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Sphingolipid Monolayers on the Air-Water Interface and Electrochemical Behavior of the Films Transferred onto Glassy Carbon Electrodes N. Wilke and A. M. Baruzzi* Instituto de Investigaciones en Fisicoquı´mica de Co´ rdoba (INFIQC), Departamento de Fisicoquı´mica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´ rdoba, 5000 Co´ rdoba, Argentina
B. Maggio Centro de Investigaciones en Quı´mica Biolo´ gica de Co´ rdoba (CIQuiBiC), Departamento Quı´mica Biolo´ gica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´ rdoba, 5000 Co´ rdoba, Argentina Received November 1, 2000. In Final Form: February 21, 2001 In the present paper the packing and dipolar properties of dimirystoylphosphatidylcholine and sphingolipid monolayers were studied at the air-water interface in correlation with the charge-transfer process of the K3Fe(CN)6/ K4Fe(CN)6 redox couple voltammetricaly analyzed on glassy carbon electrodes coated with the transferred monolayers. Lipid molecules having in common the lipophilic chain or the polar group were chosen for the analysis. Different degrees of electrochemical irreversibility in the voltammetric profile were observed depending on the lipid nature. The reversibility of the process was recovered after further addition of calcium chloride to the solution under study, indicating an increase in the monolayer permeability and consequently an increase in the rate of the charge-transfer process. The necessary amount of calcium to produce this effect depended also on the nature of the monolayer, which was evidence of some specific interaction between Ca2+ and each lipid. Surface pressure-area and surface potential-area compression isotherms at the air-water interface under different subphase composition were performed to get a better insight of this behavior. Interactions between the lipids and the ions were also observed, and a correlation between these results and the electrochemical response was found.
Introduction Extensive information about ion transfer and electrontransfer reactions has been obtained by studying these reactions on electrodes covered with organic, active, or electroinactive compounds.1 The composition and thickness of the surface layer can be varied and controlled in different manners. On homogeneous films, diffusion of reactants has a linear character and the reaction rate can be controlled by the partition of the reactant between the layer and the solution, by the permeability of ions across the film, or by electron tunneling. Nevertheless, the distribution of surfactant molecules on the electrode surface frequently has a nonuniform character and covered and bare domains can usually be present. This kind of surface is induced by strong lateral interactions between surfactant molecules or by energetic heterogeneity of the solid surface. When the reaction takes place almost only through surface patches, the monolayer film has a blocking effect. In this case adsorption affects not only the rate of the electrode reaction but also the rate of mass transfer giving rise to phenomena of nonlinear diffusion.2,3 In this case the shape of the voltammetric profiles changes with the coverage and the duration of * To whom correspondence should be addressed. Fax: +54-3514334188. E-mail:
[email protected]. (1) Lipkowski, J. In Modern Aspect of Electrochemistry; Conway, B. E., et al., Eds.; Plenum Press: New York, 1992; Vol. 23, p 1 and references therein. (2) Gueshi, T.; Tokuda, K.; Matsuda, H. J. Electroanal. Chem. 1978, 89, 247. (3) Gueshi, T.; Tokuda, K.; Matsuda, H. J. Electroanal. Chem. 1979, 101, 29.
the experiments. In other words, under some experimental conditions the diffusion toward the active centers can be equivalent to hemispherical diffusion and operationally equivalent to an array of ultramicroelectrodes.4,5 It is wellknown that ion and electron transfer through organized monolayers of amphiphilic compounds deposited on solid electrode surfaces is a most convenient way to study this type of reaction. Highly ordered films can be deposited by Langmuir-Blodgett (LB)6 or self-assembled monolayer (SAM) techniques7 described in many reviews. This kind of film facilitates control of electron transfer distances at electrodes. The redox species analyzed either is chemically bonded to the monolayer or is free in the solution. The advantages and disadvantages of both kinds of films have also been reported by different authors.1,6,7 In general, it is well recognized that SAMs can be obtained free of pinholes and of variable thickness and additionally they are easy to prepare. On the other hand, in the case of LB films the main advantage is that the monolayer organization can be externally controlled. Also, both kinds of techniques have been proposed for assembling bilayers.8 These modified electrodes have received attention not only from a fundamental point of view. They have been proposed as ion channel sensors for divalent cations as (4) Sabatini, E.; Rubinstein, I. J. Phys. Chem. 1987, 91, 6663. (5) Amatore, C.; Savean, J. M.; Tessier, D. J. Electroanal. Chem. 1983, 147, 39. (6) Roberts, G. Langmuir-Blodgett Films; Plenum Press: New York, 1990; Chapter 2. (7) Finklea, H. O. In Electroanalytical Chemistry; Bard, A. J., Rubinstein, I., Eds.; Marcel Dekker: New York, 1996; Vol. 19, p 109. (8) Guo, L. H.; Facci, J. S.; McLendon, G. J. Phys. Chem. 1995, 99, 4106.
10.1021/la0015314 CCC: $20.00 © 2001 American Chemical Society Published on Web 05/23/2001
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these ions change the permeability to different marker ions of multilayer membranes on glassy carbon electrodes.9 The authors attribute this effect to conformational changes in the membrane and/or electrostatic interaction between the lipid and marker ions. The biomembrane structure and properties have been explored by different methods. To this purpose, artificial models have been prepared with proteins and lipids of defined composition. The large amount and variety of results indicated not only that membranes are dynamic structures but also the importance of the molecular arrangement employed. Important information obtained in the past decades came from the analysis of the behavior of lipid monolayers at the water-air interface.10,11 More recently and considering the advantages already exposed, SAM and LB films have been used as simplified membrane models. It is possible to obtain information on the interaction between its constituents, electrical properties, permeability to ions and molecules, drug action mechanisms, etc.6,7,11-13 The aim of the present paper was to further study the surface properties of sphingolipids that are normal components of membranes, in comparison with a glycerophospholipid, through the effect of monolayers of these molecules on charge transfer reactions. The monolayers were prepared using the LB technique in order to control precisely the surface organization of the monolayer in terms of its molecular packing and dipole potential prior to its deposit onto the working electrode. For the analysis, cyclic voltammetry of the Fe(CN)63-/Fe(CN)64- redox couple was performed. The behavior of this couple has been very well studied and it has a quite high rate constant determined by different methods.14 The voltammetric results were correlated with the behavior of surface pressure and surface potential versus the mean molecular area compression isotherms of monolayers of dmPC, sphingomyelin, galactocerebroside, and ceramide over different air-aqueous solution interfaces. Experimental Section (A) Materials. The electrochemical measurements were carried out in a three-electrode conventional cell. Glassy carbon, used as a working electrode, was commercially available and polished with alumine 1, 0.5, and 0.03 µm. The counter electrode was a platinum mesh and the reference electrode, a Ag/AgCl/ Cl-. These experiments were performed in a solution which contains 5 mM potassium ferrocyanide (pro analysis, Merck), 20 mM sodium nitrate as supporting electrolyte (analytical reagent, Mallinckrodt), and TRIS (hydroxymethylaminomethane) buffer (Merck) (solution A). The water used for the subphase and for the electrochemical measurements was double distilled in an all-glass apparatus or was ultrapure water produced by a MilliQ system (18 MΩ). Dimirystoylphosphatidylcholine (dmPC), bovine brain sphingomyelin (Sphm), and galactocerebroside (Galcer) were from Avanti Polar Lipids, and sulfatide (Sul) was purified from bovine brain as previously described.15,16 Epifluorescence measurements were performed by doping the lipid solution with 1 mol % of 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DilC12) (Molecular Probes). (9) Sugawara, M.; Kojima, K.; Sazawa, H.; Umezawa, Y. Anal. Chem. 1987, 59, 2842. (10) Maggio, B. Prog. Biophys. Mol. Biol. 1994, 62, 55. (11) Gaines, G. L. In Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Monographs on Physical Chemistry; Prigogine, I., Ed.; Wiley-Interscience: New York, 1966; p 188. (12) Birdi K. S. Lipid and biopolimer monolayers at liquid interfaces; Plenum Press: New York, 1989. (13) Goldenberg, L. M. J. Electroanal. Chem. 1994, 379, 3. (14) Iwasita, T.; Schmickler, W.; Herrmann, J.; Vogel, U. J. Electrochem. Soc. 1983, 2026. (15) Maggio, B.; Cumar, F. A. Brain Res. 1974, 77, 297. (16) Radin N. S.; Brown, J. R. Biochem. Prepr. 1960, 7, 31.
Langmuir, Vol. 17, No. 13, 2001 3981 (B) Methods. (1) Lipid Monolayers. Lipids were spread on different subphases from solutions (approximately 1 nmol µL-1) prepared in chloroform/methanol (2:1). Details of the equipment used were given in previous publications.17,18 The subphase temperature was kept at 25 ( 1 °C with a refrigerated Haake F3C thermocirculator. Surface pressure was measured with a platinized platinum plate. Surface potential was measured with an 241Am plate as surface electrode and a calomel reference electrode. At least triplicate monolayer isotherms were obtained and averaged. The molecular dipole moment perpendicular to the interface was calculated by the parallel plate condenser model12 with the equation
µP ) A(∆V - φ0)/12π where A is the molecular area in Å2 per molecule, the surface potential (∆V) is in millivolts, and φ0 is zero for un-ionized or zwitterionic monolayers; with these units, µP is in milliDebyes (mD). For monolayers of cerebroside sulfate, the ionic double layer at the surface was taken into account by using the GouyChapman equation to calculate φ0.18-20 The reciprocal of the compressibility, denominated the surface compressional modulus κ (in mN m-1), was also used to describe monolayer properties. This is defined as
κ ) -A(∂π/∂A)T where A is in Å2 per molecule and π, the lateral surface pressure, is in mN m-1. (2) LB Film Transfer. The glassy carbon electrode was cleaned by sonication in ultrapure water. Monolayers were spread over water and taken to the transfer pressure (40 mN m-1 for dmPC, Galcel, and Sul and 45 mN m-1 for Sphm). Film transfer was accomplished by gently allowing horizontal contact of the electrode surface with the lipid monolayer with a precision step motor; the electrode was maintained in that position for 30 s before lifting.9 (3) Electrochemical Measurements. Cyclic voltammetry was used as an electrochemical technique. The potential was applied using a homemade potentiostat and ranged between -0.300 and +0.800 V with a sweep generator (LyP Argentina). After film transfer, the electrode was introduced in the solution A and few cycles were performed to attain a stationary condition of the surface. Then, the first subsequent cycle was recorded; this process was followed by a short time interval to recover the diffusion layer. (4) Epiflourescence Microscopy of Monolayers. The lipid solutions were doped with 1 mol % of the fluorescent probe DilC12 (which preferentially partitions into liquid-expanded phases). The observations were carried out at room temperature in a specially designed Teflon trough equipped with Teflon barriers and a platinized-Pt Wilhelmy plate attached to a Cahn C32 electro-microbalance. The whole trough and sensing device was mounted onto the microscope plate. An open-end Teflon mask with a lateral vertical slit, covering the objective and extending through the film into the subphase, was used in order to restrict lateral monolayer flow in the field being observed.21 A Zeiss Axioplan (Carl Zeiss, Obcrkochem, Germany) epifluorescence microscope was used, with a source of UV radiation consisting of a mercury lamp HBO 50, an objective of 20×, and a fluorescein filter set. Exposure times were typically between 0.1 and 1 s. The pictures were recorded by a CCD video camera (Micromax, Princeton Instruments, Inc., USA) commanded through Metamorph 3.0 software (Universal Imaging Corp., USA).
Results Cyclic Voltammetry. Figure 1 shows the voltammetric profiles for the Fe(CN)63-/Fe(CN)64- couple on both bare (17) Maggio, B.; Bianco, I. D.; Montich, G. G.; Fidelio, G. D.; Yu, R. K. Biochim. Biophys. Acta 1994, 1190, 137. (18) Carrer, D. C.; Maggio, B. J. Lipid Res. 1999, 40, 1978. (19) McLaughlin, S. D. M. Annu. Rev. Biophys. Biophys. Chem. 1989, 18, 113. (20) Maggio, B. J. Lipid Res. 1999, 40, 930. (21) Oliveira, R. F.; Maggio, B. Neurochem. Res. 2000, 25, 77.
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Figure 1. Cyclic voltammograms for the Fe(CN)63-/4- redox couple in 20 mM NaNO3 on the bare electrode (‚ ‚ ‚), and on the covered electrode (s) by a monolayer of the indicated lipid. ν ) 0.005 V s-1.
and modified surfaces of a glassy carbon electrode by Langmuir-Blodgett films of the different lipids studied. An increase of the potential difference between the cathodic and the anodic peaks, ∆Ep, and a decrease of the current peak, Ip, with respect to the values on the bare electrode (∆Epb and Ipb), are observed in all cases; the modification of these parameters is an indication of a decrease in the rate of the electrochemical charge transfer (vct). In other words, the process becomes more irreversible from the electrochemical point of view. It is also possible to observe that the extent of these changes depends on the nature of the lipid forming the monolayer. The largest effect is produced by the Sul monolayer, and the smallest by Sphm. Galcer and dmPC show intermediate effects with Galcer inducing greater changes than dmPC. It is important to remark that although the redox couple employed is known as a reversible one, we found that the shape of the profiles depended on the surface conditions. As written in the Experimental Section, the electrodes were only polished. They were not electrochemicaly activated to avoid an increase in hydrophilicity, improving in this way the transfer of the lipid monolayer. The rate constant of the redox couple becomes lower and not so reproducible on nonactivated electrodes. This means that even with the polished monolayer-free electrode surface there may be differences between duplicate experiments. For this reason it was very important to repeat the measurements several times to ensure reproducibility of the response with each lipid monolayer. Also, for the same reason, the voltammograms obtained with the monolayer are always compared with the corresponding measurements on the bare electrode just before the coating was done. When calcium chloride is added, vct is enhanced, that is, the interfering effect of the lipid is reversed. In Figure 2 the normalized ∆Ep and cathodic Ip values (∆Ep/∆Epb and Ip/Ipb, respectively) are plotted against the Ca2+ concentration. As can be observed, in certain cases the electrochemical process becomes even more reversible than in the absence of the lipid monolayer. Additionally, the calcium concentration necessary to produce a given change depends on the type of lipid. In the case of dmPC, 1 mM calcium is enough to reverse totally the effect of the monolayer; however, for Galcer the initial profile is not recovered with any calcium concentrations used. As in the case of dmPC, when the electrode is modified with a monolayer of Sul, small amounts of calcium increase substantially the charge-transfer rate while higher con-
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Figure 2. Dependence of the normalized cyclic voltammetric parameters (∆Ep and Ipc) with Ca2+ concentration for electrodes covered with sphm (0), dmPC (b), sul (4), and gal/cer (1). ν ) 0.005 V s-1.
Figure 3. Lateral pressure versus molecular area for the indicated lipids over water (‚ ‚ ‚), solution A (s), and solution A + 1 mM or 3 mM CaCl2 for dmPC or the other lipids respectively (- - -).
centrations are needed to induce a similar effect with a Sphm monolayer. Monolayer Isotherms. Figure 3 shows the pressurearea (Π/A) compression isotherms for monolayers of the different lipids at the air-aqueous solution interfaces employed. dmPC. At 25 °C, the film on a pure water subphase or on solution A is in a liquid-expanded state (Figure 3), with a collapse pressure of 49 ( 4 mN m-1 at a limiting mean molecular area of 40.4 Å2, in agreement with previous measurements.22 Over a subphase of solution A containing CaCl2 (0.1 mM or higher) a liquid-expanded to liquid-condensed phase transition occurs at about 38 mN/m and 47 Å2. Epifluorescence microscopy of the film doped with the fluorescent probe DilC12 revealed coexistence of phase-separated liquid-expanded and liquidcondensed domains only on the subphase of solution A with CaCl2 (Figure 5). Figure 4 shows the variation of µP as a function of the molecular area. As the liquid expanded monolayer over a pure water subphase is compressed from a molecular area of about 110 Å2, the film becomes more coherent and the molecular dipole moment increases dramatically up to a mean molecular area of about 95 Å2 (about 5 mN/m). (22) Cevc, G.; Marsh, D. Phospholipid Bilayers. Physical Principles and Models. In Cell Physiology: a series of Monographs; Bittar, E. E., Wiley-Interscience: New York, 1987; Vol. 5.
Sphingolipid Monolayers on the Air-Water Interface
Figure 4. Molecular dipole moment perpendicular to the interface versus molecular area for the indicated lipids over water (‚ ‚ ‚), solution A (s), and solution A + 1 mM or 3 mM CaCl2 for dmPC or the other lipids respectively (- - -).
Figure 5. Representative epifluorescence microphotographs (225 µm × 177 µm) of dmPC monolayers over water at 34 mN m-1 (a) and over solution A + 2 mM CaCl2 at 35 mN m-1 (b).
This probably reflects the adoption under compression of a more perpendicular orientation of the hydrocarbon chains (with their relatively large dipole moment with a positive air end20,23). Thereafter, the molecular dipole moment decreases gradually as the film is brought to a more closely packed state, revealing a contribution opposite to that of the hydrocarbon chains.18,20,23 This may be caused by reorientation of fundamental dipole moments or changes in the hydration shell. The presence of ions in the subphase induces increases of µP to more positive values due to ion-dipole interactions in the polar headgroup region which may also involve inhomogeneous location of ions in the interface layer.24 This is in keeping with the fact that the electrolytes do not introduce changes in the variation of µP with molecular packing, compared to isotherms on a pure water subphase. Also, there are negligible differences in µP between the subphase solution A with or without CaCl2. Sphm. At 25 °C this lipid shows a two-dimensional phase transition at 13.0 ( 0.8 mN m-1 at a molecular area of 70 ( 2 Å2 and it collapses at 51 ( 2 mN m-1 at a limiting molecular area of 46.9 Å2 (Figure 3). No significant changes in film compressibility are observed on water or on solution A, except that the transition pressure is slightly shifted to 10.8 ( 0.5 mN m-1 and appears more cooperative. The behavior of the monolayer on solution A + CaCl2 3 mM is very similar but the whole isotherm is shifted to larger molecular areas and the collapse pressure is increased (55 ( 2 mN m-1). The resultant dipole moment µP of Sphm (23) Vogel, V.; Mo¨bius, D. Thin Solid Films 1987, 159, 73. (24) Maggio, B.; Lucy, J. A. Biochem. J. 1976, 155, 353.
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is positive at all values of molecular area (Figure 4), but it is smaller in magnitude than that of dmPC and behaves in a different manner under compression. An initial increase of µP between 150 and 200 mD is observed, as the film becomes more coherent (up to about 5 mN/m and a molecular area near 90 Å2 ) after which it remains insensitive to compression; the two-dimensional phase transition is reflected as an upward inflection at molecular areas between 70 and 80 Å2. Over solution A, the µP is higher than that obtained over water up to about 63 Å2, and the values are even higher on solution A with CaCl2. Sphm and dmPC have the same polar headgroup, but the hydrocarbon portion is different. The sphingolipid interfacial backbone contains a carbon-carbon double bond, a hydroxyl group, and an amide linkage instead of the ester bonds present in dmPC. Besides, the hydrocarbon chains in brain Sphm are on average longer (C18-C20 in the sphingosine base and mostly C18 in the amide linked fatty acid) than in dmPC (C14). The resultant dipole moment of the hydrocarbon moieties should increase about 80-100 mD upward due to the longer chains, but a decrease compared to dmPC is observed. How the different hydrocarbon portion in Sphm may change the hydration shell properties of the phosphorylcholine polar headgroup is not known. Assuming that this is similar, the variation between both phospholipids and the insensitivity of µP to vary under compression in Sphm should be ascribed to different properties in the interfacial region. In sphingolipids an interfacial intermolecular hydrogen-bonding network is present, with both hydrogen bond donor and acceptor groups,25 while hydrogen bonding mediated by water molecules among the phosphorylcholine phosphate groups occurs deeper in the aqueous subphase in the glycerophospholipid.22 Sul. The isotherm of this lipid on pure water is liquid expanded and collapses at 52 ( 2 mN m-1 at a limiting molecular area of 37.5 Å2 (Figure 3). Over solution A, a phase transition occurs at about 3 ( 1 mN m-1 and the whole curve is shifted to slightly larger molecular areas, with no significant changes in compressibility. The collapse pressure is the same in the presence of the ions, and the limiting molecular area is 40 ( 5 Å2. Over a subphase of solution A and 3 mM CaCl2 the collapse pressure is decreased to 50.5 ( 0.5 mN m-1 and the whole isotherm is shifted to larger molecular areas. Not surprisingly, in this anionic lipid the presence of ions markedly alters the resultant molecular dipole moment µP (Figure 4). Over a pure water subphase µp is near zero at the lift-off molecular area and acquires positive values reaching about 150 mD at the closest packing. In Sul the sphingosine base is of similar length than in Sphm but the composition in the amide-linked fatty acids contains, on average, longer chains (C20-C24 and about 30% of them are hydroxylated near the interfacial backbone further contributing to the surface hydrogen bonding network25). The dramatic decrease of the perpendicular resultant dipole moment, compared to Sphm, is due to the contribution of a large resultant dipole pointing in the opposite direction (toward the aqueous subphase) than that of the hydrocarbon moiety; this was also found previously for a series of complex glycosphingolipids.18,20 Over solution A, the resultant perpendicular dipole moment is negative, reaching values close to zero near collapse. This indicates that the location and/or the interactions with ions further hyperpolarize the polar headgroup resultant dipoles. The addition of 3 mM CaCl2 to solution A partially reverses this effect, and the dipole moment is displaced about 100 (25) Boggs, J. M. Biochim. Biophys. Acta 1987, 906, 353.
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mD upward. Considerable effects induced by calcium ions on the bulk phase trantition temperature of Sul were previously reported26 indicating that the calcium interactions in the polar headgroup region are transduced to altered intermolecular packing properties of the lipid. Galcer. This lipid is liquid-condensed 27 (Figure 3), with a collapse over water at about 44 ( 2 mN m-1, at a limiting molecular area of 42 Å2. The isotherm over solution A or with CaCl2 3 mM shows a similar compressibility, but it collapses at 43 ( 2 mN m-1 and is shifted in molecular areas. The same effects are observed for µP (Figure 4). In these monolayers µp is always positive, similar to dmPC and Sphm, but completely different to Sul. Galcer and Sul are in a direct metabolically correlated pathway being Galcer the precursor molecule for the biosynthesis of Sul by the enzyme PAPS-Galcer-sulfotransferase.28 The only difference between Galcer and Sul is the presence of a -SO3 group in C3 of the galactose moiety in the polar headgroup of the anionic glycosphingolipid. The large decrease in µP of Sul is due to the contribution of the negatively charged -SO3 group.10 Different from Sul, the presence of ions in solution A leads to an increase of the resultant molecular dipole moment of Galcer indicating different location and/or polar headgroup interactions; the subsequent addition of CaCl2 (in keeping with the lack of negative charges in Galcer) induces no significant changes. It was previously shown that Ca2+ up to 100 mM induced no significant alterations of the phase transition temperature of Galcer but affected the transition enthalpy.26 Discussion Charge transfer processes on blocked surfaces can take place in different ways as considered in the Introduction. On homogeneous films, permeation of species across the film or electron tunneling is possible. If the distribution of covering molecules is not uniform, blocked and bare domains are present: in this case the charge transfer can occur not only through the above mechanisms but also through clean surface spots. Our experimental results indicate that, in general, it is likely that the processes take place through microholes or defect boundaries known to occur in Langmuir-Blodgett films.21 Simulated voltammograms based on different theoretical models that propose a microarray for nonhomogeneous blocked surfaces2,3 support this hypothesis. Additionally, the electrochemical response depends on the lipid nature and may also be correlated in a complex manner to different specific properties exhibited at the air-aqueous subphase by each type of lipid monolayer. In principle, some general considerations can be made. It is logical to assume that a low charge-transfer rate should be expected when the electrode is coated with a lipid monolayer in a liquid-condensed state. It may also be expected that the smaller the mean molecular area (i.e., when two-dimensional lipid concentration over the electrode surface is larger), the lower the charge-transfer rate. With respect to the lipid overall molecular dipole moment perpendicular to the interface (µP), it is important to remark that the electroactive species is negative (Fe(CN)64-/3-) so a charge-transfer process would be easier when the electrode is covered by a lipid monolayer contributing with a positive dipole moment toward the (26) Maggio, B.; Sturtevant, J. M.; Yu, R. K. Biochim. Biophys. Acta 1987, 901, 173. (27) Maggio, B.; Cumar, F. A.; Caputto, R. Biochem. J. 1978, 171, 559. (28) Cumar, F. A.; Barra, H. S.; Maccioni, H. J.; Caputto, R. J. Biol. Chem. 1988, 243, 3807.
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electrode surface, compared to cases in which µP is negligible or negative. Figure 6 shows the correlation between the values of the electrochemical (∆Ep and Ip, parts a and b of Figure 6) and nonelectrochemical parameters obtained with solution A (Am, µP, and κ, parts c, d, and e of Figure 6) measured for the four lipids studied. The values of these parameters in the presence of Ca2+ are included in the same figure for comparison. Only the results with solution A will be discussed because the electrochemical measurements were performed using this solution and tendency observed is the same on water. For Sul, besides (Fe(CN)64-/3-) having to transpose a negatively charged layer, the electrochemical irreversibility observed in the absence of Ca2+ (Figure 6a and Figure 6b) is well correlated to the comparatively small mean molecular area of this lipid at the transfer pressure (Figure 6c) and to the low or negative value of its molecular dipole moment (µP) (Figure 6d). On the other hand, the compressibility (Figure 6e) of the monolayer of Sul, although high enough, does not appear to have an important influence; this is opposite to the behavior of Galcer, which shows a high compressibility as the main correlate to its induction of irreversible electrochemical response. The coating of the electrode with a monolayer of Sphm results in a higher charge transfer rate than with the other two sphingolipids studied. This fact may be correlated to its reduced compressibility and to the quite large mean molecular area and resultant dipole moment vector because larger mean intermolecular spacing and favorably oriented molecular dipoles should facilitate charge transfer compared to the other lipids. The behavior of dmPC appears anomalous regarding the parameters analyzed and does not follow the same correlations as the sphingolipids, except for the mean molecular area. In other words, the glycerophospholipid modifies the electrochemical response more than Sphm and this is not in direct correlation with the surface compressibility and with the resultant molecular dipole moment shown by its monolayer over solution A. The glycerophospholipid dmPC was included in our study as a known basis for comparison since this lipid has been reported to affect electrochemical responses9 and, additionally, it contains the phosphorylcholine moiety in the polar headgroup similar to Sphm. In fact, our results are not directly comparative to those in ref 9, because these authors use more than one lipid layer in order to to get a complete blocking effect of the electrochemical response while our intention was to correlate the latter to differences in molecular packing properties among different types of lipids. The novel results found for the sphingolipids clearly indicate that the electrochemical response is not correlated in a simple manner with either the hydrocarbon portion or the polar headgroup of the lipid forming the monolayer but to synergic overall combination-compensationenhancement effects derived concomitantly from the different moieties and the intermolecular organization of the surface. Our data point out that dmPC exhibits effects out of the comparative sphingolipid series because, at variance with the latter, it contains shorter hydrocarbon chains and a different chemical structure in the interfacial backbone. Also, the field on the monolayer when potential is applied over the electrode depends on the dielectric polarization vector of the lipid interface and this affects glycerophospholipids differently than sphingolipids.20 It has been shown that electrostatic field surface effects, in combination with the lipid resultant dipole moments, are important determinants for interfacial local charge dis-
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Figure 6. Electrochemical (a and b) and nonelectrochemical (c, d, and e) parameters measured in solution A (white bars) and solution A + CaCl2 (gray bars): electrochemical parameters, normalized ∆Ep (a) and normalized Ipc (b); nonelectrochemical parameters, molecular area (c), molecular dipole moment perpendicular to the interface (d), and compressibility of the monolayer (e) near the collapse pressure point.
placement and electron rearrangement affecting interfacial enzyme reactions.20 Correlation between Voltammograms with Calcium and Isotherms over Solution A Plus Calcium. As shown in Figure 2, the response of electrodes coated with monolayers of Sul or dmPC are altered by the presence of Ca2+. Electrochemically, although the slope change is similar, more Ca2+ is necessary with Sul to revert completely the monolayer effect. The behavior of the isotherms offers some insight for interpreting the different electrochemical response in the presence of Ca2+. In the case of dmPC, the two-dimensional phase transition induced at high surface pressures in the film over solution A plus Ca2+ generates molecular packing defects in the surface monolayer26,29 and we have confirmed their existence by epifluorescence microscopy (see Figure 5). The surface microheterogeneity due to phase coexistence and lateral defects may increase the interfacial permeability30 facilitating charge transfer and accessibility of Fe(CN)63- to the electrode surface. This is in keeping with the increase in the reversibility of the electrochemical process. Figure 6 shows the values of the electrochemical parameters when the calcium concentration in the electrochemical cell is 1 mM for dmPC and 3 mM for the other lipids and of nonelectrochemical parameters of isotherms performed over solution A and the same concentration of calcium at the collapse pressure. In the case of Sul, the (29) Biltonen, R. J. Chem. Thermodyn. 1990, 22, 1. (30) Papahadjopoulos, D.; Jacobson, K.; Nir, S.; Isac, T. Biochim. Biophys. Acta 1973, 311, 330.
increase in current is well correlated with the increase of the mean molecular area and with the changes in the resultant molecular dipole moment. Similar effects were found for Sphm, but all the electrochemical and nonelectrochemical changes are smaller than those observed for Sul. Isotherms of Galcer showed practically no changes in the presence of calcium. For sphingolipids, either Sul or Sphm occupy a larger mean molecular area in the presence of CaCl2 and this should facilitate access and discharge of the electroactive ion to the electrode surface. The change in molecular area induced by Ca2+ in the Sul monolayer is larger than that for Sphm, and this is in keeping with the greater alteration of the electrochemical response. The changes of the resultant molecular dipole moment (µP) induced by calcium in monolayers of dmPC and Galcer are negligible while the divalent cation induces an increase of µP for Sphm and Sul (Figure 4). At the working surface pressures employed for monolayer transfer to the electrode surface, the absolute difference is larger for Sphm than for Sul; however, since the anionic sphingolipid shows a smaller molecular dipole moment in the absence of the cation, the relative increase of µP induced by calcium is much larger for Sul and this correlates well with the electrochemical changes observed. Summing up, in the case of sphingolipids the low reversibility of the charge-transfer process correlates with low Am and µp and high values of κ. It is therefore not unexpected that a reversion in the electrochemical response should be accompanied by increases in Am and µp, and this was observed. In addition, electrochemical
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changes correlate with changes in nonelectrochemical parameters of the lipids. Monolayers of Galcer do not appear influenced by the presence of calcium ions in the subphase; nevertheless, as shown in Figure 6, the electrochemical response is changed by different amounts of this cation. This behavior is currently under investigation. Finally, an important point to consider is the already reported effect of cations on the kinetics of the hexacianoferrate couple.31,32 In these papers the authors showed that the enhancement of the rate constants is due to interaction with cations more than to a double layer effect and conclude that the reaction mechanism of this couple is more complicated than it has been assumed. This unusual behavior was reported in solutions of high ionic strength. Although in our case this condition is not fulfilled, the already described specific interaction26,33 between the lipids and calcium, also observed in our work, could lead to increases of the interfacial concentration of this ion so as to reach the concentration necessary to cause a more reversible electrochemical response. This effect would explain why the response of the modified electrode after several additions of Ca2+ may become even more reversible than that of the bare electrode. Conclusions There is a correlation between the electrochemical and nonelectrochemical effects obtained with LB monolayers onto glassy carbon and the intermolecular organization of sphingolipids at the air-water interface. Nevertheless (31) Peter, L. M.; Du¨rr, W.; Bindra, P.; Gerischer, H. J. Electroanal. Chem. 1976, 71, 31. (32) Canpbell, S. A.; Peter, L. M. J. Electroanal. Chem. 1994, 364, 257. (33) Tocanne, J. F.; Teissie´, J. Biochim. Biophys. Acta 1990, 1031, 111.
Wilke et al.
the correlation is complex because the monolayer parameters can influence the charge transfer process in different ways. Among these parameters the lipid charge and dipole moment orientation are important, due to possible electrostatic interactions occurring between the lipid monolayer and the redox species. This is the case of the anionic monolayer of Sul shown in Figure 1. Currently the chargetransfer process of a cationic redox couple is being analyzed on this surface. Concerning the mechanism of charge transfer, the shape of the voltammograms would indicate that the process takes place through microholes formed during film transfer or after, due to possible rearrangements of the monolayer. The surface inhomogeneities would also depend on the monolayer parameters, that is, on the lipid nature. For example, the most condensed layer would give the smallest microholes. The hypothesis of the existence of microholes is supported by the comparison of the data with theoretical voltammograms calculated by using the model proposed in refs 2 and 3. A deeper analysis in this direction is being currently performed, and other theoretical models will be considered to improve the understanding of our system. Finally, we consider that a better insight on the effect of dmPC could be provided including different members of a glycerolipid family in the analysis as it was done in the present study with the sphingolipids. Acknowledgment. Financial support from the Consejo de Investigaciones de la Provincia de Co´rdoba (CONICOR), Consejo Nacional de Investigaciones Cientı´ficas y Tecnolo´gicas (CONICET), and Secretarı´a de Ciencia y Tecnologı´a (SECyT) is gratefully acknowledged. N. Wilke thanks CONICET for the fellowship granted. LA0015314
