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Characterization of Sphingosine-Phosphatidylcholine Monolayers: Effects of DNA V. Matti J. Sa¨ily,† Juha-Matti Alakoskela,† Samppa J. Ryha¨nen,† Mikko Karttunen,‡ and Paavo K. J. Kinnunen*,†,§ Helsinki Biophysics & Biomembrane Group, Institute of Biomedicine, University of Helsinki, Helsinki, Finland, and Biophysics & StatMech Group, Lab of Computational Engineering, Helsinki University of Technology, Helsinki, Finland Received February 21, 2003. In Final Form: July 30, 2003 Monolayers of the naturally occurring cationic lipid sphingosine and its mixtures with 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) were studied using a Langmuir balance. More specifically, we measured the force-area (π-A) isotherms and determined the monolayer dipole potential Ψ as a function of the mole fraction of sphingosine (XSph) with and without a charge-saturating concentration of DNA in the subphase. Both sphingosine and POPC exhibited smooth compression isotherms, indicating their monolayers to be in the liquid expanded state. Even low contents (XSph ) 0.05) of sphingosine in a POPC monolayer condensed the film dramatically, by 20% at 20 mN/m. This effect is suggested to reflect a reorientation of the P--N+ dipole of the POPC headgroup (Sa¨ily, V. M. J.; Ryha¨nen, S. J.; Holopainen, J. M.; Borocci, S.; Mancini, G.; Kinnunen, P. K. J. Biophys. J. 2001, 81, 2135), in keeping with a simultaneous and pronounced increase in Ψ. Mixed monolayers of sphingosine and POPC exhibited three critical mole fractions XSph of sphingosine, viz., 0.25, 0.6, and 0.83, at which the area/molecule reached a local minimum, followed by a pronounced expansion of the film. This suggests energetically favorable ordering, which allows the positively charged sphingosines to maximize their distance, so as to minimize the Coulombic repulsion. The presence of DNA affected the mixed POPC/sphingosine monolayers differently depending on the constituent lipid stoichiometry, yet the same discontinuities were evident as in the presence of DNA.
Introduction Sphingolipids represent one of the principal structural elements of eukaryotic biomembranes, and in the outer surface of the plasma membrane surrounding the cell they may amount to one-third of the total lipid.2 A number of active compounds have been identified and include complex glycosphingolipids,3 sphingosine,4 ceramides,5 dimethylsphingosine,6 and sphingosylphosphorylcholine.7 Sphingosine, a single-chain amphiphilic sphingolipid metabolite, has been shown to modulate diverse cellular functions, including growth and differentiation, activation of host defense systems, receptor function, and oncogenesis.8 Recently sphingosine has been reported to decrease * To whom correspondence should be addressed. Helsinki Biophysics & Biomembrane Group, Institute of Biomedicine, P.O. Box 63 Biomedicum (Haartmaninkatu 8), FIN-00014, University of Helsinki, Finland. Tel: +358-9-191 125400. Fax: +358-9-191 25444. E-mail:
[email protected]. † Helsinki Biophysics & Biomembrane Group. ‡ Biophysics & StatMech Group. § MemphyssCenter for Biomembrane Physics. (1) Sa¨ily, V. M. J.; Ryha¨nen, S. J.; Holopainen, J. M.; Borocci, S.; Mancini, G.; Kinnunen, P. K. J. Biophys. J. 2001, 81, 2135. (2) Karlsson, K. A. In Structure of biological membranes; Abrahamson, V., Pascher, I., Eds.; Plenum Press: New York, 1977. (3) (a) Nojiri, H.; Takako, F.; Terui, Y.; Miura, Y.; Saito, M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 782. (b) Nojiri, H.; Kitagawa, S.; Nakamura, M.; Kirito, K.; Enomoto, Y.; Saito, M. J. Biol. Chem. 1988, 263, 7443. (4) Zhang, H.; Buckley, N. E.; Gibson, K.; Spiegel, S. J. Biol. Chem. 1990, 265, 76. (5) (a) Ozaki, T.; Bielawska, A.; Bell, R. M.; Hannun, Y. A. J. Biol. Chem. 1990, 265, 15823. (b) Bielawska, A.; Linardic, C. M.; Hannun, Y. A. FEBS Lett. 1992, 307 (2), 211. (6) Endo, K.; Igarashi, Y.; Nisar, M.; Zhou, Q. H.; Hakomori, S. Cancer Res. 1991, 51 (6), 1613. (7) Desai, N. N.; Spiegel, S. Biochem. Biophys. Res. Commun. 1991, 181 (1), 361. (8) (a) Hannun, Y. A.; Bell, R. M. Science 1989, 234, 500. (b) Merrill, A. H., Jr.; Stevens, V. L. Biochim. Biophys. Acta 1989, 1010 (2), 131.
survival after myocardial ischemia.9 Together with the sleep-inducing lipid oleamide,10 sphingosine and its derivatives are the only naturally occurring cationic lipids. Studies on the biological roles of sphingosine and lysosphingolipids were spurred after the discovery that these molecules are potent and reversible inhibitors of protein kinase C,11 a key enzyme in cellular signaling networks. Since then, sphingosine has been shown to affect a number of other kinases12 and to be involved in electrostatically driven complex formation in model membranes.13 The molecular mechanisms by which sphingosine mediates its diverse biological actions have remained largely unresolved although evidence for the crucial role of its positive charge has been suggested.13 The interactions of sphingosine with DNA are of particular interest since sphingomyelin, a potential source of sphingoid bases, has been reported to be abundant among phospholipids present in the nuclear matrix and chromatin.14 Moreover, the amount of sphingoid bases seems to vary between active and repressed chromatin.15 It has been suggested that sphingosine could exert its actions by directly altering the chromatin structure16 and, (9) Friedrichs, G. R.; Swillo, R. E.; Jow, B.; Bridal, T.; Numann, R.; Warner, L. M.; Killar, L. M.; Sidek, K. J. Cardiovasc. Pharm. 2002, 39, 18. (10) Cravatt, B. F.; Prospero-Garcia, O.; Siuzdak, G.; Gilula, N. B.; Henriksen, S. J.; Boger, D. L.; Lerner, R. A. Science 1995, 268, 1506. (11) Hannun, Y. A.; Greenberg, C. S.; Bell, R. M. J. Biol. Chem. 1986, 261, 12604. (12) (a) Igarashi, Y.; Hakomori, S.; Toyokuni, T.; Dean, B.; Fujita, S.; Sugimoto, M.; Ogawa, T.; El-Ghendy, K.; Racker, E. Biochemistry 1989, 28, 6796. (b) McDonald, O. B.; Hannun, Y. A.; Reynolds, C. H.; Sahyoun, N. J. Biol. Chem. 1991, 266, 21773. (13) Mustonen, P.; Lehtonen, J.; Ko˜iv, A.; Kinnunen, P. K. J. Biochemistry 1993, 32, 5373. (14) Alessenko, A. V. In Nuclear Sructure and function; Harris, J. W., Zbarsky, I. B., Eds.; Plenum Press: New York, 1990; p 399. (15) Rose, H. Y.; Frenster, J. H. Biochim. Biophys. Acta 1965, 106, 577.
10.1021/la034307y CCC: $25.00 © 2003 American Chemical Society Published on Web 09/13/2003
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accordingly, contribute to the control of replication and gene expression. Complex formation by cationic lipids and negatively charged phosphates of DNA is mainly due to electrostatic attraction,17 while also other interactions seem to contribute.18 Sphingosine-containing liposomes have been employed as transfection vectors and do provide a relatively efficient and low-toxicity vehicle for in vitro gene transfer.19 The formation of ternary complexes by sphingosine, histone H1, and DNA has been documented.20 The local effects of DNA on sphingosine-containing giant vesicles have been viewed with optical microscopy, and some possible mechanisms for DNA/lipid membrane interaction and complex formation have been presented.21 We have previously characterized a cationic gemini surfactant (bearing two positive charges) with a Langmuir balance and studied its interactions with DNA in monomolecular films.1 Importantly, this method enables investigation of the lipid-lipid interactions in a systematic manner, with force-area (π-A) isotherms and interfacial elastic moduli of area compressibility (CS-1) providing precise indicators of changes in the film structure.22 Further information on the electric properties of the film can be obtained from the surface dipole potential Ψ.23 Accordingly, it was of interest to use this method to gain information on the physicochemical properties of sphingosine, on the impact of this single-chain cationic amphiphile on phospholipid monolayers, and on the interactions of this lipid with DNA. More specifically, sphingosine and its mixtures with POPC with and without DNA in the subphase were studied. Materials and Methods Materials. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), D-sphingosine (from bovine brain), calf thymus DNA, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), and ethylenediaminetetraacetic acid (EDTA) were from Sigma Chemical Co. (St. Louis, MO). The purity of lipids was checked by thin-layer chromatography on silicic acid coated plates (Merck, Darmstadt, Germany) using chloroform/methanol/water (65/25/ 4, v/v) as a solvent system. Examination of the plates after iodine staining revealed no impurities. Lipid concentrations were determined gravimetrically by using a high-precision electrobalance (Cahn, Cerritos, CA). DNA concentrations (in millimolar base pairs) were determined by absorbance at 260 nm (∈ ) 6600 L/(mol cm)). Freshly deionized filtered water (Milli RO/Milli Q, Millipore Inc., Jaffrey, NH) was used in all experiments. Monolayer Measurements. A computer-controlled Langmuir type film balance (µThrougS, Kibron Inc., Helsinki, Finland) was used to record compression isotherms (π-A). All glassware used was rinsed thoroughly with ethanol and purified water (Millipore). The indicated lipids were mixed in chloroform and spread in this solvent onto the surface of 14 mL of 5 mM HEPES, 0.1 mM EDTA, pH 7.4 buffer at ≈24 °C. To ensure complete evaporation of the solvents, the films were allowed to settle for 4 min prior to the recording of the π-A isotherms. The monolayers were compressed by two symmetrically approaching barriers at a rate of ≈4 Å2/molecule/min, so as to allow for the reorientation (16) Kinnunen, P. K. J.; Ryto¨maa, M.; Ko˜iv, A.; Lehtonen, J.; Mustonen, P.; Aro, A. Chem. Phys. Lipids 1993, 66, 75. (17) (a) Ko˜iv, A.; Kinnunen, P. K. J. Chem. Phys. Lipids 1994, 72, 77. (b) Kennedy, M. T.; Pozharski, E. V.; Rakhmanova, V. A.; MacDonald, R. C. Biophys. J. 2000, 78, 1620. (18) Ko˜iv, A.; Mustonen, P.; Kinnunen, P. K. J. Chem. Phys. Lipids 1994, 70, 1. (19) Paukku, T.; Laureus, S.; Huhtaniemi, I.; Kinnunen, P. K. J. Chem. Phys. Lipids 1997, 87, 23. (20) Ko˜iv, A.; Palvimo, J.; Kinnunen, P. K. J. Biochemistry 1995, 34, 8018. (21) (a) Angelova, M. I.; Hristova, N.; Tsonova, I. Eur. Biophys. J. 1999, 28, 142. (b) Angelova, M. I.; Tsonova, I. Chem. Phys. Lipids 1999, 101, 123. (22) Brockman, H. L. Curr. Opin. Struct. Biol. 1999, 9 (4), 438. (23) Brockman, H. L. Chem. Phys. Lipids 1994, 73, 57.
Langmuir, Vol. 19, No. 21, 2003 8957 and relaxation of the lipids in the course of the compression. Surface pressure was measured by the Wilhelmy technique with a small-diameter alloy probe that was located in the air/water interface and hung from a high-sensitivity microbalance (KBN 502, Kibron Inc.). Surface pressure π is defined as
π ) γ0 - γ
(1)
where γ0 is the surface tension of the air/buffer interface and γ is the value for surface tension in the presence of a lipid monolayer compressed at varying packing densities. When indicated, calf thymus DNA was included in the subphase. Monolayer dipole potential ψ23 was measured using the vibrating plate method (µSpot, Kibron Inc.). All experiments were repeated at least twice. Analysis of Isotherms. The value for monolayer compressibilities (Cs) for the indicated film compositions at the given surface pressures (π) were obtained from π-A data using
Cs ) (-1/Aπ) (dA/dπ)T
(2)
where Aπ is the area per molecule at the indicated surface pressure π. To identify the phase transition points, we further analyzed our data in terms of the reciprocal isothermal compressibility, that is, the elastic modulus of area compressibility (Cs-1) as described previously.24 Accordingly, the higher the value for Cs-1, the lower the interfacial elasticity. The excess free energy of mixing, ∆Gex m , at a given surface pressure π was calculated from A-π isotherms according to
∆Gex m (Π) )
∫
Π
0
(A - XPOPCAPOPC - XSphASph) dπ
(3)
where XPOPC and XSph are the mole fractions of POPC and Sph, respectively, and APOPC and ASph are the area/molecule for the pure POPC monolayer, the pure Sph monolayer, and the XPOPC/ XSph mixtures, respectively. Accordingly, eq 3 yields ∆Gex m from the area compression work difference for ideal and real mixtures. In practice, the pressure after application of lipid is rarely zero, and the lower limit for practical reasons was set at 0.55 mN/m or the highest initial surface pressure. This is unlikely to cause any significant error to the ∆Gex m values as the integrated area under the tail is negligible for all the mixtures at large separations and low pressures. Accordingly, the error can be expected to be more or less the same for each value of ∆Gex m . For numerical integration, an interpolated data set for each mixture was created and the piecewise area summation at 0.001 mN/m steps was performed.
Results Force-Area Isotherms for Sphingosine/POPC Monolayers. Sphingosine formed stable monolayers at an air/water interface, and its compression isotherms revealed smooth π-A curves, lacking indications for structural transitions and suggesting the films to be in the liquid expanded state, similarly to POPC (data not shown). Subsequently, we measured compression isotherms for mixed films of POPC and sphingosine while systematically varying the monolayer composition (Figure 1). Analysis of the mean molecular areas revealed that already at XSph ) 0.05 the films were condensed (by up to 30% at a surface pressure π ) 35 mN/m, Figure 1A). This initial decrement in the mean molecular area was similar to what was observed for mixed films of the cationic gemini surfactant SR-1 and POPC.1 When XSph ) 0.05 was exceeded, the films condensed further, reaching a minimum at XSph ) 0.25. Interestingly, after this first minimum the area/molecule increased significantly, with a relative expansion of the film toward the line representing ideal mixing and with a peak in A at XSph ) 0.38. This first XSph-dependent maximum in A was slightly more promi(24) Smaby, J.; Kulkarni, V. S.; Momsen, M.; Brown, R. E. Biophys. J. 1996, 70, 868.
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Figure 1. (A) The effect of increasing XSph on the area/molecule in compression isotherms of mixed POPC/Sph films. The values of π were 10 (9), 20 (b), 30 (2), 35 (1), and 40 (() mN/m. (B) Similar data recorded in the presence of 2.5 µM DNA (in basepairs) in the subphase. (C) The difference in the area ADNA - A (Å2/molecule) as a function of XSph. The subphase was 5 mM HEPES, 0.1 mM EDTA, pH 7.4. The temperature was ≈24 °C.
nent at lower surface pressures and was absent at π ) 40 mN/m. The total increment between XSph ) 0.25 and XSph ) 0.38 at surface pressure 35 mN/m was approximately 10 Å2/molecule, from 35 to 45 Å2. When XSph ) 0.38 was exceeded, the area/molecule decreased again, reaching a second minimum at XSph ) 0.63. This minimum was again followed by a sharp increase in A, reaching a second relative maximum at XSph ) 0.71, with the difference in area/molecule between the minimum and the peak being
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approximately 5 Å2. Subsequently, increasing the molar content of sphingosine further to XSph ) 0.83 revealed a third minimum in area/molecule after which XSph ) 1.0 was reached without further discontinuities. In the range of 0.05 e XSph g 0.25, the films collapsed at lower surface pressures (approximately 38 mN/m) and no data at π > 40 mN/m could be obtained. Effects of DNA. Binding of DNA to sphingosinecontaining cationic liposomes is well established,18,25 and its association with sphingosine-containing films is anticipated. We repeated the above experiments with 2.5 µM DNA present in the subphase to monitor the effects of this polyanion on POPC/sphingosine monolayers (Figure 1B). The amount of DNA was chosen to yield complete saturation of the positive charges of sphingosine (DNA/ sphingosine charge ratio > 1 at XSph ) 1.0). As reported previously, DNA condenses neat POPC monolayers, at 10 mN/m for example from 92 to 78 Å2/molecule.1 At XSph ) 0.05, the films were slightly expanded in the presence of DNA compared to a neat POPC monolayer; when XSph was then increased to 0.25, a condensation was observed, similarly to the isotherms measured without DNA. The shapes of the peak in the area/molecule between XSph ) 0.25 and XSph ) 0.50 were different, and the maximum in A shifted from XSph ) 0.38 to XSph ) 0.40, with the peak becoming more prominent at higher surface pressures. At π ) 10 mN/m, no discontinuity in the area/molecule versus XSph was observed. DNA condensed the films in the region of the peak as illustrated in the area difference (∆A/molecule) between isotherms measured in the presence and absence of 2.5 µM DNA. This condensation was more pronounced at lower surface pressures (Figure 1C). Following the peak at XSph ) 0.40, a slowly decreasing trend was observed in the area/molecule until at XSph ) 0.71 a small peak was evident. Upon increasing XSph from 0.83 to 0.88, a sharp decrement in the area/molecule was seen (Figure 1B). In light of the above, it was of interest to express the data also in terms of the difference in the average area occupied by a molecule between the measured values and ideal mixing of POPC and sphingosine in monolayers (Figure 2). The steep initial decrease observed upon introducing sphingosine up to XSph ) 0.05 was followed by a plateau in ∆A/molecule to XSph ) 0.25 (Figure 2A). The range of ∆A/molecule changed drastically between XSph ) 0.25 and XSph ) 0.38. At 10 mN/m, for example, this decrement was from approximately 17 to 3 Å2, respectively. When XSph ) 0.38 was exceeded, the ∆A/molecule increased until after XSph ) 0.63 a sharp decrease was observed, revealing film expansion. The peak in area/molecule at XSph ) 0.71 illustrated as ∆A/molecule also shows the films to be almost ideally mixed at higher surface pressures (from π ) 40 to π ) 30) and close to ideal at lower pressures. At XSph g 0.5, the area difference between the measured data and theoretical ideal mixing was more prominent at lower surface pressures. With 2.5 µM DNA in the subphase, the behavior of the films was somewhat different (Figure 2B). At XSph ) 0.05, the monolayers were expanded relative to the ideal mixing, and increasing XSph to 0.25 revealed a minimum in ∆A/molecule, similarly to the data measured in the absence of DNA. After XSph ) 0.25, expansion of the films was observed at surface pressures π > 10 mN/m, with peaks at XSph ) 0.33 and XSph ) 0.4. The peak at XSph ) 0.4 was followed by a sudden increase in ∆A/molecule, revealing condensation of the lipid monolayers. At π ) 10 mN/m, the films remained condensed, reaching maximal condensation at XSph ) 0.5. In the range (25) Scherer, P. G.; Seelig, J. Biochemistry 1989, 28, 7720.
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Figure 2. (A) The difference between measured values and ideal mixing Ameas - Aideal (Å2/molecule) as a function of XSph. The subphase was 5 mM HEPES, 0.1 mM EDTA, pH 7.4. The temperature was ≈24 °C. (B) Similar data recorded in the presence of 2.5 µM DNA (in basepairs) in the subphase.
of 0.5 < XSph < 0.71, an expansion of the films was observed with the measured values exceeding those expected from the ideal mixing between XSph ) 0.71 and XSph ) 0.83. Changes in Monolayer Dipole Potential ψ. Taking into account the cationic charge of sphingosine-containing monolayers and the interaction of polyanionic DNA with these films, alterations in the dipole potential23 are readily anticipated. These data were recorded as a function of XSph and are depicted at varying molecular densities (Figure 3). The presence of sphingosine had a significant impact on the monolayer dipole potential, and already at XSph ) 0.05 a pronounced increment in Ψ was evident, at 2.5 µmol/m2 from 280 to 380 mV. This steep initial increment in Ψ was followed by a moderate increase of 10 mV so that at XSph ) 0.25 and at 2.5 µmol/m2 Ψ was approximately 390 mV. Between XSph ) 0.25 and XSph ) 0.40, however, a discontinuity in surface potential was observed, similarly to the area/molecule plots (Figure 1A). A peak at XSph ) 0.38 was preceded by a decrement from XSph ) 0.25 to XSph ) 0.33, at 2.5 µmol/m2 from 390 to 360 mV, respectively. This behavior is evident also at higher surface densities and in the presence of DNA. At surface densities of 2.3 and 2.5 µmol/m2, there was a decrement in Ψ between XSph ) 0.40 and XSph ) 0.63. Subsequently, rather constant values for Ψ were measured, 340 mV up to XSph ) 0.83. After XSph ) 0.83, the monolayer dipole potential decreased so that at XSph ) 0.95 the value for Ψ
Figure 3. Values for the monolayer dipole potential Ψ derived from compression isotherms for POPC/Sph monolayers as a function of XSph and recorded both without (A) and with 2.5 µM DNA (B) in the subphase. The data are shown at varying molecular densities, at 1.8 (9), 2.3 (b), 2.5 (2), and 2.9 (1) µmol/ m2. The temperature was ≈24 °C. Also shown is the voltage difference between the above data points, representing the impact of DNA on the monolayers (C).
was 310 mV at densities of 2.3 and 2.5 µmol/m2. Thereafter Ψ reached the value recorded for a neat sphingosine monolayer. When a charge-saturating concentration of DNA was included in the subphase, the monolayer dipole potential for neat POPC increased, at 2.5 µmol/m2 from approximately 280 to 330 mV, respectively (Figure 3B). In the presence of sphingosine, Ψ increased further, up to 370 mV at XSph ) 0.05 (at 2.5 µmol/m2). A drop of
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approximately 15 mV was observed at XSph ) 0.13 irrespective of the molar density. When XSph was increased from 0.25 to 0.5, a similar pattern of changes in dipole potential was observed when compared to the data recorded without DNA, the only difference being that the values of Ψ were approximately identical for both XSph ) 0.38 and XSph ) 0.4 (400 mV at 2.5 µmol/m2). This maximum in Ψ was preceded by a local minimum in Ψ at XSph ) 0.33. After XSph ) 0.4, the values for Ψ decreased, and a minimum of 320 mV at 2.5 µmol/m2 was reached at XSph ) 0.71. Thereafter, an increase by approximately 30 mV in Ψ was evident, followed by a decrement of the same magnitude when XSph reached 0.88. For neat sphingosine monolayers, the values of Ψ were elevated by approximately 20 mV by DNA. To better illustrate the impact of DNA, these data are shown also as a function of XSph as the recorded voltage difference ∆Ψ for the monolayers with and without DNA in the subphase (Figure 3C). An initial steep decrease until XSph ) 0.13 was followed by an increase, so that when XSph was increased from 0.25 to 1.0 the values of Ψ were slightly higher in the presence of DNA compared to its absence, with exceptions at XSph ) 0.5, 0.67, and 0.71 where ∆Ψ was negative. Monolayer Compressibility Modulus. To gain further insight into the characteristics of the sphingosine/ POPC monolayers, we analyzed the above compression isotherms in terms of their compressibility modulus Cs-1 as a function of π and XSph. The values for the maxima in compressibility modulus (CS-1max) versus XSph both with and without DNA in the subphase are compiled in Figure 4A. In the absence of DNA, already low contents of sphingosine (XSph ) 0.05) decreased the CS-1max by 30 mN/ m, from ∼100 to 70 mN/m. This was followed by somewhat irregular changes, declining to ∼45 mN/m for neat sphingosine monolayers. The presence of 2.5 µM DNA reduced CS-1max for POPC significantly, from ∼100 to 70 mN/m. Yet, the changes observed with increasing XSph were again rather irregular. For neat sphingosine monolayers in the presence of DNA, the value of CS-1max was 40 mN/m. The values of π corresponding to CS-1max as a function of increasing XSph reveal a shift to lower pressures upon increasing XSph, with a local maximum at XSph ) 0.38 preceding the minimum of ∼24 mN/m at XSph ) 0.4 (Figure 4B). Thereafter, an increase in the values of πCs-1max was observed at XSph ) 0.75 followed by a drop from 29 to 21 mN/m at XSph ) 0.83. A steep increase in πCs-1max was observed after this local minimum with a maximum of ∼39 mN/m reached at XSph ) 1.0. In the presence of 2.5 µM DNA, the values of surface pressure corresponding to the compressibility modulus maxima (πCs-1max) for neat POPC decreased, from ∼33 to ∼25 mN/m, respectively. A steep increase in πCs-1max to 38 mN/m at XSph ) 0.13 was followed by an almost reciprocal decrease, with a minimum of 28 mN/m at XSph ) 0.38. Between XSph ) 0.4 and XSph ) 0.67, the values of πCs-1max were again higher, with a maximum of 38 mN/m being reached at XSph ) 0.63. A sharp decrement, however, was evident after XSph ) 0.63, and at XSph ) 0.83 the value of πCs-1max was 24 mN/m. From XSph ) 0. 83 to XSph ) 1.0, the value of πCs-1max increased to 37 mN/m. Excess Free Energy of Mixing. To analyze the interactions in the mixed POPC/sphingosine monolayers in a more quantitative manner, we calculated the excess free energy of mixing (∆Gex m , Figure 5A). Negative values of ∆Gex m for all the investigated composition ranges confirm that the mixing is energetically favorable, and
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Figure 4. (A) The dependence of CS-1 on XSph in mixed Sph/ POPC monolayers, recorded in the absence (0) and in the presence (9) of 2.5 µM DNA (in basepairs) in the subphase. (B) Surface pressures π corresponding to the compressibility modulus maxima CS-1 (πCs-1max) measured in the absence (0) and in the presence of 2.5 µM DNA (9) in the subphase (5 mM HEPES, 0.1 mM EDTA, pH 7.4) at ≈26 °C.
accordingly, there should be no phase separation of the individual compounds. The minimum values, indicating the highest stability of the mixed phase, occurred in regions where the monolayer was the most condensed with respect to the ideal mixing (Figure 2), from XSph ) 0.05 to XSph ) 0.25 and from XSph ) 0.6 to XSph ) 0.63, respectively. ∆Gex m was pressure dependent so that for higher pressures the values of ∆Gex m decreased. This is expected as at higher packing densities the interactions between the molecules are stronger and also the differences derived from molecular characteristics for different compound molecules are larger. When XSph was increased, the free energy became less negative, indicating the interactions between the two components became weaker. The presence of DNA altered the mixing energies dramatically (Figure 5B,C). Accordingly, the values for ∆Gex m shifted from a highly negative value observed in the absence of DNA to close to zero, indicating the mixing of the components becoming less favorable. At XSph ) 0.05, interestingly, ∆Gex m was positive, from 0.3 kJ/mol at π ) 10 mN/m to 1.0 kJ/mol at π ) 40 mN/m, respectively. Between XSph ) 0.13 and XSph ) 0.25, ∆Gex m was in the range of -0.1 to -0.7 kJ/mol with the lowest values reached at highest pressures. At XSph ) 0.33 and at XSph ) 0.41, peaks with positive ∆Gex m were observed, similarly
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Notably, the most positive values were again measured for the films at highest pressure. When XSph ) 0.71 was exceeded, the values for ∆Gex m became negative, reaching a local minimum at XSph ) 0.88 where ∆Gex m was approximately -1.0 kJ/mol. To better illustrate the effects of DNA on mixing properties of the components in the monolayer, we calculated the difference between ∆Gex m measured both with and without DNA (Figure 5C). With a chargesaturating concentration of DNA in the subphase, the mixing of POPC and sphingosine became less favorable with the differences in ∆Gex m being largest at low XSph. At XSph ) 0.37, however, practically no difference in ∆Gex m was observed, and at XSph ) 0.33 and XSph ) 0.41, respectively, the difference was very small. When XSph was increased from 0.37, an increase in ∆Gex m became evident, with the largest values measured at higher surface pressures. At XSph ) 0.67, a decrement in the ∆Gex m was seen, after which an increment to the original level preceded a decrement upon increasing XSph to 1.0. Discussion
Figure 5. (A) Values for excess free energy of mixing derived from compression isotherms for POPC/Sph monolayers as a function of XSph. The values of π were 10 (9), 20 (b), 30 (2), 35 (1), and 40 (() mN/m. (B) Similar data recorded in the presence of 2.5 µM DNA (in basepairs) in the subphase. Also shown is the difference between the above data points, representing the impact of DNA on the monolayers (C). The subphase was 5 mM HEPES, 0.1 mM EDTA, pH 7.4. The temperature was ≈24 °C.
to the peaks in Figure 2B. At XSph ) 0.37, ∆Gex m was in the range of -0.25 to -0.5 kJ/mol. From XSph ) 0.5 to 0.67, the value for ∆Gex m remained close to -0.5 kJ/mol, and at XSph ) 0.71 a peak with positive ∆Gex m was evident.
Sphingosine as such formed stable monolayers exhibiting smooth, continuous π-A isotherms similar to those of POPC, indicating the film to be in the liquid expanded state. As observed in our previous study1 on the effects of a dicationic gemini surfactant, already low amounts of the cationic lipid sphingosine (XSph ) 0.05) caused a pronounced condensation of POPC monolayers. Our results indicate that this effect is of electrostatic origin. This notion is supported by the fact that condensation was not observed when sphingosine was added into monolayers composed of either saturated or unsaturated diacylglycerol (data not shown). The effect of sphingosine on the monolayer dipole potential also supports the above interpretation (Figure 3). In addition, when 200 mM NaCl was present in the aqueous subphase to screen the charges in the monolayer, the observed minima at XSph ) 0.25, 0.63, and 0.83 (Figure 1A) were nearly completely absent (data not shown). Although this intriguing behavior with several condensation minima is likely to have an electrostatic origin, the detailed microscopic physical mechanisms remain unresolved. There are several mutually nonexclusive possibilities, as follows: (i) reorientation of phosphocholine dipoles, (ii) condensation of counterions with their release increasing entropy, (iii) formation of hydrogen-bonded networks, and (iv) lateral diffusion and reorganization of the components in the monolayer. These possibilities are discussed in more detail below. Reorienting the dipole (P--N+) of the phosphocholine moiety from a parallel to a more vertical orientation with respect to the membrane plane would maximize the distance between the positive charges of sphingosine and the choline moiety. This reorientation has been observed in NMR studies of liposomes25 and in molecular dynamics simulations26 as well as in differential scanning calorimetry (DSC) and Langmuir balance studies. 1,27 Reorientation alone is not enough to explain multiple condensation minima, however. The POPC headgroup is relatively rigid, and it is not obvious why it should return back to its original, approximately plane parallel orientation upon (26) Bandyopadhyay, S.; Tarek, M.; Klein, M. L. J. Phys. Chem. B 1999, 103, 10075. (27) (a) Zantl, R.; Baicu, L.; Artzner, F.; Sprenger, I.; Rapp, G.; Ra¨dler, J. O. J. Phys. Chem. B. 1999, 103, 10300. (b) Ryha¨nen, S. J.; Sa¨ily, M. J.; Paukku, T.; Borocci, S.; Mancini, G.; Holopainen, J. M.; Kinnunen, P. K. J. Biophys. J. 2003, 84, 578.
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Figure 6. Putative mean lateral arrangements of acyl chains in Sph/POPC monolayers with corresponding unit cells. White and gray hexagons represent the acyl chains of POPC and the alkyl chains of sphingosine, respectively.
further increase in XSph. In addition to the former process, counterion condensation and release from the membrane surface are possible. When the content of Sph is increased, counterions see the surface becoming more positively charged. Due to this, some of them may condense, thus resulting in the destabilization of the initially prevailing energetically favorable structure. Because of the increased negative counterion content in the vicinity of the surface, the locally stable Sph-POPC complexes would dissociate. Both Sph and POPC thus become on the average more mobile, the average area per lipid increasing. In other words, counterion condensation would result in partial demixing of the two lipid species and thus cause reorientation of the P--N+ dipole from vertical to parallel to the plane of the monolayer. Condensation of counterions, or a part of them, does not come without a price. The reduction in energy is contrasted by a decrease in entropy due to condensation, continuing after each minimum up to the next local maximum. After that, no free energy can be gained by further condensation. Instead, the phosphocholine dipoles should again flip up, leading to a reduction in the area per lipid as the negatively charged phosphate moiety of the P--N+ dipole of POPC can associate electrostatically with protonated sphingosine. As a consequence, some of the negative counterions are released, leading to a favorable increase in entropy and thus a decrement in free energy. This mechanism clearly allows for several condensation minima in the area per lipid curves, as observed. To confirm the validity of the above scenarios warrants both theoretical and experimental studies. A possible starting point is to analyze for the isoelectric instability shown by Bruinsma28 for DNA condensation in lamellar systems and the two-fluid theory by Lau et al.29 The latter is of particular interest, since it distinguishes between two populations of ions: the condensed ones and the ones that remain in the bulk solution. These two populations have very different electrostatic characteristics (in terms of interactions) leading to the two-fluid description. Further aspects involve mobility of lipids30 and bridging by water and ions. In addition, our previous molecular dynamic simulations of cationic gemini surfactants and zwitterionic lipids with and without DNA support the possibility of the above mechanism where counterions have an important role.31 Possible consequences of hydrogen bonding need to be considered as well. Sphingosines in mixed monolayers should maximize their mutual distances due to Coulombic repulsion. Accordingly, we may assume miscibility of POPC and sphingosine based on the negative values of ex ∆Gex m for mixtures of POPC and Sph. Interestingly, ∆Gm reveals shifts in excess free energy of mixing toward less (28) Bruinsma, R. Eur. Phys. J. B 1998, 4, 75. (29) Lau, A. W. C.; Lukatsky, D. B.; Pincus, P.; Safran, S. Phys. Rev. E 2000, 65, 051502. (30) Kim, Y. W.; Sung, W. Europhys. Lett. 1998, 58, 147. (31) Karttunen, M.; Pakkanen, A. L.; Kinnunen, P. K. J.; Kaski, K. Cell. Mol. Biol. Lett. 2002, 7, 238.
negative values with increasing XSph. To reduce the free energy increase caused by increased positive surface charge density upon increasing XSph, the extent of protonation of the NH2 moiety should decrease. It is possible that the protonated and deprotonated forms of sphingosine associate via hydrogen bonding and segregate laterally as a regular lattice, similar to that suggested for acidic phospholipids.32 The interactions between POPC and sphingosine are enhanced at higher surface pressures, ∆Gex m ∼ -4.0 kJ/mol at XSph ) 0.13 and at π ) 35 mN/m, compared to ∼1.0 kJ/m at π ) 10 mN/m, for example (Figure 5A). This problem can be analyzed from the point of view of lipid lateral packing. To illustrate this, we calculated if the lipid stoichiometries corresponding to the critical mole fractions (XSph ) 0.25, 0.6, and 0.83) would allow for regular lateral patterns of lipid distribution, maximizing the distance between sphingosine and thus minimizing their mutual Coulombic repulsion. One possible way of ordering sphingosine and POPC is represented in Figure 6. At XSph ) 0.25, a superlattice can be assembled consisting of a unit cell with one acyl chain of sphingosine being surrounded by six acyl chains of POPC (i.e., three POPC molecules), yielding the maximal distance between the positively charged sphingosines. The effects of DNA on the sphingosine/POPC monolayer are of interest. Intriguingly, the inclusion of DNA underneath POPC monolayers had a significant condensing effect, in keeping with our previous results.1 We assume that similarly to the condensing effect of sphingosine, the strongly anionic DNA associates with the POPC monolayer due to electrostatic interactions, causing the P--N+ dipole to reorient with respect to the membrane plane, thus decreasing its projected surface area in the plane of the film. The initial increment in the area/molecule at XSph ) 0.05 could arise from efficient electrostatic association of the cationic sphingosine with DNA, which would diminish the average extent of the orientation of the P--N+ dipole of POPC parallel to the membrane plane. Increasing XSph above 0.05, however, results in condensation of the film with the first minimum in area/molecule appearing at XSph ) 0.25. This is in keeping with the data obtained in the absence of DNA. The discontinuities observed in area/ molecule (Figure 1B) occur at the same values of XSph both in the absence and presence of DNA and are thus likely to have the same origin. DNA seems to expand the mixed monolayers at critical mole fractions of sphingosine (Figure 1C), suggesting diminished stability of the otherwise highly condensed monolayers. In the regions of the peaks in area/molecule, however, a condensation due to DNA was evident. This can be explained by a decrement in Coulombic repulsion between sphingosine molecules caused by the negative charges of DNA. (32) (a) Boggs, J. M.; Moscarello, M. A.; Papahadjopoulos, D. Biochemistry 1977, 16 (25), 5420. (b) Subramanian, M.; Jutila, A.; Kinnunen, P. K. J. Biochemistry 1998, 37, 1394.
Sphingosine Monolayers and Binding of DNA
Diminished monolayer stability in the presence of DNA is evident in Figure 5B,C. With DNA, the mixing of sphingosine and POPC seems not to be favorable as ∆Gex m changes from highly negative (in the absence of DNA) to close to zero or even positive at certain values for XSph. This suggests that despite the favorable mixing between POPC and Sph, binding of DNA to the monolayer induces local demixing of the two lipids in the film, even if the ∆Gex m value for the whole film remains negative. Accordingly, the free energy of binding for DNA overcomes the unfavorable free energy contribution from local demixing of lipids. This effect is likely to arise from local charge densities of the film and DNA, thus allowing for, for example, more complete counterion release from DNA and the film. At XSph ) 0.05, the value for ∆Gex m increases from 0.3 kJ/mol at 10 mN/m to over 1.0 kJ/mol at 40 mN/m. At this mole fraction of sphingosine, the change induced by DNA is the largest (Figure 5C). One possible explanation is that at the low surface density of sphingosine and, accordingly, minimal Coulombic repulsion between the sphingosines allow for the DNA to easily reorganize the positively charged sphingosines so as to screen the charges of the anionic phosphates of DNA. Interestingly, at XSph ) 0.37 DNA has practically no impact on ∆Gex m (Figure 5C). In other words, the organization in the monolayer should be the same regardless of DNA. Importantly, calculating the charge density of DNA projection versus that of the monolayer revealed that at XSph ) 0.37 the density of sphingosine molecules and thus also the density of cationic charges equal the charge density of DNA projection. Yet, this explanation fails, as the XSph at local minima of ∆Gex m for different surface pressures does not vary between XSph ) 0.3 and 0.45 (Figure 5C). As discussed above, the finding of highly sensitive mole fraction dependent behavior of mixed monolayers of sphingosine and POPC is intriguing and is unlikely to be
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explained by simple electrostatic arguments only. The interaction energies derived from collapse pressures of the mixed films differ for the different mixtures (data not shown). The mixing of these two lipids is far from ideal, and adding DNA to the system further complicates the interpretation of the data. Further studies on the characterization of the nature of these complex interactions are in progress in our laboratory. Acknowledgment. The authors thank Kaija Niva for technical assistance. The Helsinki Biophysics & Biomembrane Group is supported by the Finnish State Medical Research Council. V.M.J.S. is supported by the Finnish Medical Foundation, and J.-M.A. and S.J.R. are supported by the Finnish Medical Foundation and the M.D./PhD program of Helsinki Biomedical Graduate School. Memphys is supported by the Danish National Research Council. Abbreviations A, area per molecule ADNA, area per molecule recorded with DNA in the subphase CS-1, elastic modulus of area compressibility EDTA, ethylenediaminetetraacetic acid HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid PC, phosphatidylcholine POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Sph, sphingosine XA, mole fraction of compound A ∆Gex m , excess free energy of mixing π, surface pressure πCs-1max, surface pressure corresponding to the compressibility modulus maximum ψ, dipole potential LA034307Y
