© Copyright 1999 American Chemical Society
SEPTEMBER 29, 1999 VOLUME 15, NUMBER 20
Letters Transbilayer Movement of Sodium Dodecyl Sulfate in Large Unilamellar Phospholid Vesicles M. Co´cera,† O. Lo´pez,† J. Estelrich,‡ J. L. Parra,† and A. de la Maza*,† Departamento de Tensioactivos, Centro de Investigacio´ n y Desarrollo (C.I.D.), Consejo Superior de Investigaciones Cientı´ficas (C.S.I.C.), C/ Jordi Girona, 18-26, 08034 Barcelona, Spain, and Departament de Fisicoquı´mica, Facultat de Farma` cia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain Received June 2, 1999. In Final Form: July 29, 1999 The ability of sodium dodecyl sulfate (SDS) to traverse phosphatidylcholine bilayers was examined by fluorescence spectroscopy. To this end, we measured the interaction of the anionic fluorescent probe 2-(ptoluidinyl)naphthalene-6-sodium sulfonate present in the outer vesicle leaflet with the SDS monomers incorporated in this structure. The surfactant transbilayer movement, or “flip-flop”, was measured from the fluorescence intensity changes due to the interaction of the liposome/probe with SDS versus incubation time. This effect was quantified as the resulting variations in the surface potential (ψo) of liposomes. When the SDS concentration increased, ψo rose due to the electronegative contribution of the sulfate group incorporated in the bilayer surface. Increased periods of incubation resulted in a decreased ψo and, consequently, in a fall in the number of surfactant molecules in the outer vesicle leaflet. This variation was associated to the SDS “flip-flop”. The maximum “flip-flop” (of about 50%) was always detected at a very low surfactant concentration, and the effective molar ratio of surfactant to PC for this maximum was always a constant value (0.02 mol/mol). Although the incorporation of SDS monomers to the bilayer surface was a very rapid process, the “flip-flop” rate of these monomers across lipid bilayer was very slow and time dependent with an enhanced kinetics between 10 and 90 min after mixing.
Introduction The interaction of surfactants with phopholipid vesicles leads to the solubilization of these structures via formation of mixed micelles. However, the mechanisms of this interaction and the means by which it is generated remain relatively obscure. In general terms, this complex process involves a partition equilibrium of surfactant monomers between bilayers and water1-5 and, consequently, the transbilayer migration or “flip-flop” of surfactant mol† ‡
Consejo Superior de Investigaciones. Universitat de Barcelona.
(1) Liu, Y.; Regen, S. L. J. Am. Chem. Soc. 1993, 115, 708-713. (2) Paternostre, M.; Meyer, O.; Gabielle-Madelmont, C.; Lesieur, S.; Ghanam, M.; Ollivon, M. Biophys J. 1995, 69, 2476-2488. (3) Inoue, T., Interaction of Surfactant with Phospholipid Vesicles. In Vesicles; Rosoff, M., Ed.; Surfactant Science Series, Volume 62; Marcel Dekker Inc.: New York, 1996; Chapter 5.
ecules. Liposomes have been used as simple membrane models to study the ability of different lipids and surfactants to undergo transbilayer flip-flop.6-10 Thus, Cabral et al.11 and Donovan and Jackson12 reported the transbilayer movement of bile salts in egg phosphatidylcholine (4) Wenk, M. R.; Alt, T.; Seelig, A.; Seelig, J. Biophys J. 1997, 72, 1719-1731. (5) Wenk, M. R.; Seelig, J. Biophys J. 1997, 73, 2565-2574. (6) Eastmann, S. J.; Hope, M. J.; Cullis P. R. Biochemistry 1991, 30, 1740-1745. (7) Wimley, W.; Thompson, T. E. Biochemistry 1991, 30, 1702-1709. (8) Bhattacharya, S.; Moss, R. A.; Ringsdorf H.; Simon, J. J. Am. Chem. Soc. 1993, 115, 3812-3813. (9) Jezek, P.; Modriansky, M.; Garlid, K. D. FEBS Lett. 1997, 408, 161-165. (10) le Marie, M.; Møller, J. V.; Champeil, P. Biochemistry 1987, 26, 4803-4810. (11) Cabral, D. J.; Small, D. M.; Lilly, H. S.; Hamilton, J. A. Biochemistry 1987, 26, 1801-1804.
10.1021/la9906929 CCC: $15.00 © 1999 American Chemical Society Published on Web 09/11/1999
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unilamellar liposomes. Furthermore, Lentz et al.13 reported the transbilayer lipid redistribution by treatment of phospholipid vesicles with poly(ethylene glycol). In earlier papers we reported the kinetic studies and the structural changes resulting in the interaction of alkyl sulfates with phosphatidylcholine liposomes.14-17 Here, we seek to extend these investigations by studying the ability of sodium dodecyl sulfate to undergo transbilayer flip-flop. To this end, we have developed a fluorescent assay utilizing the probe 2-(p-toluidinyl)naphthalene-6sodium sulfonate (TNS), which reports on the surface potential (ψo) of membranes.6,18,19 The “flip-flop” of surfactant molecules is associated to the ψo changes of vesicles as a function of the time of incubation of the liposome/ probe with surfactant. The use of this simple spectroscopy technique may shed light on the not very well understood mechanism of transbilayer movement of this biologically active surfactant in the initial steps of its interaction with phosphatidylcholine vesicles. Materials and Methods Phosphatidylcholine (PC) was purified from egg lecithin (Merck, Darmstadt, Germany) by the method of Singleton20 and was shown to be pure by thin-layer chromatography (TLC). Sodium dodecyl sulfate (SDS) was supplied by Lancaster Synthesis Ltd. (Strasbourg, France) and further purified by column chromatography.21 Tris(hydroximethyl)aminomethane (TRIS) was obtained from Merck. TRIS buffer was prepared as 5.0 mM TRIS buffer adjusted to pH 7.4 with HCl and containing 100 mM NaCl. The fluorescent agent 2-(p-toluidinyl)naphthalene6-sodium sulfonate (TNS) purchased from Sigma was prepared as 100 µM TNS in TRIS buffer and stored at 4 °C. Polycarbonate membranes were purchased from Nucleopore (Pleasanton, CA). Preparation of PC Liposomes and Interaction with SDS. Unilamellar vesicles (LUV) of a defined size (of about 200 nm) were prepared from large unilamellar vesicles obtained by means of a reverse phase evaporation technique14 followed by a 10-fold passage through 800-200 nm polycarbonate membranes.22 To incorporate the probe on the vesicle surface TNS was added to liposomes. A period of about 30 min was needed to obtain a complete and stable incorporation of TNS on the vesicle surface (measured as a constant fluorescence intensity value of the liposome/probe). Afterward, equal volumes of appropriate surfactant solutions were added to liposome/probe suspensions (PC concentration ranging from 0.5 to 2.5 mM). After that, fluorescence measurements were performed at 25 °C. Fluorescence Measurements. TNS is a probe of membrane surface potential introduced by Eisenberg and co-workers.18 The main pathway in the excited state of TNS is a twisted intramolecular charge transfer, which is quite fast in the highly polar aqueous media. Hence, the quantum yield of fluorescence of this probe is extremely low in water.23 The addition of TNS to liposomes led to the adsorption of these molecules on the bilayers surface. This adsorption produces a marked increase in their (12) Donovan, J. M.; Jackson, A. A. Biochemistry 1997, 36, 1144411451. (13) Lentz, B. R.; Talbot, W.; Lee, J.; Zheng, L.-X. Biochemistry 1997, 36, 2076-2083. (14) de la Maza, A.; Parra, J. L. Langmuir 1995, 11, 2435-2441. (15) de la Maza, A.; Parra, J. L. Langmuir 1996, 12, 3393-3398. (16) Lo´pez, O.; Co´cera, M.; Pons, R.; Azemar, N.; de la Maza, A. Langmuir 1998, 14, 4671-4674. (17) Co´cera, M.; Lo´pez, O.; de la Maza, A.; Parra, J. L.; Estelrich, J. Langmuir 1999, 15, 2230-2233. (18) Eisenberg, M.; Gresalfi, T.; Riccio, T.; McLaughlin, S. Biochemistry 1979, 18, 5213-5223. (19) Mukerjee, P.; Moroi, Y.; Murata, M.; Yang, Y. S. Hepatology 1984, 4, 61S-65S. (20) Singleton, W. S.; Gray, M. S.; Brown, M. L.; White, J. L. J. Am. Oil Chem. Soc. 1965, 42, 53-57. (21) Rosen, M. J.; Hua, X. Y. Colloid Interface Sci. 1982, 86, 164168. (22) Dorovska-Taran, V.; Wich, R.; Walde, P. Anal. Biochem. 1996, 240, 37-47. (23) Chang, L.; Cheung, H. C. Chem. Phys Lett. 1990, 173, 343-346.
Letters fluorescence, which is quenched by the presence of negative charges as those of SDS. This variation allows calculation of the surface potential (ψo) of the charged vesicles. This probe has been used as alternative to other markers to characterize the incorporation of anionic surfactants as alkyl sulfates to bilayers as well as the self-association of bile salts in water.17,19 Fluorescence measurements were performed on a spectrofluorometer (Shimadzu RF-540, Kyoto Japan) with excitation wavelength (λexc) at 321 nm and emission (λem) at 446 nm. The surface potential of vesicles (ψo) was calculated on the basis of the ratio of fluorescence of pure liposomes and those containing surfactant molecules at the same lipid concentration using the relation
f(-)/f(0) ) exp{Fψo/RT}
(1)
where f(0) and f(-) are the fluorescence intensity in the absence and in the presence of quencher, F is the Faraday constant, R is the gas constant, and T is the absolute temperature. This equation is useful when a small fraction of binding sites are occupied by TNS at the liposome surface, and when the aggregates provide very similar surface environments for TNS to give equal lifetimes, as occurred in our experimental conditions. From the surface potential values (ψo) (expressed in volts), the surface charge density (σo) expressed in µC cm-2 for symmetrical electrolytes may be calculated by means of24
σo ) 11.74xc sin h(zeψo/2kT)
(2)
where c is the electrolyte concentration in mol L-1, z the valence of ions, e the elementary charge, k the Boltzmann constant, and T the absolute temperature. The number of charged molecules (n) can be obtained from:
n)
σo 1.60219 × 10-19
Sext × 10-22
(3)
where Sext is the external surface of a vesicle expressed in Å2.
Results and Discussion Detection of the SDS Transbilayer Migration Using TNS. The transbilayer migration of SDS molecules was measured from the fluorescence intensity changes due to the interaction of the liposome/probe with SDS versus time. This effect was quantified as the resulting variations in the surface potential (ψo) of liposomes. We first checked the fluorescence lifetime of the probe and the optimal ratio lipid/probe. Fluorescence intensity was almost constant during 6 h after mixing liposomes with the probe, the molar ratio PC/TNS 100 being the most appropriate. Around this ratio, the net TNS fluorescence was proportional to the number of probe molecules adsorbed to the membrane and its fluorescence in aqueous solution was negligible with respect to that of the TNS bound to lipid vesicles. Given that no changes in the fluorescence intensity of the liposome/probe occurred during 6 h, we may assume that under our experimental conditions (buffered system adjusted to pH 7.4) no migration of the probe to the interior of vesicles occurred. These findings are in line with the assays reported by Eastman et al., in relation to the partitioning of TNS into the interior lipid monolayer as a function of the pH gradient (transmembrane pH gradient).6 This partitioning would be expected to result in variations in the fluorescence intensity due to the different aqueous to lipid volume ratio in the LUV interior. To study the surface potential (ψo) variations of liposomes due to the incorporation of SDS and its ability to undergo flip-flop (variations of ψo versus the incubation (24) Hunter, J. R. Zeta potential in Colloid Science; Academic Press: London, 1981; pp 28
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Figure 1. Variation in the fluorescence intensity of PC bilayers (PC concentration 1.0 mM and PC/TNS molar ratio 100) containing increasing amounts of sodium dodecyl sulfate after different times of incubation (10 s ([), 30 min (9), and 270 min (2), respectively). In each curve fluorescence intensity 100% corresponded to that of liposomes/probe lacking surfactant after the same period of time.
time liposome/probe-surfactant) the anionic probe TNS was used. Thus, fluorescence changes of liposomes (PC/ TNS molar ratio 100) containing increasing amounts of SDS after different times of incubation are plotted in Figure 1. The assays were carried out in triplicate, and the results given are the averages. The standard deviations of the data for each point were lower than 1.1%. In each curve 100% fluorescence intensity corresponded to that of liposomes/probe lacking surfactant after the same period of time. An abrupt decrease in the fluorescence intensity occurred (even after 10 s of mixing) due to the incorporation of a very low concentration of surfactant monomers (SDS concentration markedly lower than its cmc, which was 0.75 mM). This finding is in agreement with our previous electrokinetic studies of the interaction of alkyl sulfates with PC liposomes using TNS.17 Increasing periods of incubation liposome/probe with SDS led to a relative rise in the fluorescence intensity values. Given that in the period investigated no migration of the probe to the interior of vesicles occurred (no changes in ψo of the liposome/ probe versus time) the rise in the fluorescence intensity detected with time of incubation (Figure 1) was only attributed to the transbilayer movement of surfactant molecules present in the outer vesicle leaflet or “flip-flop”. This surfactant migration is expected to reduce the quenching of the fluorescent probe inserted in the outer monolayer. The theoretical surface potentials (ψo) calculated from the fluorescence intensity values applying eq 1 allows determination of the variations in charge surface density (σo) of liposomes (eq 2) and, consequently, the variation in number of molecules of surfactant present in the outer membrane leaflet per vesicle (eq 3). This variation quantifies the transbilayer migration of surfactant molecules per vesicle as a function of the time of incubation. Given that the liposomes used were unilamellar and formed by spherical vesicles with a diameter of 190 nm,17,25 and assuming that the surface area on the lipid molecules was 70 Å2 and the thickness of the bilayers was 4 nm,26 an outer vesicle surface of 11.3 × 106 Å2 (Sext, eq 3) was obtained with 160 000 molecules of lipid in this surface. This value is consistent with the data reported by Lasic.26 (25) Lo´pez, O.; de la Maza, A.; Coderch, L.; Lo´pez-Iglesias, C.; Wehrli, E.; Parra, J. L. FEBS Lett. 1998, 426, 314-318. (26) Lasic, D. D. In Liposomes: from Physics to Applications; Elsevier Science Publishers: Amsterdam, 1993; p 554.
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Figure 2. Variation in the number of surfactant molecules present in the bilayer surface per vesicle (PC concentration 1.0 mM) versus the sodium dodecyl sulfate concentration in the system after different times of incubation (10 s ([), 30 min (9), and 270 min (2), respectively).
The variations of the number of SDS molecules present in the bilayer surface (per vesicle) versus the surfactant concentration for different times of incubation are plotted in Figure 2. The incorporation of surfactant monomers to the outer bilayer leaflet was a very rapid process, in which a great number of molecules were already incorporated 10 s after the surfactant addition in the range of SDS concentration investigated (from 0 to 0.5 mM). Furthermore, the number of molecules incorporated increased almost linearly with the surfactant concentration up to 0.15 mM SDS, especially for 270 min of incubation. Hence, a linear incorporation of surfactant molecules on the bilayer surface occurred in this surfactant concentration range. Increasing SDS amounts led to a relative decrease in this incorporation possibly due to the increasing electrostatic repulsion between the incorporated molecules and those in process of incorporation. It is noteworthy that for the same SDS concentration the number of surfactant monomers present in the outer vesicle leaflet decreased with the time of incubation due to their transbilayer migration. Figure 3 shows the variation in the percentage of surfactant flip-flop versus the effective molar ratio of surfactant to PC (Re) for three PC concentrations (0.5, 1.0, and 2.5 mM). The “flip-flop” percent is defined as the difference between the number of molecules present in the outer monolayer after 30 and 270 min of incubation and those present 10 s after the surfactant addition to the liposome/probe. It is noteworthy that the maximum “flipflop” (about 50%) always occurred at a very low surfactant concentration and that the Re for this maximum was the same in all cases (0.02 mol/mol). This finding underlines the influence of the physicochemical characteristics of the liposomes on this process. The fact that increasing Re values resulted in a decrease in their transbilayer movement shows that an optimum number of surfactant molecules per vesicle is needed to obtain the maximum flip-flop. It is interesting to note that this optimum Re (0.02) was clearly lower than the ReSAT (effective surfactant to phospholipid molar ratio for bilayer saturation) reported for this surfactant-liposome system (1.1 mol/mol).14 Hence, the ability of SDS to undergo transbilayer movement in unilamellar PC vesicles did not seem to be corrrelated with its capacity to saturate these bilayer structures. An interesting question is how fast surfactant molecules move through bilayers. To clarify this question we determined the variation in the percentage of flip-flop at the optimum Re versus the time of incubation using a PC
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Figure 3. Variation in the percentage of flip-flop of surfactant molecules versus the effective molar ratio of surfactant to PC (Re) for three PC concentrations (0.5, 1.0, and 2.5 mM). This percentage is defined as the number of surfactant molecules present in the outer monolayer after 30 and 270 min of incubation in relation to those present 10 s after the surfactant addition to the liposome/probe. Symbols: 0.5 mM PC/30 min ([), 0.5 mM PC/270 min (]), 1.0 mM PC/30 min (2), 1.0 mM PC/270 min (4), 2.5 mM PC/30 min (b), 2.5 mM PC/270 min (O).
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Afterward, a progressive flip-flop plateau was achieved with time. From these findings we may assume that the flip-flop rate of SDS molecules across lipid bilayer was slow and very dependent on the time of incubation. These findings are in accordance with those reported by various authors,27,28 who demonstrated for different surfactants and using black membrane experiments that this effect was fairly slow. Comparison of Figures 2 and 4 reveals that although the incorporation of SDS monomers to the bilayer surface was a very rapid process, the flip-flop rate of these monomers across lipid bilayer was very slow and time dependent with an enhanced kinetics between 10 and 90 min after mixing. Hence, although the SDS monomers exhibit a great affinity by PC bilayers, the slow transbilayer migration of these monomers appears to be the rate-limiting step in the adsorption of SDS on these bilayers. This assumption is in line with the recent studies reported by Kragh-Hansen et al.,29 who using radioactive experiments attributed the extremely slow process of solubilization of PC liposomes by SDS to the slow transbilayer migration of this surfactant in closed vesicles. Hence, the use of the TNS probe is proposed as a new and simple fluorescence spectroscopy technique to study the transbilayer migration of anionic amphiphiles as SDS in biological membranes. Abbreviations PC, phosphatidylcholine SDS, sodium dodecyl sulfate TRIS, tris(hydroximethyl)aminomethane TNS, 2-(p-toluidinyl)naphthalene-6-sodium sulfonate ψo, surface potential Re, effective molar ratio of surfactant to PC cmc, critical micellar concentration TLC, thin-layer chromatography
Figure 4. Variation in the percentage of flip-flop of sodium dodecyl sulfate molecules at the optimum surfactant to PC molar ratio (Re 0.02 mol/mol) versus time of incubation using a liposomes/probe at a PC concentration 1.0 mM.
concentration 1.0 mM. The curve obtained is plotted in Figure 4. Although in the initial 10 min of incubation no “flip-flop” was detected, a subsequent state of enhanced flip-flop occurred up to approximately 90 min after mixing.
Acknowledgment. We are grateful to Mr. G. von Knorring for expert technical assistance. This work was supported by funds from DGICYT, Prog No PB94-0043, Spain. LA9906929 (27) Van Zutphen, H.; Merola, A. J.; Brierly, G. P.; Cornwell, D. G. Arch. Biochem. Biophys. 1972, 152, 755-766. (28) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 2979. (29) Kragh-Hansen, U.; le Maire, M.; Møller, J. V. Biophys. J. 1998, 75, 2932-2946.
