The relations between the composition of liposomes and their interaction with triton X-100

The Relations between the Composition of Liposomes and Their Interaction with Triton X-IO01 RACHEL HERTZ Department of Biochemistry, The Hebrew University Hadassah Medical School, Jerusalem, f srael AND

YECHEZKEL BARENHOLZ Department of Biochemistry, The Hebrew University Hadassah Medical School, Jerusalem, Israel, and Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, Virginia Received June 20, 1976; accepted August 13, 1976 The interaction of Triton X-100 with phospholipid bilayers of multilamellar liposomes formed from mixtures of spinal cord sphingomyelin and egg yolk lecithin in various mole ratios (all containing 1 mole of dicetylphosphate per 10 moles of phospholipid) was studied. The results indicate that the process is time dependent and is much slower than the formation of simple micelles. The time to reach the final equilibrium state is dependent on the SPM to PC mole ratio, on the Triton to phospholipid mole ratio, and on the Tirton concentration. Titration with increasing Triton concentration shows that the behavior of Triton is biphasic for all the various lipid compositions tested. For low Triton to phospholipid mole ratio there is no mass formation of mixed micelles; in addition, the Triton seems to radically increase the leakage of glucose without reducing the turbidity. This range is limited by a turning point where most of the phospholipids and about half of the Triton coprecipitate. Above this Triton to phospholipid mole ratio formation of mixed Triton-phospholipid micelles occurred followed by a drastic decline in turbidity. This turning point as well as the exact profile of the Triton effect are strongly related to the SPM:PC mole ratio. The higher the mole fraction of SPM in the membrane, the less Triton is required to reach the turning point and to cause a complete solubilization. These effects can be explained by tighter packing and stronger phospholipid-phospholipid interactions imposed by SPM and expressed as apparent microviscosity which increases upon increasing the mole fraction of SPM in the bilayer. INTRODUCTION

Detergents are used extensively for solubilization of biological membranes, isolation and purification of membrane-bound proteins, and reconstitution of lipids and proteins to form functional membranes (1, 2, 3). Interaction of enzymes with their lipid substrates also requires the addition of detergents (4, 5, 6). 1 Presented at the 49th National Colloid Symposium, Clarkson College of Technology, Potsdam, N. Y. 13676, June 16-18, 1975.

The detergents can be divided into two main groups: the ionic and nonionic detergents. One might expect a different mechanism for the interaction of these two groups with lipids or proteins since only the ionic detergents have ionogenic groups which contribute an electrostatic expression. In general, the ionic detergents have higher CMC values and their micelles are smaller than those of nonionic micelles (7, 8). These differences are also reflected in the nature of the interaction with lipids and proteins (1, 9, 10). Of all the nonionic 188

Journal of Colloid and Interface Science, Vol. 60, No. I, June 1, 1977 ISSN 0021-9797

Copyright ~ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

LIPOSOMES AND TRITON X-100 detergents in use for biochemical and biophysical studies, Triton X-100 is one of the most effective membrane solubilizers and in lipid enzymology, especially so, since its effect on protein structure is mild (11, 12, 13a). It seems that the primary effect of Triton X-100 on the solubilization of membranes and on the interaction of enzymes with their lipid substrates is through its effect on the organization of the lipids (1, 5, 6). Triton X-100 was successfully used for the solubilization of erythrocytes, Mitochondria, lysosomes, semilik forest virus (10, 14 16), other biological membranes (1, 12), and liposomes (17, 18). In the present communication we studied the relations between the Triton X-100 as a membrane solubilizer and the lipid composition of a lipid bilayer using the change in the sphingomyelin to lecithin mole ratio as the only variable in membrane composition. This question has to be answered since biological membranes differ drastically in their lipid composition (19). This compositional heterogeneity is reflected in many physical properties of the membranes and as is indicated by this paper, such differences in lipid composition might affect membrane solubilization by detergents. MATERIALS AND METHODS Materials Egg yolk lecithin and bovine spinal cord sphingomyelin were prepared, purified, and analyzed as described previously (20). Both phospholipids were chromatographically pure; their fatty acids and sphingosine base composition are described by Hertz and Barenholz (33). 3H lecithin (0.5 #c/~mole) was prepared and purified as described by Gatt (21). The lecithin was more than 99% pure. Its analysis shows that 90% of the label is in position 1 of the lecithin. Dicetylphosphate and glucose oxidase from Aspergillus niger type V 1090 u/ml were purchased from Sigma. Glucose and Triton X-100 were purchased from BDH. Perylene and diphenylhexatriene were a gift of Dr. M. Shinitzky, the Weizmann Institute

189

of Sciences, Israel. All other reagents were of analytical grade. Experimental Procedure Osmotically active multibilayered liposomes were prepared from the desired mixture of lecithin and sphingomyelin containing 1 mole of dicetylphosphate per 10 moles of choline phospholipids as described by Bangham et al. (22). The liposomes were prepared in 0.3 M glucose. The untrapped glucose was replaced by dialysis against potassium phosphate buffer pH 6.5 in isoosmotic concentration (23) at 20°C. Usually four to five changes are required to bring the glucose concentration of the solution below 0.05% of its original value without affecting much the glucose concentration inside the liposomes (23). Glucose concentration inside and outside the liposomes and the release of glucose from liposomes were measured as described by Hertz and Barenholz (23). The Effect of Triton X-IO0 on the Turbidity of the Liposomes Suspension A liposome dispersion (usually 0.01-0.1 ml containing 1 umole of phospholipids) was injected into a thermally equilibrated (37 +0.2°C) cuvette (2-cm light path) containing 8 ml of isoosmotic buffer using continuous rapid stirring at constant rate. The turbidity of the stirred dispersion was recorded as optical density at 450 nm. Various amounts of Triton X-100 in isoosmotic buffer were injected into the cuvette and the change in turbidity with time was monitored using a Servagor potentiometric logarithmic recorder with a chart speed of 1 cm/sec. The initial velocity of the turbidity change was calculated from the slope of the initial linear portion of the trace. The osmotic beharior of the liposomes was measured using the same procedure except that the liposomes were injected into a buffer solution of different osmotic pressures. The electrophoretic mobility of the multibilayered liposomes in 0.16 M potassium phosphate buffer pH 6.5 was measured by the method of Bangham et al. (25) at 37°C.

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Electron micrographs were done on liposomes prepared in 0.145 M ammonium acetate using negative staining with 0.5% ammonium molybdate (26). Determination of Triton concentration. The concentration of Triton was determined from its fluorescence intensity using the PerkinElmer MDF-3 spectrofluorimeter. The excitation spectra show three maxima: 260, 290, and 310 nm. The emission spectra show two distinct maxima, one at 302 nm and the second at 330 nm. To prevent any overlap between excitation and emission the Triton solutions were excited at 260 nm and the emission was measured at 330 nm using an instrument filter which cut the emission below 290 nm. This determination is based on the calibration curve using Triton X-100 solutions of known concentrations. Fluorescence polarization. Two fluorescent hydrocarbons were used as probes in this study : the 1,6-diphenyl 1,3,5-hexatriene (DPH) and perylene. Labeling of liposomes or rnicelles with D P H or perylene was carried out as described previously (20). The fluorescence method for evaluation of dynamic properties with D P H or perylene was described in previous papers (20, 28) and was used here without any alterations. The method is based on simultaneous determinations of the degree of fluorescence anisotropy, r (see Eq. [2]), and the excitedstate lifetime of the DPH- or perylene-labeled system and translating the results into microviscosity using the Perrin equation (20, 28). ro/r = 1 + C(,~(Tr/n),

[13

where the anisotropy r is defined as r = (I,, -- I . ) / ( I , , + 2I~),

[23

where IN and I~ are the fluorescence intensities detected through an analyzer with a direction of polarization oriented parallel (11) and perpendicular (3_) to the direction of polarization of the excitation beam (15), r0 is the limiting anisotropy, T is the absolute temperature, ~/is the apparent microviscosity, r is the lifetime of the fluorophore and C(r3 is a parame-

ter of the fluorophore shape and is dependent on r. Data were calculated and analyzed as described elsewhere (20). Correlation factors of all measurements were above 0.98 with standard deviations of less than 2.5%. RESULTS CMC of Triton X-IO0 The CMC of Triton X-100 in 0.16 M potassium phosphate buffer, pH 6.5, was determined by the method of Yedgar et al. (27) and found to be in the range of 0.1-0.2 mM. Interaction of Triton X-IO0 ~ith Liposomes The interaction of Triton X-100 with liposomes composed of mixtures of lecithin and sphingomyelin containing 1 mole of DCP per 10 moles of phospholipid was followed using two different parameters: (a) the integrity of the liposomal membrane based on the release of glucose trapped in the liposomes, and (b) the change in turbidity at 450 nm which will reflect the solubilization of the phospholipids and the formation of mixed micelles with the Triton X-100 The Effect of Triton X-IO0 on the Eilayer Integrity Membrane permeability is one of the best parameters for membrane integrity. As shown elsehwere (33), at 20°C the membranes of the liposomes made of sphingomyelin and lecithin in various ratios are practically impermeable to glucose when incubated in isoosmotic buffer (33). Liposomes containing glucose were prepared from sphingornyelin and lecithin in different mole ratios (containing 1 mole of DCP per 10 moles of choline phospholipids) as described in the Methods section. The kinetics of the release of trapped glucose from the liposomes by Triton X-100 were studied by incubation of liposomes in fixed concentration with increasing concentration of Triton. It seems that the effect of Triton X-100 is dependent on at

Journal of Colloid and Interlace Science, Vol. 60, No. 1, June 1, 1977

LIPOSOMES AND TRITON X 100 least three parameters: (i) The effect of Triton is time dependent especially in the low Triton concentrations; whereas for 0.6 /finale (0.6 mM) of Triton (per 1/~mole of lecithin) all the trapped glucose was released at once. For 0.3/~mole (0.3 raM) the release was only 25% after 1 rain, 54% after 15 rain, and the final value of 62% was reached after 45 rain. (ii) The release of glucose is strongly dependent on the composition of the liposomes. As shown in Fig. 1, sphingomyelin liposomes are more sensitive to Triton X-100 than lecithin liposomes, and the liposomes made of mixtures of the two choline phospholipids represent intermediate sensitivity. This is summarized by Fig. 2, which describes the amount of Triton X-100 required for the release of 50% of the glucose trapped inside liposomes of different sphingomyelin-to-lecithin ratio. (iii) The effect of Triton X-100 was dependent mainly on the ratio of Triton X-100 to phospholipids, since increasing the volume of the incubation mixture up to 8 times, without changing the amounts of both Triton I

I 100

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FIG. 1. The effectof increasing Triton to phospholipid ratio on the release of glucose from multibilayered liposomes made of SPM (O) or PC (0).

191

LECITHIN(molar fraction x 100) 100 75 50 25

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FIG. 2. The effect of sphingomyelin to lecithin mole ratio in the multibilayered liposomes on the Triton amount which releases 50% of the glucose trapped in these liposomes (Triton X-100~0). or phospholipids, had no effect on the release of glucose. On the other hand, keeping the concentration of Triton X-100 constant and increasing the concentration of liposomes reduces the release of glucose. The integrity of the Triton-treated liposomes was also studied using electron microscopy. Figure 3 demonstrates electron micrographs of liposomes made of sphingomyelin and lecithin in 3:1 mole ratio (containing 1 mole of DCP per 10 moles of phospholipids). One can see the multibilayer onion-like structures for the untreated liposomes (Fig. 3a). When treated with Triton X-100 (Fig. 3b) using 1.25 (0.156 raM) /zmole per 1 #mole of choline phospholipid in 8 ml, which is the Triton concentration where the turbidity starts to decline (see Fig. 4), drastic changes in the structure of the liposomes are observed. Most of the lipid bilayers were damaged and seem to have fused. The product of this fusion is a complex network of lipid layers. Similar data were obtained for liposomes made of sphingomyelin and lecithin in other mole ratios when the right ratios of Triton of choline phospholipids were used (Fig. 4).

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HERTZ AND BARENHOLZ

FIG. 3. Electron micrograph of multibilayered liposomes made of P C : S P M 3:1 (mole ratio) containing 1 mole of D C P per 10 moles of choline phospholipids before (a) and after (b) the t r e a t m e n t with Triton. T h e final magnification for both was 77.500. A 2000-_~ rod is shown on both. For details see text.

Solubilization of the Liposomes by Triton X-IO0 The multilamellar liposomes have high specific turbidity2 values at 450 nm due to the fact that these suspensions are not obeying the Rayleigh-Gans approximation and the change

of turbidity by changing wavelength is much smaller than expected for Rayleigh scatters. The wavelength dependence change in turbidity for micelles or single-walled liposomes (which are Rayleigh scatters) is described by (29),

2 Turbidity per t~mole of phospholipids. Journal of Colloid and Interface Science, Vol. 60, No. 1, June 1, 1977

zl/r2 = (X2/~,1)',

[3]

LIPOSOMES AND-TRITON X-100 1

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FI6.4. The effect of increasing Triton X-100 concentration on the turbidity at 450 nm of liposomes made of various lecithin to sphingomyelin mole ratios. O, egg yolk lecithin (PC); ID, beef spinal cord sphingomyelin (SPM) ; 1:3, PC: SPM 3:1 ; A, PC: SPM 1: 1; i , PC: SPM 1: 3. For details see text. where rl and r2 are turbidities measured at wavelengths Xl and k2, respectively. For multilamellar liposomes, this power is always less than 4.0 and usually around 1.7 (Barenholz, unpublished). This big difference enables one to follow the solubilization of the multilamellar liposomes either to single bilayered liposomes or to Triton X-100 phospholipids mixed micelles using either the sharp drop in turbidity at 450 nm or using the turbidity ratio at two given wavelengths. The effect of Triton X-100 was measured at 450 and 350 nm since in these wavelengths neither Triton X-100 nor the phospholipids have any absorption bands and the only contribution to the optical density is the turbidity. The effect of Triton X-100 on the turbidity of multilamellar liposomes composed of sphingomyelin and lecithin in various ratios (all containing 1 ~mole of DCP per 10 ~mole of choline phospholipids) was studied as described in "Methods." The liposomes were prepared in 0.3 M glucose. The change in turbidity caused by the various Triton concentrations was followed with time. For all samples the change in turbidity was time dependent; the time to reach equilibrium increased with the reduction in the Triton-to-phospholipid mole ratio. Under our experimental conditions a

plateau was reached after 2 hr and the plateau values were used to determine the final turbidity change. In order to compare all the lipid mixtures, the results are described as the measured optical density at 450 or 350 nm divided by the optical density of the same liposomes obtained without the presence of Triton X-100 (OD/OD isoosmotic). The turbidity studies at 450 nm are summarized by Fig. 4 and show the following. (i) For liposomes of all compositions there are two ranges for the interaction with the Triton X-100. In the first range (smaller amounts of Triton X-100) the turbidity is either increasing or unaffected. In the second range (higher amounts of Triton) the turbidity is declining. The ratio of turbidity 450 to 350 nm in the first range was 1.7 while this ratio was increased continuously in the second range and finally reaches a ratio close to 4, which demonstrates that all particles became Rayleigh scatters. (ii) Quantitatively both effects are dependent on the ratio between lecithin and sphingomyelin in the liposomes. For sphingomyelin liposomes there is no increase in turbidity in the range of low Triton concentration and the turbidity remains constant in this range. For liposomes containing lecithin and

Journal of Colloid and Interface .Science, V o l . 6 0 , N o . 1, J u n e

1, 1 9 7 7

194

HERTZ AND BARENHOLZ L ECITHIN(molar frac lion x 100) 100 75 50 25 5.0

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FIG. 5. The effect of liposome composition on the Triton amount which causes a 50% fall in the initial turbidity of liposome dispersion (Triton X-10050). sphingomyelin the increase of turbidity caused by the presence of Triton is enhanced when the content of lecithin in the membrane is increased, and it is maximal for lecithin liposomes. (iii) The concentration of Triton X-100 where the turbidity starts to decline is also increased with the mole fraction of lecithin in the liposomes (3.0 ~mole (0.375 mM) of Triton per 1 ~mole of lecithin, but only 1.0 ~mole (0.125 mM) Triton X-100 per 1 ~mole of sphingomyelin). (iv) Figure 4 also clearly demonstrates that the higher the mole fraction of lecithin in the bilayer the more Triton is required for the solubilization of the membranes. This is demonstrated by Fig. 5, which describes the amount of Triton X-100 required for reducing the turbidity at 450 nm to 50°-/o of the turbidity of the untreated liposomes. The amount of Triton X-100 required for SPM liposomes was about one-third of that required for lecithin liposomes; the mixtures of the two gave intermediate (but not weight-average) values; thus it seems that the effect of sphingomyelin is dominant. A better insight into the mechanism of interaction between the Triton and the phospholipid Journal of Colloid and Inlerface Science, Vol. 60,

bilayers was obtained using liposomes loaded with 0.3 M glucose and made of 3H-lecithin (Methods) containing 1 mole of DCP per 10 moles of lecithin. The above interaction was monitored using the following parameters: (a) turbidity at 450 mm, (b) the amount of glucose released from the liposomes, (c) the amount of lecithin which does not precipitate by 2-hr centrifugafion at 160,000g, and (d) the amount of Triton in the 160,000g supernatant. The exact procedures are described in "Methods." This experiment (summarized in Table I) confirms the previous observations: (1) The interaction is dependent on Triton concentration and on Triton to phospholipid ratio. (2) There are two ranges with respect to Triton concentration. (3) There is a turning point between the two ranges where almost all the lecithin precipitates at 160,000g (2 hr) together with 52% of the Triton. At this Triton concentration all the glucose trapped inside the liposomes was released to the medium (as measured by its presence in the supernatant and its availability to glucose oxidase (23). Increasing Triton concentration above this turning point causes a decline in the turbidity, and an increase in the amount of the lecithin and Triton in the supernatant. Complete solubilization occurred between 1.5 and 3 mM of Triton (for 1 mM lecithin). The Relations between the Interaction with Triton X-IO0 and the Osmotic Behazior of the Liposomes The osmotic activity of the liposome is one of the parameters used to indicate membrane integrity. The following experiment was designed to study the effect of Triton X-100 on the osmotic activity of the liposomes. Lecithin liposomes were used since they have two distinct regions for the interaction with Triton X-100 with respect to the Triton concentration. Liposomes were prepared in 0.3 M glucose and dialyzed against isoosmotic potassium phosphate buffer pH 6.5 to remove the free untrapped glucose. After the dialysis the lipo-

No. 1, June 1, 1977

LIPOSOMES A N D T R I T O N X-100

195

TABLE I Effect of Triton X-100 on Lecithin Liposomesa Triton X-100 (raM)

OD/ OD isoosmotic (450 r i m )

% Triton X-100in supernatant

0 0.18 0.3 0.6 0.9 1.2 1.5 3

1 1.050 1.090 0.965 0.708 0.600 0.452 0.01

-90.0 97.0 48.0 66.6 78.0 100.0 98.0

Triton concentration in supernatant (mM)

% 3H Lecithin in supernatant

0 10.8 21.2 1.0 34.4 56.0 66.4 100.0

0.162 0.29 0.285 0.57 0.94 1.5 2.94

Lecithin % Glucose concentration releasedby in supernatant Triton X-100 (raM) to the supernatant

0.11 0.21 0.01 0.34 0.56 0.66 1.0

0 28.5 62.0 100.0 100.0 100.0 100.0 100.0

Triton to lecithin mole ratio in supernatant

1.47 1.38 1.68 1.68 2.27 2.94

Incubation mixtures in a final volume of 1 ml of isoosmotic potassium phosphate buffer p H 6.5 contains multilamellar llposomes made of 1 ~mole 3H-lecithin and 0.1/zmole DCP. For details see text.

somes were incubtaed with Triton X-100 in various concentrations from 0 up to 6 umole per 1 #mole of choline phospholipids. The osmotic behavior was studied as described in Methods. The liposomes were transferred to hypoosmotic or hyperosmotic potassium phosphate buffer pH 6.5. The relative change in turbidity at 450 nm which is proportional to the relative change in volume (22) was identical for the treated and untreated liposomes in the range of 0-2.5 umole of Triton per 1 ~mole of lecithin (in 8-ml buffer). In this range the turbidity of the dispersion in isoosmotic medium is higher or identical to that of untreated liposomes. Increasing the Triton above 3.0 #mole per 1 ~mole of lecithin caused reduction in the turbidity, paralleled by a rapid decline in the osmotic activity of the liposomes.

Effect of Triton X-IO0 on the Electrophoretic Mobility of the Liposomes Liposomes were made of lecithin and dicetylphosphate in a molar ratio of 10 to 1, in 0.3 M glucose solution. The external glucose was removed as described in "Methods" and replaced by isoosmotic (0.155 M) potassium phosphate buffer pH 6.5. The liposomes were treated with Triton X-100 in a concentration where the decline in turbidity is minimal (3.0 #mole of Triton per 1 /~mole of lecithin in volume of 8 ml; Fig. 4), and the electrophoretic

mobility of the Triton-treated liposomes was measured as described in "Methods" and compared to that of the untreated liposomes. The measurements for both were performed at 37°C. At least 15 measurements were carried out for each of the samples. Both samples showed anodic mobility but the untreated liposomes had higher zeta potential: -19.6 4-1.2 inV. The value of the Triton-treated liposomes was lower and the standard deviation much greater (±25°f0), which indicates change in the homogeneity of the liposome population with respect to the charge.

Measurements of the Apparent Microviscosity of the Hydrophobic Region of the Liposomes One of the parameters affecting the interaction with a detergent is the packing density of the phospholipids. This will be reflected in the apparent microviscosity of the hydrophobic regions of the bilayer. The microviscosity of the hydrophobic region of the bilayer was measured using the fluorescence polarization of DPH as described elsewhere (20). Figure 6 demonstrates the effect of temperature on the microviscosity of lecithin multilamellar liposomes and sphingomyelin liposomes (both containing 1 mole of DCP per 10 moles of phospholipids). It is clear that there are two main differences between the two phospholipids. The micro-

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HERTZ AND BARENHOLZ 20

10

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1.0

0.5

,

0.2

3.0

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pholipids (1). The hydrodynamic properties of mixed micelles containing Triton X-100 and sphingomyelin were studied by Yedgar et al. (5). We studied the effect of Triton X-100 on the apparent microviscosity of the hydrophobic region of the Triton-sphingomyelin mixed micelles in comparison to the microviscosity of multilamellar sphingomyelin liposomes. Multilayered liposomes were made of sphingomyelin in 0.1 M sodium acetate pH 5.0 as described in "Methods." Mixtures of sphingomyelin and Triton X-100 in various mole ratios and 1 mM solution of Triton X-100 were prepared as described by Yedgar et al. (5) in 0.1 M sodium acetate pH 5.0. The fluorescence polarization of perylene in these dispersions was measured as described elsewhere (20). Table II demonstrates that the microviscosity of the hydrophobic region is decreased with increasing Triton concentration.

1 / T x 10 3 (OK-'1)

DISCUSSION FIG. 6. Temperature dependence of microviscosity of the hydrophobic region of mulfilamellar SPM Is the solubilization of a membrane by a liposomes (/k) and PC liposomes (0). For details detergent related to the lipid composition of the see text. The lipid concentration was 0.5 raM; I mole membrane? The present communication was of DPH was used per 1000 moles of phospholipid. aimed toward a better insight into this problem. We used the nonionic detergent Triton X-100 viscosity of liposomes made of sphingomyelin is higher than that of lecithin liposomes for and multilamellar liposomes made of various the temperature range 0-60°C. The spinal sphingomyelin-to-lecithin mole ratios as a cord sphingomyelin liposomes have a broad model system for answering the above question. The process of bilayer solubilization rethermotropic transition which starts around 25°C and continues at least up to 55°C, while quired an association between the detergent no thermotropic transition is observed for T A B L E II lecithin liposomes. The liposomes made of mixtures of the two (not shown in the figure) Microviscosities of Sphingomyelin-Triton X-100 Dispersions • have intermediate behavior, although the effect is not a simple additive one (Barenholz Physical state b Co m p o s itio n Mole r a tio Microviscosity and Shinitzky, in preparation). Phase separaa t 22°C tion of regions enriched in SPM becomes ap(poise) parent when the mole fraction of SPM exceeds T r ito n * 1 Micelles 1.58 0.5. Triton:SPM a 1:0.3 M i c e d micelles 2.89 M icroviscosity of S phingomyelin- Triton X-IO0 Mixed Micelles The final step in the solubilization if biological membranes is the formation of mixed micelles containing the detergent and the phosJournal of Colloid and Interface Science, Vol. 60, No. 1, J u n e 1, 1977

Triton:SPM ~ Triton:SPM a SPM a

1:1 1 :5 1

M ix e d micelles M a i n l y liposomes Multibilayered liposomes

4.33 6.25 7.17

aFor details, see te x t. T h e definition of th e physical s t a t e is based on Refs. (5, 27). Triton concentration was 1 raM. a S P M c o n c e n t r a t i o n was 1 m M .

197

I,IPOSOMES A N D T R I T O N X 100 TABLE I I I Dynamic Properties of the Hydrocarbon Region in Mixed Liposomes Containing Sphingomyelin and Lecithin at Different Mole Ratios ~,b Sphingomyelin/ (Sphingomyelin + lecithin) (mole/mole)

5 °C

Microviscosity (poise) at 25 °C

37 °C

45 °C

0 0.25 0.50 0.75 1

2. i 3.4 7.1 9.3 10.4

0.8 1.1 2.7 .5.1 8.5

0.4 0.6 1.3 2.3 4.4

0.3 0.4 0.8 1.3 2.0

Temperature (°C), transition starts

No transition ~ No transition c Diffused transition 14.0 ° , 25.0 ° 25.0 °

D a t a are based on Barenholz and Shinitzky (in preparation) using single-walled liposomes. b All liposomes containing 1 mole DCP per 10 moles choline phospholipids. c No transition was observed at the range of 0-60°C.

and the lipids. Assuming that the free energy of micellization (AF) is calculated AF = RT

In CMC,

[4]

where R is the gas constant and T is the absolute temperature, one can calculate the free energy of micellization of Triton X-100. Based on CMC values of 0.1-0.2 raM, F is in the range of (--5.3) (--4.9) kcal/mole. The interaction of the Triton with the phospholipid bilayer is explained by having the free energy required to interact with the lipid bilayer lower than the free energy of micellization. Our data demonstrate that the solubilization of a lipid bilayer by Triton is a time-dependent process for which the equilibrium state is dependent on the lipid composition and on the phospholipidto-Triton mole ratio. Triton X-100 demonstrates a biphasic behavior with respect to the membrane-detergent interaction. With low Triton-to-phospholipid molar ratios the turbidity of the liposomal dispersions is either unaffected or shows an increase; however, the membrane demonstrates leakage of glucose without losing its capability of being osmotically active. We refer to this effect as the membrane integrity. There exists a turning point where all the lipid together with about half of the Triton coprecipitate. Electron micrographs of negative-stained liposomes at this point (Fig. 5) show formation of a network of bilayers, probably by a fusion-like

process. Increasing the Triton-to-phospholipid mole ratio above the turning point (the second phase) causes the formation of mixed Tritonphospholipid micelles. This effect is defined as membrane solubilization. The exact profile of the Triton effect is strongly dependent on the lipid composition. For example, under the experimental conditions shown in the legend of Table I, complete solubilization of lecithin liposomes occurred when the mole ratio of Triton to Lecithin was above 2.0. Similar results were obtained using the proton N M R technique (7). Under the same conditions liposomes made of sphingomyelin required less than 0.8 umole of Triton per 1 #mole of phospholipid. Sohn et al. (13) show a similar pattern for Triton interaction with the rat liver plasma membrane. At low Triton-to-phospholipid mole ratio he reports an increase of the turbidity of the dispersion followed by a decline at higher Triton-to-phospholipid mole ratio. The authors claim that the increase in turbidity is due to formation of a ternary complex of Mg2+-membrane-detergent. However, in our system Mg 2+ was not present. Moreover our preliminary results investigating the effect of Triton on rat erythrocytes (Barenholz, unpublished results) also showed the same behavior in the absence of Mg 2+ ions. The increase in turbidity might simply be explained by a change in size, shape, and refractive index of the dispersion. Our results (i.e., electron

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micrograph and partial specific volume measurements) demonstrate the change in the above parameters. The sensitivity of the lipid bilayer towards Triton increases in parallel with increasing the mole fraction of sphingomyelin in the membrane. The higher the SPM mole fraction the smaller is the number of Triton molecules required to affect membrane integrity and solubilization. The main issue of this study is aimed at an understanding of the forces in the membrane which control its interaction with the detergent. We found a very good correlation between the detergent effect and the apparent microviscosity of the hydrophobic region of the membrane (as measured by fluoresence polarization of either perylene or 1,6-diphenylhexatriene (20)). The higher the mole fraction of SPM in the bilayer the more viscous is the membrane. A comparison between the microviscosity of PC (Fig. 6) in either multilamellar or single-walled vesicles indicates that the hydrophobic region of a bilayer composed of SPM is 5 to 10 times higher than the microviscosity determined for PC bilayers throughout the temperature range of 0-60°C. Sphingomyelin is also unique among lipids commonly present in biological membranes in that the thermotropic behavior of SPM has a broad phase transition which starts at 25°C (20, 34). When the mole fraction of SPM in the bilayer is 0.5 or above 0.5 a phase separation of regions either enriched or containing only SPM molecules occurs. Similar phenomena were observed in ghosts derived of sheep red blood cells which are very rich in SPM, but not in ghosts derived of rat red blood cells, in which the relative concentration of SPM is low. The phenomenon of phase separation (Barenholz and Shinitzky, in preparation) caused by SPM was further confirmed in multilamellar liposomes composed of mixtures of N-stearoyl sphingosine-phosphorylcholine and 1-palmitoyl 2-oleyl lecithin (34). The values of apparent microviscosity obtained for single-walled liposomes are summarized in Table III using data obtained by

Barenholz and Shinitzky (in preparation). Is the correlation between the apparent microviscosity and the sensitivity towards Triton X-100 relevant? We assume that the answer is positive since the apparent microviscosity is strongly related to the interactions among the lipid molecules in the bilayer. This includes hydrophobic as well as polar interactions. Comparison between the two molecules leads one to the conclusion that although the two choline phospholipids have the same ionogenic group, phosphorylcholine, the two molecules are very different. Lecithin is symmetrical with respect to the two acyl chains which are very similar in length whereas sphingomyelin is asymmetric in this region (33). They differ in degree of saturation; the average number of cis double bonds per mole is four times larger for egg PC than for beef spinal cord SPM (33). In the polar region the main difference is that SPM is capable of participating in hydrogen bonding as both donor and acceptor (due to its amide bond and the free hydroxyl group), but the lecithin molecule can serve only as an hydrogen acceptor (20, 33). All the above differences enable strong SPM-SPM and probably SPMPC interactions which explain the effect of SPM on the apparent microviscosity and pecking density. In SPM bilayers the high microviscosity and rigidity will reduce the free volume and prevent the Triton molecules from being evenly distributed throughout the bilayer plain. Regions enriched in Triton were formed. This will cause fragmentation of the lipid bilayer, and formation of regions with high curvature will impose the membrane solubilization by formation of micelles (5, 27). In the case of lecithin, due to the less rigid and less dense structure, there is more free volume for the Triton. It will be evenly distributed by lateral diffusion over the bilayer plain; therefore much greater Triton concentrations are required to reach a high enough local Triton concentration to cause membrane solubilization. Bilayers made of mixtures of the two choline phospholipids have intermediate apparent

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LIPOSOMES AND TRITON X-100 microviscosity, and therefore their sensitivity toward Triton will be intermediate and dependent on the SPM-to-PC mole ratio in the bilayer. The effect of Triton on SPM-SPM interactions was studied using a dispersion of the two compounds made by colyophilization of the two from a benzene solution and dispersing them in 0.1 M sodium acetate buffer pH 5.0. Using this method equilibrium is reached in a very short time. At mole ratio 1:5, Triton to SPM, the physical state of the dispersion exists as a mixture of large micelles and of liposomes of various sizes (5, 27) and the apparent microviscosity was similar to that of liposomes made of pure SPM, probably since the SPM-SPM interactions are dominant. Increasing the mole ratio of Triton-SPM to 1 : 1 and 1 : 3.0 affects a change in the physical state of the system to mixed micelles only. The micelle size is dependent on the Triton to SPM mole ratio and for each ratio the population is homogeneous in size. The apparent microviscosity of the hydrophobic region of the micelle is much lower, indicating the weakening of the SPM-SPM interactions. Similar results were obtained using proton NMR (Lichtenberg and Yedgar, preliminary results). It is of interest to note that the interior micellar microviscosity in micelles made with cationic detergents (such as cetyl trimethyl ammonium bromide) measured by Shinitzky et al. using perylene depolarization (28) was much lower than what we found for the Triton micelles. This may be explained by the following lines of reason: the contribution of the repulsion between the ionogenic head groups of the ionic detergents reducing the interactions between the hydrophobic chains, and on the other hand by the strong interactions between the hydrophobic residues of the Trition due to the presence of the phenyl rings and the high degree of branching, which increase the microviscosity. The interrelationship between the bilayer solubilization and its apparent microviscosity is also supported by the results of Inove and Kitagawa (18). These authors show that at

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21°C multilamellar liposomes made of various lecithins show different sensitivity toward Triton X-100 in the following order: the highest sensitivity is for dipalmitoyl lecithin > dimyristoyl lecithin > egg yolk lecithin > rat liver lecithin, and it is following the differences in the apparent microviscosity. Is the Triton effect as solubilizer in concentrations below its CMC? Inoue and Kitagawa (17) show release of glucose from multilamellar liposomes by Triton X-100 below its CMC although there is a threshold concentration of Triton which is dependent on the lipid composition. Our results using the Triton fluorescence for egg lecithin liposomes and the results obtained by Yedgar et al. (27) using 3H Triton and SPM liposomes show that when the Triton concentration is below 1 X 10-4 M most of the Triton (90%) is not associated with the lipid, and membrane solubilization does not occur although the membrane integrity was affected. This step requires only a small number of Triton molecules which depends on the lipid composition. Since the Triton concentration in solution will exceed its CMC only in cases of association with the phospholipid as mixed micelle and no micelles of pure Triton exist in the concentration range used by us. It is of interest that the solubilization of phospholipids and cholesterol from erythrocyte membranes by Triton is differential (31). From all lipids present in this membrane, sphingomyelin and cholesterol are preferentially retained in the pellet and require much higher Triton concentrations for their solubilization. This might indicate that in the plane of the membrane there are domains enriched in sphingomyelin and cholesterol (or both). These domains have much higher microviscosity (Barenholz and Shinitzky, in preparation) while the regions enriched in lecithin have a much lower microviscosity. The Triton molecules "prefer" to penetrate the latter regions since their microviscosity (which is a parameter of the free volume and the lipid packing) is lower, and only then, to interact with sphingomyelin.

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I t should be stressed t h a t the solubilization of biological m e m b r a n e s is a more c o m p l i c a t e d process t h a n solubilization of a p h o s p h o l i p i d b i l a y e r since the m e m b r a n e s contain proteins and their lipids m i g h t be organized in a nonr a n d o m i z e d fashion (35), b u t there are m a n y similarities between the effect of T r i t o n X-100 on biological m e m b r a n e s a n d on lipid bilayers. Since liposomes represent a s y s t e m in which the composition can be controlled a n d whose a s y m m e t r i e s can be studied, this m a y increase our u n d e r s t a n d i n g of the m e c h a n i s m of solubilization of m e m b r a n e s , as well as l i p i d lipid and lipid p r o t e i n interactions. ACKNOWLEDGMENTS This work was supported in part by the U.S. Public Health Service, Grants HL17576 and NS02967.

13a. GATT,S., in "Metabolic Inhibitors" (G. H. Questl and M. Kates, Eds.), Vol. 3, p. 349. Academic Press, New York, 1972. 14. WEISSMAN,G., Biochem. Pharmacol. 14, 525 (1965). 15. WEISSMAN, G., AND KEISER, H., Biochem. Pharmacol. 14, 97 (1965). 16. DE DovE, C., WATTIAUX, R., AND WIBO, M., Biochem. Pharmacol. 9, 97 (1962). 17. WEISSMAN, G., SESSA, G., AND WEISSMAN, S., Nature 208, 649 (1965). 18. INOUE, K., AND KITAGAWA,T., Biochim. Biophys. Acta 426, 1 (1976). 19. ROUSER, G., NELSON, G. J., FISCHER, S., AND SIMON, G., in "Biological Membranes" (D. Chapman, Ed.), Vol. 1, p. 69. Academic Press, New York, 1968. 20. SHINITZK¥,M., ANDBARENHOLZ,Y., J. Biol. Chem. 249, 2652 (1974). 21. GATT, S., Biochim. Biophys. Acta 159, 304 (1968). 22. BANGHAM, A. D., DE GIER, J., AND GREVILLE, G. D., Chem. Phys. Lipids 1, 225 (1967). 23. HERTZ, R., AND BARENHOLZ,Y., Biochim. Biophys. Acta 330, 1 (1973).

AVECILLA,L. C., AND SMALL, D. M., J. Lipid Res. 15, 124 (1974). 25. BANGHAM,A. D., FLEMANS, R., HEARD, D. H., AND SEEMAN, G. V. F., Nature (London) 182,

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