Equilibrium and Kinetic Studies of the Solubilization of Phospholipid

1960

Langmuir 2000, 16, 1960-1968

Equilibrium and Kinetic Studies of the Solubilization of Phospholipid-Cholesterol Bilayers by C12E8. The Influence of the Lipid Phase Structure Asier Saéz-Cirioń,† Alicia Alonso,† Fe´lix M. Gon˜i,*,† Todd P. W. McMullen,‡ Ronald N. McElhaney,‡ and Emilio A. Rivas§ Unidad de Biofı´sica (Centro Mixto CSIC-UPV/EHU) and Departamento de Bioquı´mica, Universidad del Paı´s Vasco, P.O. Box 644, 48080 Bilbao, Spain, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, and Instituto de Biologıá Celular y Neurociencias, Facultad de Medicina, Paraguay 2155, 1121 Buenos Aires, Argentina Received July 7, 1999. In Final Form: October 4, 1999 The influence of the lipidic phase structures on their susceptibility to solubilization by the nonionic detergent C12E8 has been explored. To this aim, a phase diagram for dimyristoylphosphatidylcholine/ cholesterol in excess water has been constructed in which phase boundaries were derived from highsensitivity differential scanning calorimetry. Six different lamellar phases can be obtained with this system, namely crystalline (Lc′), gel (Lβ′ or Lβ), rippled (Pβ′), liquid crystalline or fluid (LR), gellike liquid ordered (Loβ), and fluidlike liquid ordered (LoR). The solubilization of samples in each of these phases by C12E8 has been studied through changes in suspension turbidity under equilibrium conditions and also using a stopped-flow time-resolved technique. We find that variations in temperature and cholesterol content within a single phase can affect the equilibrium and kinetic parameters of detergent solubilization somewhat, particularly in the former case. However, much larger variations in the equilibrium and kinetics parameters of C12E8 solubilization are noted between different phases, particularly those phases containing cholesterol. In general, the presence of cholesterol potentiates the solubilization of DMPC vesicles at lower temperatures and inhibits their solubilization at higher temperatures. Moreover, in the more fluid phases (LR for DMPC alone or Loβ and LoR for DMPC-cholesterol mixtures), vesicle turbidity was not affected by detergent concentration until concentration near the solubilization concentration was reached, at which point an increase in turbidity attributed to vesicle lysis and reassembly occurs prior to vesicle solubilization. In contrast, this effect is markedly reduced in the various gel phases of DMPC alone, where vesicle turbidity generally decreases monotonically with detergent concentration. Cholesterol-induced liquid crystallinelike liquid ordered phases (LoR), that are presumed to coexist with the LR phase in animal cell membranes, are much more resistant to solubilization than the predominant fluid disordered LR phase, present in such membranes.

* Corresponding author. Fax +34-944648500. E-mail gbpgourf@ lg.ehu.es. † Unidad de Biofi´sica. ‡ University of Alberta. § Instituto de Biologıá Celular y Neurociencias.

ordered) may coexist in biomembranes.5 The recent observation6 that detergent-insoluble membrane fractions, enriched in sphingomyelin and cholesterol, may constitute microdomains of liquid-ordered lipids in an otherwise liquid-disordered bilayer has greatly increased interest in these kinds of studies. Until recently, the fact that cell membrane lipids were thought to exist exclusively in the fluid disordered state has largely limited the studies of detergent solubilization of both cell and model membranes to liquid-crystalline systems. Even simple cases, such as the solubilization of phospholipids in the gel state, appear to have been largely neglected until recently.7 In the present work, we have undertaken a detailed investigation of the phase diagram of dimyristoylphosphatidylcholine (DMPC)-cholesterol (Ch) in excess water by examining the solubilization properties of a variety of lipid mixtures at different temperatures. Changing lipid composition or temperature allows us to explore several lamellar phases, namely the crystalline (Lc′), gel (Lβ′ or Lβ), liquid ordered gellike (Loβ), liquid ordered liquid crystalline-like (LoR), rippled (Pβ′), and liquid crystalline or fluid (LR) phase. [The prime (′) sign denotes tilted hydrocarbon chains in the various DMPC ordered phases. Certainly for the pure DMPC systems, Lc′, Lβ′ and Pβ′ are

(1) Tanford, C. The Hydrophobic Effect; Wiley: New York, 1980. (2) Cevc, G. and Marsh, D. Phospholipid Bilayers: Physical Principles and Models; Wiley: New York, 1987. (3) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 29. (4) Lichtenberg, D.; Robson, R. J.; Dennis, E. A. Biochim. Biophys. Acta 1983, 737, 285.

(5) Welti, R.; Glaser, M. Chem. Phys. Lipids 1994, 73, 121. (6) Ahmed, S. N.; Brown, D. A.; London, E. Biochemistry 1997, 36, 10944. (7) Patra, S. K.; Alonso, A.; Gon˜i, F. M. Biochim. Biophys. Acta 1998, 1373, 112.

Introduction Soluble amphiphiles, commonly known as detergents, are standard tools in membrane biology where they find application in the solubilization and reconstitution of membrane proteins. In addition, they constitute an intriguing object of study, either alone or in combination with nonsoluble amphiphiles, because of their amazing capabilities of complex interaction with one another and with the solvent. Although the foundations for the study of membrane solubilization by detergents were laid long ago,1-4 many of the fine details of the molecular interactions involved are still to be solved. One aspect that has received comparatively little attention is the influence of the phase structure of the membrane lipids on the solubilizing capacity of the detergent. Apart from obvious theoretical interest, the subject is also physiologically relevant, since lipids in different bilayer phases (gel, fluid, fluid disordered, fluid

10.1021/la9908889 CCC: $19.00 © 2000 American Chemical Society Published on Web 12/28/1999

Solubilization of Phospholipid-Cholesterol Bilayers

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Table 1. The Influence of Phase Structures on the Solubilization of Phospholipid and Phospholipid/Cholesterol Aqueous Dispersions by the Nonionic Detergent C12E8; Equilibrium Studiesa phase

system

solubilization patternb

DSOLc (µM)

DMAXd (µM)

∆Ae (%)

Lc′ Lβ′ Lβ′ Pβ′ LR Loβ LoR Lβ + Loβ

DMPC, 5 °C DMPC, 5 °C DMPC, 16 °C DMPC, 18.5 °C DMPC, 50 °C DMPC/Ch (70:30), 10 °C DMPC/Ch (70:30), 65 °C DMPC/Ch (90:10), 16 °C

A A A A B B B B

1250 950 933 485 1670 500 5300 200

s s s s 950 50 1670 144

s s s s 990 180 1400 790

a Average values of at least three independent experiments. Lipid concentration was 1 mM in all cases. b Solubilization patterns: A, suspension turbidity decreases monotonically with increasing detergent concentrations (e.g., Figure 2A); B, suspension turbidity first increases, then decreases with increasing detergent concentrations (e.g., Figure 2C). c Minimum detergent concentration (µM) that produces ≈0 suspension turbidity. d Detergent concentration (µM) that produces maximum suspension turbidity. e Percent increase in turbidity of the lipid-detergent-water system at the turbidity maximum (100% is the suspension turbidity in the absence of detergent).

Figure 1. Phase diagram for DMPC/cholesterol mixtures in excess water. The phase diagram was constructed using highsensitivity DSC and FTIR on cholesterol/DMPC mixtures annealed at low temperatures.11 The midpoint of the phase transitions of the pure DMPC (cholesterol-poor) domains are denoted by the open figures with (∆) representing the main, chain-melting (LR/Lβ′) phase transition and (3) representing the crystalline to gel (Lc′/Lβ′) phase transition (this transition is found only in mixtures annealed at low temperatures, it is useful because it clearly indicates that pure DMPC domains exist up to 20 mol % cholesterol). When the mixtures are not annealed the phase diagram is identical except that the Lc′/Lβ′ phase transition is replaced by the pretransition (Lβ′/Pβ′) at almost exactly the same temperature. In both cases the cooperativity of these transitions does not vary significantly with increasing cholesterol levels but by 20 mol % the enthalpy of both of these transitions decreases to zero.The chain-melting phase transition of the cholesterol-rich domains in cholesterol/ DMPC mixtures is denoted by the filled symbols. The upper and lower boundaries of the chain-melting phase transition (Loβ/LoR) of the cholesterol-rich domains are shown by (9) while the transition midpoint is shown by (b) (Loβ represents the gellike cholesterol-rich phase and LoR represents the liquidcrystalline-like cholesterol-rich phase).

appropriate. Also, since cholesterol is excluded from the crystalline lamellar phase entirely, Lc′ is also appropriate even in the presence of cholesterol. However, above 5 mol % cholesterol, the pretransition disappears because both the Lβ′ and Pβ′ phases are replaced by an Lβ phase. Of course, the partitioning of detergent into the bilayer may also abolish hydrocarbon chain tilt, an effect that has not been studied up to now.] Lipids have been dispersed in the form of large unilamellar vesicles obtained by extrusion, that are believed to represent rather closely the physical properties of cell membranes. Octa(oxyethyl-

Figure 2. Equilibrium measurements of the C12E8 solubilization of large unilamellar vesicles of pure DMPC in different phases, obtained by equilibration at the appropriate temperatures. The turbidity (A500) of the detergent-treated suspensions is plotted as a function of detergent concentration. Average of three measurements; S. E. M. are approximately the size of the dots.

eneglycol) dodecyl monoether (C12E8) has been used as the soluble amphiphile. C12E8 is a nonionic detergent, widely used in biochemical studies, that is available in highly purified form.8-10 A combination of equilibrium and time-resolved measurements provides evidence for important differences in the solubilization of lamellar systems according to their phase structure. (8) Edwards, K.; Almgren, M. J. Colloid Interface Sci. 1991, 147, 1. (9) Otten, D.; Lo¨bbecke, L.; Beyer, K. Biophys. J. 1995, 68, 584. (10) Kragh-Hansen, U.; le Maire, M.; Møller, J. V. Biophys. J. 1998, 75, 2932.

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Sa´ ez-Cirio´ n et al.

Figure 3. Parameters of DMPC solubilization as a function of temperature. Equilibrium conditions. The predominant phases in the pure lipid are indicated in each case. (3) DSOL, the minimum detergent concentration that produces ≈0 turbidity. (9) Maximum turbidity attained by the suspension at subsolubilizing C12E8 concentrations, shown only for the cases in which such an increase in turbidity occurs. (b) DMAX, detergent concentration producing a maximum in turbidity. The data are derived from plots as shown in Figure 2.

Materials and Methods Detergent C12E8 was a kind gift from Dr. S. Paredes (Brussels). Dimyristoylphosphatidylcholine was from Avanti Polar Lipids (Alabaster, AL). Cholesterol was from Sigma (St. Louis, MO). The phase diagram for DMPC-Ch in excess water was constructed from high-sensitivity differential scanning calorimetry and infrared spectroscopy measurements exactly as described by McMullen and McElhaney11 only using DMPC instead of dipalmitoylphosphatidylcholine. For liposome preparation the lipids were dissolved in chloroform, mixed as required, and the solvent evaporated exhaustively. Lipids were hydrated in 0.04% (w/w) sodium azide, 20 mM Tris HCl buffer, pH 7.4, at a temperature above the gelto-liquid crystalline transition temperature of the mixture. Large unilamellar vesicles were prepared by the extrusion method with filters 0.1 µm in diameter.12 Vesicle size and homogeneity were routinely checked by quasielastic light scattering, using a Malvern Zeta-Sizer spectrometer. Average vesicle diameter was ca. 100 nm, irrespective of bilayer composition. Polydispersity was usually in the 0.08-0.12 range, indicating a sufficiently homogeneous vesicle size distribution. Liposome suspensions were mixed with the same volumes of the appropriate detergent solutions, in the same buffer. The final lipid concentration was always 1 mM. Both liposomes and detergent had been previously equilibrated to the desired temperature. For equilibrium measurements, the mixtures were left to equilibrate for 2 h at the appropriate temperature, and solubilization was assessed from the decrease in turbidity.13 Changes in the structure of lipid aggregates, other than solubilization, may give rise to fluctuations in turbidity. However, all structural changes described up to now (e.g., the gel-to-liquid crystalline transition) produce effects on turbidity that are much smaller than those caused by solubilization (see, e.g., ref 14). Vesicle aggregation may induce large changes in turbidity (see below) but in these cases turbidity is increased, opposite to what happens with solubilization. Turbidity was measured as absorbance at 500 nm in a Cary Bio 3 spectrophotometer, equipped with thermoregulated cell holders. Turbidity values were normalized by setting 100% as the turbidity of the LUV suspension, 1 mM (11) McMullen, T. P. W.; McElhaney, R. N. Biochim. Biophys. Acta 1995, 1234, 90. (12) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161. (13) Alonso, A.; Villena, A.; Gon˜i, F. M. FEBS Lett. 1981, 123, 200. (14) Aranda, F. J.; Go´mez-Fernańdez, J. C. Biochim. Biophys. Acta 1985, 820, 19.

in lipid, in the absence of C12E8, while 0% turbidity corresponded to pure buffer. Total, rather than effective,4 C12E8 concentrations have been used for convenience. This simplification is acceptable because lipid concentration is kept constant (at 1 mM) in all cases. For stopped-flow measurements, a Hi-Tech (Salisbury, U.K.) spectrometer was used, consisting of the following elements: SF51 sample handling unit, LS-10 visible light lamp, M-300 monochromator, PM-60 photomultiplier, connected to a SU-40 amplifier. At least six transients were averaged for each measurement. The optical path for absorbance measurements was 1 cm. The signal was transferred through an A/D converter to a PC computer, through which the machine was operated. Curves were fitted to exponential equations using a GaussNewton algorithm. The dead time of the stopped-flow system was 2.3 ms, after measurements with 2-dichloroindophenol + ascorbate. The contents of the syringes, loaded respectively with detergent and liposome suspensions, were mixed in a 1:1 ratio.

Results The DMPC/Cholesterol Phase Diagram. A phase diagram for DMPC/cholesterol mixtures in excess water is shown in Figure 1. It has been constructed on the basis of a previously published phase diagram for dipalmitoylphosphatidylcholine/cholesterol.11 The different phases present in these systems were identified by X-ray scattering long ago (see ref 15 for a review of the early work). In our case, phase boundaries have been determined mainly from high-sensitivity differential scanning calorimetry data. A short description is included in the figure legend. From 0 to 20% cholesterol the chain-melting transitions of the cholesterol-poor and the cholesterolrich DMPC domains overlap, while above 25 mol % cholesterol only cholesterol-rich phases exist. In addition, increasing levels of cholesterol will increase the temperature and decrease the cooperativity of the cholesterolrich chain-melting phase transition. At 42 mol % the transition is weak and very broad, and at 50 mol % no cooperative phase transition is discernible. For details on the construction and justification for this phase diagram, the reader is referred to McMullen and McElhaney.11 Unlike prior phase diagrams, we do not use the socalled β-phase. We believe that the properties of the (15) Phillips, M. C. Prog. Surf. Membr. Sci. 1972, 5, 139


Figure 4. Equilibrium measurements of the C12E8 solubilization of LUV of DMPC/Ch mixtures at 15 °C. The predominant phases are indicated in each case. The turbidity (A500) of the detergent-treated suspensions is plotted as a function of detergent concentration. Average of three measurements; S. E. M. are approximately the size of the dots.

cholesterol-rich DMPC domains depend on temperature, even at cholesterol levels of approaching 45 mol % cholesterol. That is why we use Loβ and LoR instead of a single β-phase. However, the changes in the properties of

Langmuir, Vol. 16, No. 4, 2000 1963

the bilayer are continuous and gradual, as suggested by the broad transition. Thus many physical techniques may not discern changes in the physical properties of the bilayer at high cholesterol levels.11 Solubilization. Equilibrium Studies. The term solubilization is used in the customary way in biochemistry, meaning the conversion of lipids in the lamellar state into lipid-detergent mixed micelle suspensions (see, e.g., refs 3,4). Aqueous pure DMPC can be found in either the Lc′, Lβ′, Pβ′, or LR phases according to temperature and preincubation times (see previous section and legend to Figure 1). DMPC solubilization by C12E8 is shown in Figure 2 as the decrease in turbidity of lipid dispersions at 5 °C (Lβ′), 18.5 °C (Pβ′) and 50 °C (LR). Samples preincubated at 4 °C so as to induce formation of the lamellar crystalline (Lc′) phase gave very similar results to those of the Lβ′ phase. The data at 5 °C and 50 °C (Figure 2A,C) show the two most common patterns of solubilization as detected through changes in suspension turbidities. In one case (Figure 2A), corresponding to the gel Lβ′ phase, turbidity remains invariant up to a certain detergent concentration, above which it decreases gradually until a minimum (near zero) turbidity is reached. A different pattern is seen for the solubilization of DMPC in the liquid crystalline LR phase (Figure 2C): the turbidity remains invariant, then increases steeply by several fold, finally the suspension becomes optically transparent. This phenomenon was first observed with sonicated DMPC vesicles and Triton X-10013 and was explained in terms of lysis and reassembly of the sonicated vesicles into large multilamellar assemblies, that are later solubilized. In the rippled Pβ′ phase (Figure 2B) the situation resembles the Lβ′ pattern, although a small increase in turbidity is seen. The lowest detergent concentration that produces ≈ 0 turbidity is defined as the solubilizing detergent concentration, DSOL′ and the corresponding values for the various lipidic phases are collected in Table 1. For every observed increase in turbidity, the detergent concentration producing the maximum turbidity (DMAX) and the extent of the turbidity increase (% ∆A) have also been recorded in Table 1. The results of DMPC solubilization assays performed

Figure 5. Parameters of DMPC/Ch solubilization as a function of bilayer compositions. Equilibrium conditions. The predominant phases are indicated in each case. (3) DSOL, the minimum detergent concentration that produces ≈0 turbidity. (9) Maximum turbidity attained by the suspension at subsolubilizing C12E8 concentrations, shown only for the cases in which such an increase in turbidity occurs. (b) DMAX, detergent concentration producing a maximum in turbidity. The data are derived from plots as shown in Figure 4.

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Figure 6. A. Time-resolved change in turbidity of a DMPC LUV suspension when treated with C12E8. Final concentrations: 1 mM DMPC, 1 mM C12E8. Temperature: 50 °C. The increase in turbidity is interpreted as vesicle lysis and reassembly. Bottom: residuals after fitting to two exponentials, see text for details. B. Kinetic parameters of lysis and reassembly of DMPC (LR phase) vesicles as a function of temperature. Lipid and detergent concentrations as in A. (b) Amplitude of exponential 1, ∆A1. (O) Rate constant of exponential 1, K1. (9) Amplitude of exponential 2, ∆A2. (0) Rate constant of exponential 2, K2. (2) Turbidity (absorbance) at infinite time. Average values of three independent experiments, each performed in triplicate. The errors are the size of the dots.

at a variety of temperatures in the 5-50 °C range are summarized in Figure 3. The lowest detergent concentration that produces total solubilization (DSOL) is minimal at the border between the Lβ and Pβ′ phases, and increases steeply with decreasing temperatures in the Lβ gel phase. A similar situation was observed for Triton X-100 and a variety of phospholipids.7 An increase in turbidity is seen for certain C12E8 concentrations at or above the Pβ′ - LR transition temperature, as described in the previous paragraph (see also Figure 2). Once the LR phase is formed, i.e., in the 35-50 °C range, the solubilization parameters appear to be rather independent from temperature. Addition of cholesterol to DMPC bilayers notoriously complicates the phase diagram (Figure 1) and modifies accordingly the detergent effects. To mention but one example, Figure 4 shows the solubilization patterns of bilayers with three different compositions, namely pure DMPC, DMPC/Ch (90:10 mole ratio), and DMPC/Ch (70:30 mole ratio), at 15 °C. The presence of 10 mol % cholesterol (Figure 4B) modifies drastically the behavior of the phospholipid, and a pattern is seen that mimics the

one of pure DMPC at 50 °C (Figure 2C), only solubilization occurs at a much lower C12E8 concentration. When cholesterol is present at 30 mol %, a pure fluid ordered Loβ phase exists (Figure 1) and the solubilization pattern changes again (Figure 4C), with an increase in turbidity at very low surfactant concentration, followed by a slow decrease. An additional example, at a different cross section of the phase diagram, is shown in abbreviated form in Figure 5, for solubilization data at 50 °C, i.e., when only fluid phases exist. The parameters do not vary much between the pure LR (pure DMPC) and the LR + LoR (DMPC + cholesterol) phases. However, the presence of the gellike ordered Loβ phase above 22% cholesterol reduces significantly the extent of the increase in turbidity, and, more importantly, increases by almost 3-fold the detergent concentration required for solubilization. Some selected results of our equilibrium studies on the solubilization of DMPC-Ch mixtures are summarized in Table 1. The solubilization patterns and parameters for the six lamellar phases under study are listed there.


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Figure 7. A. Time-resolved change in turbidity (solubilization) of a DMPC LUV suspension when treated with C12E8. Final concentrations: 1 mM DMPC, 1.67 mM C12E8. Temperature: 46 °C. Bottom: residuals after fitting to two exponentials, see text for details. B. Kinetic parameters of DMPC (LR phase) vesicle solubilization as a function of temperature. Lipid and detergent concentrations as in A. (b) Amplitude of exponential 1, ∆A1. (O) Rate constant of exponential 1, K1. (9) Amplitude of exponential 2, ∆A2. (0) Rate constant of exponential 2, K2. (2) Turbidity (absorbance) at infinite time. Average values of three independent experiments, each performed in triplicate. The errors are the size of the dots.

Important differences are seen between the phases both in the patterns of turbidity vs detergent concentration curves and in the solubilization parameters. What we have called pattern A, i.e., suspension turbidity decreasing monotonically with detergent concentration, is favored by low temperatures and low cholesterol concentrations. In general, within a given phase, changes in temperature and/or composition do not greatly affect the solubilization parameters (with the above-mentioned exception of the influence of temperature in the solubilization of the gel phase, see in Table 1 the data for Lβ at 5 and 16 °C). Both the detergent concentrations required to produce full solubilization (DSOL) and those producing a maximum in turbidity (DMAX) may change by over 1 order of magnitude with the different lamellar phases. The liquid ordered LoR phase is, among those studied in this work, the most resistant to C12E8. By contrast, we have also included in the Table the system DMPC/Ch (90:10) at 16 °C, that is solubilized at the lowest detergent concentration among all samples tested. Note that the latter corresponds to a region of the phase diagram in which two phases, Lβ and Loβ, both rather sensitive to solubilization, coexist.

Solubilization. Time-Resolved Studies. One inherent limitation of the equilibrium studies presented above is that the detergent itself modifies the phase structure of the lipids and changes the phase boundaries once it becomes inserted in the bilayer.16-18 Thus the equilibrium data have been complemented by a series of stopped-flow rapid kinetic measurements that reveal the first stages of detergent-bilayer interaction19 when the bilayer architecture remains intact. We have examined conditions in which either the phenomenon of the increase in turbidity (vesicle reassembly or fusion) or the progressive vesicle solubilization prevails. The equimolar mixture of DMPC and C12E8 at 50 °C (LR phase) leads to a maximum of turbidity (Figure 2C). The corresponding time-resolved curve of a stopped-flow (16) Alonso, A.; Gon˜i, F. M. J. Membrane Biol. 1983, 71, 183. (17) Jackson, M. L.; Schmidt, C. F.; Lichtenberg, D.; Litman, B. J.; Albert, A. D. Biochemistry 1982, 21, 4576. (18) Gon˜i, F. M.; Urbaneja, M. A.; Arrondo, J. L. R.; Alonso, A.; Durrani, A. A. Eur. J. Biochem. 1986, 160, 659. (19) Alonso, A.; Urbaneja, M. A.; Gon˜i, F. M.; Carmona, F. G.; Cańovas, F. G.; Go´mez-Fernańdez, J. C. Biochim. Biophys. Acta 1987, 902, 237.

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Figure 8. A. Time-resolved change in turbidity (solubilization) of a DMPC/Ch (94:6 mole ratio) LUV suspension when treated with C12E8. Final concentrations: 1 mM lipid, 3 mM C12E8. Temperature: 50 °C. Bottom: residuals after fitting to two exponentials, see text for details. B. Kinetic parameters of DMPC/Ch LUV solubilization as a function of bilayer composition. Final concentrations: 1 mM lipid, 3 mM surfactant. Temperature: 50 °C. (b) Amplitude of exponential 1, ∆A1. (O) Rate constant of exponential 1, K1. (9) Amplitude of exponential 2, ∆A2. (0) Rate constant of exponential 2, K2. (2) Turbidity (absorbance) at infinite time. Average values of three independent experiments, each performed in triplicate. The errors are the size of the dots.

experiment can be seen in Figure 6A. The data are best fitted to two exponentials, a fast one, with amplitude ∆A1 ) 0.424 ( 0.0036 au and rate constant K1 ) 3.25 ( 0.037 s-1, and a slow one, with ∆A2 ) 0.617 ( 0.0039 au and K2 ) 0.7367 ( 0.0035 s-1. The physical meaning of these two exponentials cannot be ascertained at present, but the fact that turbidity vs time curves obtained under different conditions can be analyzed similarly, with exponential parameters remaining constant or varying gradually with temperature or lipid composition (see below), suggest that at least the two-exponential fit can be used with internal consistency in our analysis. Essentially similar data to those of Figure 6A are obtained for experiments performed in the 38-50 °C range, all of them within the domain of the pure LR phase (Figure 6B), confirming that within a given phase, changes in temperature do not modify greatly the detergent-induced processes. Solubilization of DMPC in the LR phase is achieved at higher detergent concentrations than those producing maximum turbidity. An example of fast solubilization caused by 1.67 mM C12E8 at 46 °C is shown in Figure 7A.

A summary of similar experiments at 38-46 °C (Figure 7B) indicates that the kinetic parameters do not vary between 38 and 42 °C, while the exponential constants K1 and K2 increase rapidly between 42 and 46 °C, indicating a large increase in the rate of this process. The diagram in Figure 7B may be suggesting a transition from a situation under thermodynamic control below 42 °C, to one under kinetic control at higher temperatures. Both fusion and solubilization of cholesterol-containing mixtures have been examined by stopped-flow rapid kinetic methods. Fusion, or, more rigorously, lysis and reassembly of the vesicles, occurs as a kinetically complex process in the presence of cholesterol at 50 °C, i.e., when the LR and LoR phases coexist (Figure 1). Under those conditions, the process cannot be easily fitted to a small number of components (data not shown), thus this phenomenon was not investigated any further. In the same region of the phase diagram, however, solubilization curves at 3 mM C12E8 (e.g. Figure 8A) can be fitted to the usual two exponentials, and the kinetic parameters do not change much with cholesterol concentrations in the 0-10 mol % range (Figure 8B). Samples containing higher


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Table 2. Kinetic Parameters for the Solubilization of Lipidic Dispersions with Different Phase Structures by C12E8 phase

system

[C12E8] (µM)

∆A1 (a.u.)

K1 (s-1)

∆A2 (a.u.)

K2 (s-1)

Lβ′ Pβ′ LR Loβ LoR

DMPC, 12 °C DMPC, 18.5 °C DMPC, 46 °C DMPC/Ch (70:30),12 °C DMPC/Ch (70:30),62 °C

950 485 1670 500 5150

-0.0443 -0.0506 -0.0680 -0.108 -0.446

55.1 2.35 72.2 30.3 2.19

-0.0469 -0.0198 -0.0430 -0.0234 -1.35

0.120 0.402 5.30 0.133 1.02

a Final lipid concentration was 1 mM in all cases. The absorbance vs time curve after rapid mixing of LUV with surfactant has been fitted to two exponentials, of which ∆A1, ∆A2, K1 and K2 represent, respectively, the total changes in absorbance and the rate constants.

cholesterol concentrations again undergo complex kinetics in the presence of 3 mM C12E8, probably the result of concurrent fusion and solubilization phenomena. Representative results of the above kinetic studies are collected in Table 2, for the solubilization step only. Important differences in the extent of the change in turbidity, and particularly in the rate constants, can be seen between the different phases. The differences cannot be attributed merely to thermal effects, because sometimes an increase in temperature is accompanied by a decrease in rate (e.g., K1 for Lβ at 12 °C and Pβ′ at 18.5 °C), nor to different lipid compositions, because differences are seen even with pure DMPC at various temperatures. Solubilization of Liquid-Ordered Phases. In view of the interest elicited by the so-called detergent-resistant membranes, that have been attributed to the presence of microdomains rich in sphingolipids and cholesterol, putatively in a liquid-ordered phase,6 we have studied comparatively the solubilization of the fluid disordered LR phase (pure DMPC above 23 °C) and the fluidlike liquidordered LoR phase (DMPC/Ch, 70:30 mole ratio, at 65 °C). Equilibrium results are shown in Figure 9A. In both cases a B-type curve is observed, with a large increase in turbidity that precedes solubilization. However the patterns are clearly different, mainly because the LR phase is completely solubilized at 1.67 mM C12E8, while the LoR displays a maximum of turbidity at such detergent concentration, full solubilization being achieved at a much higher detergent concentration, ca. 5 mM. The timeresolved curves shown in Figure 9B and C, respectively for the LR and LoR phases, also indicate clearly the inherent resistance to solubilization of the fluid ordered phase (note the different turbidity scales in Figure 9B and 9C). Discussion The present study was intended to answer the question of whether the lipid phase structure had an influence on the phenomenon of bilayer solubilization by detergents. For this purpose, a phase diagram was built for DMPC/ Ch mixtures in excess water, that showed as many as six different phases, all of them maintaining the bilayer disposition (Figure 1). For a more detailed information on the structure of these phases see refs 11,15. After extensive equilibrium and time-resolved experiments in virtually all regions of the phase diagram, it is apparent that the phase structure imposes quantitative and qualitative differences on the interaction of C12E8 with the lipid bilayer. Studies as a function of temperature, both at equilibrium (Figure 3) and time-resolved (Figures 6B, 7B), show that within the boundaries of a given phase, as expressed in Figure 1, the parameters of detergentmembrane interaction change relatively little. The same can be said from studies in which the DMPC/Ch ratio is changed (Figures 5, 8B). However, when the experimental conditions are such that the phase structure is changed, then the solubilization parameters change dramatically as well, as summarized in Tables 1 and 2. To the authors knowledge, this is the first observation of such phase dependence of membrane solubilization by

Figure 9. Solubilization of a liquid crystalline-like liquid ordered phase (LoR). A. Solubilization pattern of DMPC/Ch (70: 30, mole ratio) at 65 °C. Average of three measurements, errors are approximately the size of the dots. Broken line: data for pure DMPC at 50 °C (LR phase) replotted from Figure 2C. B. Time-resolved change in turbidity of a DMPC LUV suspension (62 °C, LR phase) when treated with C12E8. Final concentrations: 1 mM DMPC, 5.15 mM C12E8. C. Time-resolved change in turbidity of a DMPC/Ch (70:30, mole ratio) LUV suspension (62 °C, LoR phase) when treated with C12E8. Final concentrations: 1 mM DMPC, 5.15 mM C12E8.

detergents. Previous studies of the interaction of C12E8 with phospholipid bilayers or cell membranes8,10 were carried out only with lipids in the fluid disordered state (LR). Our observations for the LR phase are in good agreement with those. However, the differences observed among the various phases remain to be explained. A simple

1968


hypothesis would involve geometrical considerations of the disposition of lipids in the different phases, in relation to the entry of detergent monomers into the bilayer. Detergent partitioning appears to precede cooperative binding and solubilization,10 and lipid phase structure is an obvious factor that will influence detergent partition. Apart from the quantitative differences between phases summarized in Tables 1 and 2, an important qualitative difference occurs as well, namely the detergent-induced increase in turbidity that occurs at subsolubilizing surfactant concentrations in all phases except Lc, Lβ and Pβ′ (class B patterns in Table 1). This phenomenon was first observed with DMPC and Triton X-100 by Alonso et al.,13 and attributed to vesicle lysis and reassembly into multilamellar structures. Otten et al.9 did perform some experiments with a 2:1 (mole ratio) DMPC/C12E8 system in the 10-45 °C temperature range, and observed an increase in vesicle size above the phase transition temperature of the pure lipid. The facilitation of pattern B by cholesterol-induced ordering of the lipid chains had already been put forward by Saéz et al.20 The Lβ gel phase is peculiar in that changes in temperature within the said phase affect considerably the detergent solubilization, lower temperatures making the bilayer more resistant to solubilization. This is seen for DMPC and C12E8 in Figure 3 and Table 1, and the observation is in agreement with the data for Triton X-100 and a variety of phosphatidylcholines published by Patra et al.7 In that publication the temperature-sensitivity of Lβ phase solubilization was attributed to the fact that bilayers in the gel phase exhibit, apart from a large increase in order when cooling below the gel-fluid transition temperature, a small but progressive ordering, (20) Saéz, R.; Gon˜i, F. M.; Alonso, A. FEBS Lett. 1985, 179, 311.


once in the gel state, with decreasing temperatures.21 An increase in lipid order is likely to decrease the membrane/ water partition coefficient of the detergent, and thus to increase the resistance of the gel phase toward detergents. The same phenomenon is probably operating in the observed resistance to detergent solubilization by a fluid ordered phase (LoR) as compared to LR (Figure 9). This observation provides clear support to the idea that the so-called detergent-resistant membranes, actually fractions derived from animal cell plasma membranes, resist solubilization by Triton X-100 because they are in the liquid-ordered state.6 Highly ordered lipid domains would inhibit the insertion of detergent monomers, commonly accepted as the first step in membrane solubilization.3,10 In fact, in the extreme case of the purple membrane of Halobacterium, in which lipids form two-dimensional crystalline assays, we have proposed that detergent insertion is virtually impossible, with the surfactant binding only the periphery of the purple membrane patches.22 Acknowledgment. This work was supported in part by grants from CICYT (PB96/0171), the Basque Government (Ex98/28) and the University of the Basque Country (G03/98). A.S.C. is a predoctoral student supported by the Basque Government. E.R. is a Career Investigator from CONICET (Argentina). The authors are grateful to Dr. S. Paredes (Brussels) for his gift of C12E8. LA9908889 (21) Bartucci, R.; Pa´li, T.; Marsh, D. Biochemistry 1993, 32, 274. (22) Viguera, A. R.; Gonza´lez-Manãs, J. M.; Taneva, S.; Gon˜i, F. M. Biochim. Biophys. Acta 1994, 1196, 76.

Equilibrium and Kinetic Studies of the Solubilization of Phospholipid

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