders, L. (1958) J. Phurm. Pharmacol. 10,755-761 2. Hamori, E. & Michaels, A. M. (1971) Biochim. Biophys. Acta 231, 496-504 3. Reiss-Husson, F. (1967) J. Mol. Biol. 25, 363-382 4. Robinson, N. & Saunders, L. (1959) J. Pharm. Plmrmucol. Sirppl. I1, 115T- 119T 5. Junger, E., Hahn, M. H. & Reinauer, H. (1970) Biochim. Biophys. Actu 211, 381-388 6. Reman, F. C., Demel, R. A. & Be Gier, J. (1969) Cklcm. Phys. Lipids 3, 221-233
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7. Haydon, D. A. & Taylor, J. (1963) J. Tlreor. Biol. 4, 281-296 8. Van Zutphen, H. & Van Deenen, L. L. M. (1976) Chem. Phys. Lipids 1, 389-391 9. Chapman, D., Kamat, V. B., De Gier, J. & Penkett, S. A. (1968) J. Mol. Biol. 31, 101-614 10. Collier, H. B. & Cken, H. L. (1950) Cast. J. Res. Sect. E 28, 289-297 11. Poole, A. R., Howell, J. 1. & Lucy, J. A. (1970) Nature 227, 810-814 12. Lucy, J. A. (1970) Naare 227, 815-817 13. Croce, C. M., Sawicki, W., Kritchevsky, D. & Koprowski, H. (1971) Exp. Cell Res. 67, 427-435 14. Gledhill, B. L., Sawicki, W., Croce, C. M. & Koprowski, H. (1972) Exp. Cell Res. 73, 33-40 15. Deamer, D. W. (1973) J. Biol. Chem. 248, 5477-5486 16. Dervichian, D. G. (1969) Mol. Cryst. 2, 55-62 17. Rand, R. P., Pangborn, W., Purdon, A. D. & Tinker, D. 0. (1975) Can. J. Biockem. 53, 189-195 18. Tinker, D. 0. & Saunders, L. (1968) Clrem. Plys. Lipids 2, 316-329 19. Wu, T.-W. & Tinker, D. 0. (1969) Biochemistry 8, 1558-1 568 20. Kuksis, A. (1966) Chromatogr. Rev. 8, 172-207 21. Keana, J. F. W., Keana, S. B. & Beatkam, D. (1967) J. Am. Cltem. Soc. 89, 3055-3056 22. Waggoner, A. S., Kingzett, T. S., Rottsckaefer, S., Griffith, 0.H. & Keith, A. D. (1969) Chem. Plrys. Lipids 3, 245-253 23. Faber, R. J. & Fraekel, G. K. (1967) J. Clrem. Phys. 47,2462-2476 24. Hsia, J. C., Sckneider, H. & Smith, I. C. P. (1971) Can. J. Biocltem. 49, 614-622 25. Tinker, D. 0. & Pinteric, L. (1971) Biochemistry 10, 860-865 26. Pinteric, L., Tinker, D. 0. & Wei, J. (1973) Biochim. Biophys. Actu 293, 630-638 27. Boggs, J. M. & Hsia, J. C. (1972) Biochim. Biophys. Acfa 290, 32-42 28. Hamilton, C. L. & McConnell, H. M. (1968) in Str~ictwalChemistry aitd Molecular Biology (Rich, A. & Davidson, N., eds), pp. 115-149, W. H. Freeman & Co., San Francisco, Calif. 29. McConnell, H. M. & McFarland, B. G. (1970) Q. Rev. Biophy~.3, 91-136 30. Libertini, L. J. & Griffith,0.H. (1970) J. Chem. Phys. 53, 1359-1367 31. Seelig, J. (1970) J. Am. Chem. Soc. 92, 3881-3887 32. McConnell, H. M. & McFarland, B. G. (1972) Ann. N. Y . Acad. Sci. 195, 207-217 33. Rand, R. P., Tinker, B. 0.& Fast, P. 6 . (1971) Chem. Phys. Lipids 6, 333-342 34. Lapper, R. D., Paterson, S. J. & Smith, I. C. P. (1972) Can. J. Biockem. 50, 969-981 35. Kellaway, I. W. & Saunders, L. (1969) J. Pharm. Pharmacol. 26, 189s-194s 36. Blasckko, H., Firemark, H., Smith, A. D. & Winkler, H. (1967) Bioclrem. J. 104, 545-549 37. Portman, 0.W. & Alexander, M. (1969) J. Lipid Res. 10, 158-165
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Appendix bTibe-FI~sr9 Model sf the $truelure of Thie Lipid Films The minimum stable area of lysolecithincholesterol lamellar phase is represented by a square 'tile9 of unit area, and the lipid film is considered as a 'floor' of area N which is to be covered with tiles of two kinds, namely, black, representing lysolecithin-cholesterol lamellar complex, and white, representing pure lysolecithin phase (if lysolecithin is in excess) or cholesterol (if cholesterol is in excess). The floor is first ruled off into M contiguous squares of unit area, and these squares are covered at
random with tiles randomly drawn from a pile containing N13 black tiles and Nw white tiles (MB Mw = N ) initially.
+
This process was simulated by digital computation wing a Monte Carlo technique. Figure 10 shows a drawing of a typical random matrix containing 900 elements (30 tile by 30 tile 'floor'), half of which are black. The perimeter to area ratio of the black areas (not including perimeters at the edges of the 'floor') was computed for various percentage coverages with black tiles, corresponding to various proportions of thin lipid films occupied by lysolecithin-cholesterol lamellar phase. These ratios are shown in Fig. 11.
