9940
J. Phys. Chem. 1995, 99, 9940-9951
Adsorption of Tetraphenylphosphonium and Tetraphenylborate in Self-Assembled Phosphatidylcholine and Phosphatidylserine Monolayers Deposited on Mercury Electrodes Maria Rosa Moncelli, Roberto Herrero,” Lucia Becucci, and Roland0 Guidelli” Department of Chemistry, Florence University, Florence, Italy Received: December 14, 1994; In Final Form: March 7, 1995@
The adsorption of tetraphenylborate (TPhB-) and tetraphenylphosphonium (TPhP+) ions in self-assembled monolayers of phosphatidylcholine (PC) and phosphatidylserine (PS) deposited on a mercury electrode was estimated by measuring the time dependence of the charge which flows as a consequence of potential steps causing ion translocation across the monolayer. In the time scale of 100 ms, the translocation of TPhP+ ions is controlled by diffusion from the bathing solution, while that of TPhB- ions is primarily controlled by the adsorption into the polar head region. Deviations from the Henry isotherm behavior were interpreted on the basis of an adsorption isotherm which accounts both for discreteness-of-charge effects and for the presence of two regions of different dielectric constant 6, Le., the hydrocarbon tail region ( E M 2) and the polar head region (€2 = 8-30). This isotherm also interprets a number of data for ionic adsorption in bilayers available in the literature using reasonable values for the parameters of lipid layers. The pH dependence of the adsorption coefficient of TPhP+ and TPhB- ions in PS monolayers points to changes in the conformation and in the acidic properties of the polar heads of this lipid induced by ionic adsorption.
Introduction The transport of lipophilic ions across bilayer lipid membranes (BLMs) has been extensively studied both from a kinetic point of view and with the aim of determining the amount of ions adsorbed within the two polar head regions of BLMs.’-~ In general, this transport is assumed to require the passage over a central energy barrier separating two deep energy wells located within the two polar head regions. If the membrane is symmetric, the energy barrier is also assumed to be symmetric. The kinetics of transport in relaxation experiments is often tackled by solving the generalized time-dependent NemstPlanck equation under the simplifing assumption that the lipophilic ions are almost completely located within the energy wells; the additional assumptions that the transmembrane potential is small with respect to RT/F’,5,8and that the electric field varies linearly between the two energy wells are also frequently made.2.3 The potential difference between the two energy wells is usually considered to be a constant fraction of the transmembrane potential, although Andersen et aL6 used a three-capacitor model to show that this fraction depends on the amounts of ions in the two energy wells and hence varies during their translocation. The kinetics of translocation of ions through the membrane in relaxation experiments is generally assumed to involve three steps: namely, (i) adsorption of the ions in the polar head region on one side of the membrane, (ii) surmounting of the intermediate energy barrier with the resulting translocation to the polar head region on the other side of the membrane, and (iii) desorption from the latter polar head region into the adjacent bathing solution. In the case of dipicrylamine (DpA-) and tetraphenylborate (TPhB-), translocation across a dioleoylphosphatidylcholine bilayer, potential step experiments have shown that the rate-determining step is step iii, namely, ion desorption into the bathing solution.’ However, by using potential steps of short duration and very low concentrations of the liphophilic ions in the bathing solutions, the kinetic study can be focused
’
On leave from the Departamento de Quimica Fundamental e Industrial, Universidade da Corufia, A Corufia, Spain. @Abstractpublished in Advance ACS Abstracrs, May 1, 1995.
exclusively on the translocation step ii, since step iii becomes too slow to be operatives3 Under these experimental conditions, only a capacitive current flows across the membrane and the number of ions adsorbed in it remains constant, but at the end of the potential step the current has not yet attained its minimum stationary value. So far ion transport systems have been usually studied using symmetric BLMs. A more recent model of biomembranes is represented by a phospholipid coated mercury The phospholipid coating is provided by spreading a solution of the phospholipid in a suitable solvent (e.g., pentane) on the surface of an aqueous electrolyte, allowing the solvent to evaporate, and immersing a hanging mercury drop electrode in the electrolyte. This procedure gives rise to half a self-assembled bilayer, with the hydrocarbon tails directed toward the metal and the polar heads directed toward the solution. Self-assembled phospholipid monolayers on mercury electrodes offer some advantages over BLMs, when used as biomimetic membranes. Thus, the electric potential and the ion flux across these monolayers can be controlled more accurately and more directly than across BLMs. Moreover, these half-membranes provide an inherent mechanical stability and a resistance to high electric fields which is not shared by BLMs. Over the region of minimum capacity, the film is impermeable to inorganic metal ions, whereas it becomes permeable outside this region. By measuring the differential capacity C of these lipid monolayers supported on mercury, we have recently determined the intrinsic protonation constants of the ionizable groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS).I2 It was thus shown that PS monolayers bear only a small negative charge at the physiological pH, are practically neutral around pH 6, and become slightly positively charged at lower pH values. These results, obtained with PS monolayers with a rigorously constant cross-sectional area per lipid molecule and in the absence of any incorporated lipophilic molecules, contrast with literature data according to which PS mono- or bilayers are negatively charged at pH > 2.I3-l6 This led us to conclude that even slight perturbations may induce conformational changes in the PS polar heads which are
0022-3654/95/2099-9940$09.00/0 0 1995 American Chemical Society
Ion Adsorption in Lipid Monolayers
J. Phys. Chem., Vol. 99, No. 24, I995 9941
accompanied by appreciable changes in the acidic properties of the corresponding ionizable groups. The present note reports an investigation of the adsorption of the TPhB- anion and of the tetraphenylphosphonium cation (TPhP+) within the polar head region of self-assembled monolayers of PC and PS. To this end, the differential capacity C of the monolayers was measured as a function of the applied potential E; moreover, the charge Q(t)flowing along the external circuit as a consequence of a series of potential steps AE of different width was measured as a function of time t using a wholly computerized chronocoulometric apparatus.
time t, this quantity was found to increase progressively with z attaining a maximum limiting value for z values greater than a certain minimum value zmin. Owing to mass transport control of ionic adsorption, zminwas found to decrease rapidly with an increase in the bulk ionic concentration, c. Each series of chronocoulometric measurements at constant c was carried out after stirring the solution at Ei for a time period greater than the corresponding zminvalue. At the lowest ionic concentration investigated, c = 1 x lo-' M, zminwas found to be about equal to 4 min.
Results Experimental Section The adsorbed monolayers of PC and PS on mercury were prepared as described by Nelson and A ~ f f r e t . ' ~ , 'The * water used was obtained from light mineral water by distilling it once, and then distilling the water so obtained from alkaline permanganate, while constantly discarding the heads. Merck reagent grade KC1 was baked at 500 "C before use to remove any organic impurities. Dioleoyl PC and dioleoyl PS were obtained from Lipid Products (South Nutfield, Surrey, England). All measurements were carried out in aqueous solutions of 0.1 M KC1 at 25 k 0.1 "C. The solutions were buffered with PS and unbuffered with PC; in the latter case, the pH was constantly found to range from 4.8 to 5.3. The desired pH values were realized with Merck suprapur HCl over the pH range from 2 to 5, with a 1 x M HP042-/H2P04- buffer over the pH range from 6.5 to 7.5 and with a 1 x M H3B03NaOH buffer over the pH range from 8.5 to 9. The home-made hanging mercury drop electrode (HMDE) employed in our measurements, the cell, and the detailed procedure to produce self-organized lipid monolayers deposited on mercury are described elsewhere.'* Differential capacity measurements were carried out using a Metrohm Polarecord E506 (Herisau, Switzerland). The ac signal had a 10 mV amplitude and a 75 Hz frequency. The system was calibrated using a precision capacitor. All potentials were measured versus a saturated calomel electrode (SCE). The computerized chronocoulometric apparatus is described e1se~here.I~The microprocessor used to control all the operations was a Model NOVA 4X from Data General (Westboro, MA), whereas an Amel Model 551 (Milano, Italy) fast rise potentiostat with a rise time 50.1 ps was employed for the potentiostatic control of the three-electrode system. The detailed scheme of the home-made electronic current integrator working under microprocessor control is described in ref 20. The chronocoulometric measurements consisted of a series of consecutive potential steps of progressively increasing width from a fixed initial value E, to different final values E; each series was carried out on a single lipid coated mercury drop. The initial potential E, was chosen to be positive (negative) enough to prevent adsorption of TPhP+ (TPhB-) directly on the mercury surface, while allowing ionic adsorption in the polar head region under equilibrium conditions. After 100 ms from each E, E potential step, the potential was stepped back from E to E, and kept at E, for 10 s, in order to restore the initial equilibrium conditions; the current i(E,t) flowing as a consequence of each forward potential step E, E was integrated electronically, and the resulting charge Q(E,t) was stored in the microprocessor memory as a function of the time t elapsed from the instant of the corresponding E, E potential step. To ensure the attainment of adsorption equilibrium of the lipophilic ions in the polar head region at E,, the bathing solution was first subjected to mild stirring for different time lengths z, and the corresponding charge Q(E,t) was then measured; at any given
-
-
-
In the absence of lipophilic ions, the differential capacity C at the lipid coated electrode varies only very slightly over the potential region of minimum capacity, namely, from -0.2 to -0.8 V. Conversely, in the presence of these ions C shows a hump which develops on the negative side of this potential region in the case of TPhP+ in PC and PS monolayers, and on the positive side in the case of TPhB- in PS monolayers. The presence of TPhB- in PC monolayers gives rise to a shoulder simply because the hump merges with the steep rise exhibited in the proximity of -0.2 V by the differential capacity versus potential curve in the absence of TPhB-. The capacity humps obtained with the TPhP+ and TPhB- ions in PS monolayers are shown in Figure lA, whereas those obtained in PC monolayers are shown in Figure 1B. Along these humps the in-phase component of the ac current was constantly found to be entirely negligible with respect to the quadrature component, thus excluding the presence of any Faradaic contributions. The hump in the presence of TPhPf ions was obtained by scanning the applied potential E in the negative direction after keeping the mercury drop under mild stirring at a potential positive to the hump for a time period t > zmln. It is clearly due to the charge displacement resulting from TPhP+ translocation from the polar heads to the mercury surface and backward across the hydrocarbon tail region, following the ac signal superimposed on the bias potential E. Naturally, the to and fro movement of TPW+ cations following the ac signal is only possible at bias potentials E at which these ions are present both in the polar head region and on the mercury surface; this condition is not satisfied at potentials positive to the hump, where TPhP+ is only present in the polar head region. In the case of the TPhB- anions, the applied potential E was scanned in the positive direction after keeping the mercury drop under mild stirring at a potential negative to the hump for a time period z >
tmin.
Chronocoulometric curves of Q(E,t) versus r for the translocation of 1 x M TPhP+ across a PS monolayer at pH 8.9 are shown in Figure 2; they were obtained by stepping the applied potential from a fixed initial value E, = -0.200 V to different, regularly spaced final values. At final potentials E positive enough to prevent TPhP+ translocation, the charge density Q(E,t) undergoes an abrupt rise and then remains constant, since the capacitive current required to charge the whole interphase flows in less than 1 ms just after the El E potential step. At potentials E at which TPhP+ translocation starts to take place, the initial abrupt rise in charge is followed by a gradual increase in time, which becomes more pronounced as E is made progressively more negative. Ultimately, at sufficiently negative potentials, the Q(E,t) versus t curves shift regularly upward with a further negative shift in E, while maintaining a constant shape. The Q(E,t) versus t curves for TPhP+ translocation across a PC monolayer exhibit the same behavior as those across a PS monolayer. All these curves provide linear Q(t) versus ti/* plots whose slopes S increase
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9942 J. Phys. Chem., Vol. 99, No. 24, 1995 I
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Figure 3. Q(t) vs r1l2plots for the translocation of 0 (a), 3
x (b), 7 x (c), 9 x (d), 1 x (e), 1.2 x (9, 1.6 x (g), and 2 x M (h) TPhP+ across a PC monolayer following a potential step from E, = -0.200 V to E = -0.900 V. The inset shows a plot of S = Q(t)/t”’ vs c, whose constant slope equals -160 C cm SKI!* mol-’.
6
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concentrations, the Q(t) versus t l / * plots start to deviate from linear behavior at progressively shorter times t with an increase in c (see Figure 3). According to the Cottrell equation, a charge density Q(t) which is diffusion controlled under limiting conditions is given by :
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Figure 1. (A) C vs E curve for PS monolayers in contact with 1
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M TPhP+ at pH 8.9 (a) and with 6 x
M TPhB- at pH 2.7 (b). The dashed curve was calculated from the d l vs E curve corresponding to the C vs E curve (a) using eq 10; Co = 1.74 pF cm-2 and u)= 0.47 pC cm-2. (B) C vs E curve for PC monolayers in contact with 7 x M TPhP’ (a) and with 1 x lo-’ M TPhB- (b) in unbuffered solutions of 0.1 M KC1.
-2.0
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where n is the number of faradays flowing along the extemal circuit in concomitance with the translocation of 1 mol of the lipophilic ion and D is the diffusion coefficient of the latter. If we apply this equation to the constant slope of the plot of S = Q(t)/t1/2versus c (see the inset of Figure 3), we obtain a reasonable value of x2 x cm2/s for the diffusion coefficient D provided we set n = -1. The justification for the charge Q(t) flowing along the extemal circuit being almost equal in magnitude and opposite in sign to that translocating across the lipid monolayer will be deffered to the Discussion. The present result points to a high rate of both the adsorption step of TPhP+ ions into the polar head region and the subsequent translocation step, such that diffusion control is achieved in less than 1 ms from the instant of the potential step. The deviation plots from linear behavior at c values of the Q(t) versus greater than about 1 x M is simply due to the saturation of the adsorption sites on the mercury surface, which prevents a complete depletion of the polar head region after the negative potential step. Extrapolation of the linear Q(t) versus tl’* plots in Figure 3 to tl/* = 0 yields an intercept which measures the charge along the extemal circuit due to the charging of the interphase following the potential step E, E and to the complete translocation of the TPhP+ ions originally present in the polar head region. The intercepts for different bulk TPhP+ concentrations c, as obtained by adopting a fixed potential step Ei E negative enough to ensure complete translocation of the TPhP+ ions initially adsorbed at Ei = -0.200 V, were measured relative to the intercept provided by the supporting electrolyte alone and then plotted against c, thus yielding the charge involved in this translocation; this is practically a measure of the charge q of the TPhP’ ions adsorbed at Ei as a function of c, namely, the adsorption isotherm of these ions in the polar head region
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Figure 2. Q(E,r) vs r curves for the translocation of 1 x
M TPhP+ across a PS monolayer at pH 8.9, as obtained by stepping the potential from the fixed initial value E, = -0.200 V to final values of E varying from -0.200 to -0.900 V by -20 mV increments.
proportionally to the bulk TPhP’ concentration c, at least up to c values of the order of 1 x M. At higher TPhP+
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Ion Adsorption in Lipid Monolayers
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J. Phys. Chem., Vol. 99, No. 24, 1995 9943 1-
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