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The complex formation of ions with a phospholipid monolayer adsorbed at an aqueous|1,2-dichloroethane interface
Journal of
Electroanalytical Chemistry Journal of Electroanalytical Chemistry 578 (2005) 17–24 www.elsevier.com/locate/jelechem
The complex formation of ions with a phospholipid monolayer adsorbed at an aqueous|1,2-dichloroethane interface Yumi Yoshida
a,*
, Kohji Maeda a, Osamu Shirai
b
a
b
Department of Chemistry, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan Division of Nuclear Engineering Science, Research Reactor Institute, Kyoto University, Kumatori, Osaka 590-0494, Japan Received 21 September 2004; received in revised form 7 December 2004; accepted 7 December 2004 Available online 8 February 2005
Abstract The complex formation of phosphatidylcholine adsorbed at a water|1,2-dichloroethane, W|DCE, interface with ions was evaluated based on the desorption behavior of lipids at the interface accompanied by an ion transfer and was confirmed by measuring voltammograms for the ion transfer facilitated by the complex formation with lipids. The desorption behavior of lipids was measured according to the Wilhelmy vertical plate method, the cell of which was improved to achieve polarization of the W|DCE interface. Voltammograms for the ion transfer at the W|DCE interface facilitated by the complex formation with lipids were recorded at a micro W|DCE interface in order to avoid the influence of convection caused by the desorption of lipids at the interface. Lipids þ adsorbed at the W|DCE interface showed complex formation with alkali metal ions, NHþ 4 ; ðCH3 ÞNH3 ; ðCH3 Þ2 2 þ þ þ NH2 or ðCH3 Þ3 NH , but not with ðCH3 Þ4 N or anions such as SO4 , CH3COO , Cl , Br , NO3 , I or ClO4 . Lipids also showed strong complex formation with a basic amino acid such as an arginine cation, which has two amino groups in the molecule. The þ þ þ þ strength of the complex formation with lipids was Argþ > Liþ ; Naþ > Kþ > NHþ 4 > CH3 NH3 > ðCH3 Þ2 NH2 > Cs > ðCH3 Þ3 NH . 2005 Elsevier B.V. All rights reserved. Keywords: Phospholipid; Liquid|liquid interface; Association constants; Amino acid; Voltammetry; Interfacial tension
1. Introduction The evaluation of binding of an ion or an ionic functional group to the surface of a phospholipid membrane is very important to understand not only the ion transport through a biomembrane but also the orientation of a membrane protein on the lipid membrane [1]. The binding of an ion to a bilayer lipid membrane, BLM, has been investigated by NMR measurement, IR spectroscopy, electrophoresis, the ion-selective electrode method, the monolayer-radiotracer technique, neutron diffraction, etc. [2]. Results obtained by these techniques indicate that the binding strength of an ion
*
Corresponding author. Tel./fax: +81 75 724 7518. E-mail address: [email protected] (Y. Yoshida).
0022-0728/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2004.12.016
with the BLM was large for a large surface charge density of the cation, e.g., lanthanide > transition metals > alkali earth metals > alkali metals organic cation, and for a hydrophobic anion, e.g., tetra phenylborate > ClO 4 > I > SCN > NO3 > Br > 2 Cl > SO4 . The binding strength of an ion with a BLM has been discussed mainly based on the following interactions [2]; (1) the long-range electrostatic force, which is governed by charges located on an ion and phospholipids and the local dielectric constant near the water|membrane interface, (2) the complex formation of an ion with the polar head group of a phospholipid, and (3) the hydrophobic interaction caused by breaking of the water structure. In previous studies, the location of an ion or the orientation of a protein on the BLM surface has been mainly discussed based on interaction (1) in the absence of an inorganic salt irrespective of
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the objective ion. However, the inorganic salt at high concentration such as 0.1 M NaCl is present under biological conditions. Under biological conditions, the contribution of interaction (2) to the binding to the BLM is more important than interaction (1) in order to understand the location of an ion or the orientation of a protein on the BLM surface because interaction (1) is masked by a large amount of the inorganic salt. The purpose of the present work is to evaluate interaction (2) and to elucidate the selectivity of the complex formation of lipids with ions. According to the previous method using the BLM, interaction (2) is difficult to evaluate in distinguishing it from other interactions. Using a phospholipid monolayer adsorbed at the liquid|liquid interface, only interaction (2) can be estimated. When the phospholipid monolayer forms a complex with an ion, the phospholipid monolayer desorbs with the transfer of the ionic complex realized by applying an interfacial potential [3–7]. In addition, the complex formation can be confirmed by voltammetry for the ion transfer at the liquid|liquid interface facilitated by complex formation with the phospholipid [4,5,8]. Desorption of the phospholipid monolayer and the facilitation of ionic transfer at the liquid|liquid interface can be measured even in the presence of inorganic salts and is not influenced by interaction (3). In measurements using the phospholipid monolayer at a liquid|liquid interface, the strong complex formation of lipids with alkali earth metal ions [4,9,10], H+ [6,8] or Li+ [3,4,7] has been reported. In the case of other alkali metal ions or ammonium ions, complex formation has not been demonstrated in previous studies though we pointed out the possibility of complex formation with other alkali metal ions and ammonium ion [5]. In order to estimate the weak complex formation with these ions, we improved the measurement of the interfacial tension based on the Wilhelmy vertical plate method and confirmed the complex formation by voltammetry for the ion transfer at a micro water|1,2-dichloroethane, W|DCE, interface, where convection caused by desorption of lipids is small.
dence of the solubility of the salt in ethanol. Na+TFPB was synthesized according to the procedure described previously [11,12]. Tetraphenylarsonium sulfate, ðTPhAsþ Þ2 SO2 4 , was prepared by the titration of TPhAs+Cl (Aldrich) with Ag2SO4 in an aqueous solution [13]. The salt of tetraphenylborate with TPhAs+, TPhAs+TPhB, was prepared according to the previous studies [13]. The DCE used was purified according to the method described in the literature [14] and was saturated with distilled water. All other chemicals used were of reagent grade quality. 2.2. Measurements of the interfacial tension at a polarized W|DCE interface The interfacial tension, c, at a polarized W|DCE interface was measured by the Wilhelmy vertical plate technique [15] applying the potential difference, E, at the W|DCE interface. Fig. 1 shows the cell used for the measurement of c. The W|DCE interface was formed in a glass beaker (35 mm diameter). The objective cation or anion was added to W as a chloride or sodium salt. The pH of W was not controlled by any buffer and was confirmed to be almost 6–7. The DCE contained PC and 0.01 M BTPPA+TFPB as a supporting electrolyte. A glass plate (23.85 ± 0.05 mm width, 0.3 ± 0.01 mm thickness, Kyowa Interface Science Co., Ltd.) silanized with dimethyldichlorosilane was placed on the W|DCE interface, and connected to an electrobalance. The interfacial potential difference at the W|DCE interface was applied as the potential of W referred to that of DCE using two reference electrodes of an Ag|AgCl electrode in W and a tetraphenylarsonium ion-selective electrode, TPhAsE, in DCE and two counter electrodes of a Pt wire in W and a Pt net in DCE. The configuration of TPhAsE is given by Eq. (1).
2. Experimental 2.1. Chemicals The phospholipid employed was L-a-phosphatidylcholine dioleoyl, PC, of analytical grade (SIGMA, Lot No. 32k5202). Bis(triphenylphosphoranylidene)ammonium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, BTPPA+TFPB, was obtained as a precipitate by mixing a methanol solution of BTPPA+Cl with a methanol solution of Na+TFPB, and the precipitate was purified by recrystallization based on the temperature depen-
Fig. 1. Cell for the measurement of interfacial tension at a polarized W|DCE interface: (1) aqueous phase, (2) DCE phase, (3) a Pt wire used as a counter electrode in W, (4) a Ag|AgCl electrode used as a reference electrode in W, (5) TPhAsE used as a reference electrode in DCE, (6) a Pt net used as a counter electrode in DCE and (7) a silanized glass plate.
Y. Yoshida et al. / Journal of Electroanalytical Chemistry 578 (2005) 17–24
Ag AgCl 0.1 M LiCl (W)
5 × 10–4 M
19
10–3 M
(TPhAs+)2SO42– TPhAs+TPhB– (W)
(DCE) (M = mol dm-3)
ð1Þ Here, E was related to the Galvani potential difference at the W|DCE interface, D/, as, E ¼ D/ þ Eref ;
ð2Þ
where Eref is the potential of the reference electrodes employed. According to the same procedure described in a previous study [16], the Eref of TPhAsE was determined to be 0.350 V referred to the standard potential, where the Gibbs energy for the transfer of an ion from W to DCE, DGtr, is recognized to be zero based on the extrathermodynamic assumption of Parker [17]. 2.3. Voltammetry for ion transfer at a micro W|DCE interface The voltammogram for ion transfer at the interface between W and DCE was measured by scanning E applied at a micro W|DCE interface and measuring the current due to the ion transfer [18,19]. The cell consisted of W and DCE separated by a polyester film, 16 lm thick with a micro hole 50 lm in diameter [19]. The W|DCE interface was formed at the micro hole. In the measurements, the objective cation or anion was added to W as a chloride salt or sodium salt, respectively. The supporting electrolyte added to DCE was BTPPA+TFPB. All electrochemical measurements were carried out at 25 ± 0.5 C. 2.4. Apparatus The potentiostat/galvanostat for voltammetry at the micro W|DCE interface and that for c measurement were Model HECS-318 (Huso Electro Chemical System) and Model HA 1010MIS (Hokuto Denko Co.), respectively. The function generator and X–Y recorder used were Model HB-111 (Hokuto Denko Co.) and Type 3086 (Yokogawa Electric Works, Ltd.), respectively. The electrobalance and a electrobalance lift were HD-60 (A&D Company, Ltd.) and SYLB-7K (Sanyu Tech., Ltd.).
3. Results and discussion 3.1. Polarization at a W|DCE interface in the cell for the c measurement The polarization of the rather large W|DCE interface formed in the cell of Fig. 1 was confirmed by measuring
Fig. 2. Voltammograms in the presence (a) and in the absence (b) of 2 · 104 M (CH3)4N+Cl in W and the potential dependence of the interfacial tension in the absence of (CH3)4N+Cl measured at the W|DCE interface formed in the cell of Fig. 1. W and DCE contained 0.1 M NaCl and 0.01 M BTPPA+TFPB as supporting electrolytes, respectively. scan rate: 50 mV s1.
the transfer of (CH3)4N+. Curve (a) in Fig. 2 is the voltammogram for the transfer of (CH3)4N+ at the W|DCE interface formed in the cell. The middle potential between the positive and negative peaks agreed with the reported value [20]. The peak separation was 0.075 V. It was recognized that the influence of the IR drop on the polarization of the W|DCE interface in the cell was not serious. The electrocapillary curve in Fig. 2 was measured by using the cell of Fig. 1. The c values were reproducible by about ±0.3 mN m1 on repeated runs. The electrocapillary curve was expressed as a quadratic function, and the differential capacitance was 0.054 F m2. The c value of the potential of zero charge, pzc, was 28.1 mN m1, which agreed with the reported value at the W|DCE interface estimated by the quasi-elastic laser light scattering method [3]. 3.2. Adsorption of the PC monolayer at the W|DCE interface Fig. 3 shows the c value at the W|DCE interface measured as a function of D/ in the presence of PC in DCE. The D/ was held at 0.2 V until the decrease in c stopped before starting the potential sweep. The adsorption equilibrium of PC was achieved at 0.2 V, where strong adsorption of PC was observed in our preliminary results for c. The potential dependence of c was measured on changing D/ from a negative to a positive potential because the reproducibility of c became poor at more positive potentials than about 0.1 V as was pointed out by Kakiuchi et al. [7]. The difference between c in the presence and in the absence of PC was significant when D/ was more negative than around 0.1 V, indicating that the amount of adsorbed PC in this D/ range (Range A) was large, but the difference was less significant when D/ was more
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30
γ / mN m-1
25 20 15 10 5 0 -0.4
-0.2
0.0 0.2 ∆f / V
0.4
0.6 Fig. 4. The interfacial tension at 0 V in Fig. 3 as a function of logarithmic concentration of PC in DCE.
Fig. 3. The potential dependences of the interfacial tension in the presence of x M PC + 0.01 M BTPPA+TFPB in DCE and 0.1 M NaCl in W: x = 0 (- - - -), 2 · 106 (d), 5 · 106 (s), 105 (.), 2 · 105 (,), 5 · 105 (j) or 104 (h).
positive than around 0.1 V, indicating that the amount of adsorbed PC in this D/ range (Range B) was small in accordance with the literature [3–7,9,10]. The c value at the polarized W|DCE interface was expressed by the Gibbs adsorption equation taking into account adsorption of supporting electrolytes in both phases and PC species in DCE. X dc ¼ Ciz d~ liz þ CPC dlPC ; ð3Þ iz ¼ Naþ ; Cl ; BTPPAþ ; TFPB : ~a are the surface excess concentraHere, Ca, la and l tion relative to both solvents, the chemical potential and the electrochemical potential for the a species in the bulk phase. When the composition of the supporting electrolytes in W and DCE is not changed, Eq. (3) can be written as dc ¼ Q dD/ þ CPC dlPC ;
ð4Þ
where Q is the surface charge density. When D/ is constant, Eq. (5) can be derived from Eq. (4). Here, aPC is the activity of PC in DCE. 1 oc CPC ¼ : ð5Þ RT o ln aPC D/;li Fig. 4 is a plot of c at 0 V, which corresponds to the potential zero charge, as a function of the bulk concentration of PC in DCE. When the concentration of PC was in the range from 2 · 106 to 2 · 105 M, the slope was constant, indicating that the adsorption of PC at the W|DCE interface was saturated. The CPC was calculated to be 2.31 lmol m2 from the slope based on Eq. (5). The value of CPC agreed with the reported values of the phospholipid monolayer, 2.71 lmol m2 [21] and 2.5 lmol m2 [3]. When the concentration of PC was more than 5 · 105 M, the slope decreased, indicating that this concentration of PC was more than the critical aggregation concentration.
3.3. Complex formation of Na+ with a PC monolayer adsorbed at the W|DCE interface Previous literature references have suggested that the desorption of PC in Range B is caused by the transfer of the complex following complex formation of a hydrophilic cation with PC [3,5,8]. In order to confirm the complex formation of Na+ with PC, voltammograms for the transfer of Na+ were measured at the micro W|DCE interface. The reason for the measurement at the micro interface is to decrease the effect of convection of the interface due to desorption of PC. Fig. 5 shows voltammograms for the transfer of Na+ in the presence of PC at high concentration in DCE. The positive current appeared at a positive potential greater than 0.2 V. The positive current increased with the increase in the concentration of PC. The positive current, however, was not proportional to the concentration of PC since the PC concentration was higher than the critical aggregation concentration, 5 · 105 M, as described in Section 3.2. In the concentration range, the positive current might be controlled by diffusion of the dissociated PC and the aggregated PC. When a concentration
Fig. 5. The voltammograms measured at the micro interface between W containing 0.1 M NaCl and DCE containing (1) 0, (2) 104, (3) 2 · 104, (4) 5 · 104 or (5) 103 M PC + 0.01 M BTPPA+TFPB. Scan rate was 1 mV s1.
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of Na+ in W increased by one order, the positive wave shifted by about 0.060 V to a more negative potential. This result indicates that the positive current is due to the facilitated transfer of Na+ by the complex formation with PC. The potential range for the positive current in the voltammogram agreed with Range B in the potential dependence of c. The PC desorption in Range B is caused by the transfer of the complex of PC with Na+. The association constant of an ion with PC should be estimated by voltammetric analysis of the positive current due to the facilitated transfer of Na+ according to previous studies for the facilitated ion transfer at a liquid|liquid interface [22]. From analysis of the present voltammograms, it is difficult to evaluate the association constant. Marecˇek et al. [8] estimated the association constant by analyzing the peak current for desorption of protonated PC in the presence of a dilute PC which was recorded based on the fast scan voltammetry. We had also tried to measure the peak current for desorption of PC accompanied by the transfer of Na+ at a conventional interface according to MarecˇekÕs procedure. The peak current for the PC desorption, however, could not be observed. The rate of the PC desorption following complex formation with Na+ might be slower than that of the protonated PC. 3.4. Association constant of PC with Na
+
We attempted to estimate the association constant of PC with a hydrophilic cation, M+, from the potential dependence of c. The mechanism of the desorption of PC preceded by complex formation of PC with M+ can be expressed as Eq. (6) [3,8]. þ PCðOÞ ¡PCðadsÞ þ Mþ ¡MPCþ ðadsÞ ¡MPCðOÞ
ð6Þ
Here, the subscripts (O) and (ads) denote the species in the bulk organic phase and the adsorbed species at the interface, respectively. Samec et al. proposed the theory for the potential dependence of c by assuming the mechanism of Eq. (6) and using two Frumkin isotherms for adsorption of PC and MPC+ [3]. The theory suggests that the onset of the increase in interfacial tension, D/desorb, is determined mainly by the half-wave potential, D/1/2, of the facilitated transfer of M+ by PC.
1=2
1=2
D/0M ðRT=zFÞ lnðK ass c0MX DMPC DPC Þ; 0
Table 1 The association constants, Kass, of PC with the cations in DCE evaluated from the onset of the increase in the potential dependence of c, D/desorb Ions +
Li Na+ K+ Cs+ NHþ 4 ðCH3 ÞNHþ 3 ðCH3 Þ2 NHþ 2 (CH3)3NH+ Arg+
D/desorba (V) 0.13 0.13 0.13 0.10 0.08 0.03 0.05 0.10 0.15
D/0i (V) c
0.58 0.58c 0.54c 0.39c 0.45d 0.36d 0.27d 0.17d 0.64d
log Kassb 8.6 8.6 7.9 5.9 7.3 6.6 6.4 5.6 9.3
In the presence of 105 M PC in DCE. Association constant in DCE defined in Section 3.4. c Ref. [25]. d The value of D/0 was calculated based on the potential difference between the final rise for the transfer of a cation and that for the transfer of Cs+ in the voltammogram recorded in the absence of PC. a
b
Na+ with PC is shown in Table 1. In the calculation, D of MPC+ is assumed to be equal to that of PC. 3.5. The complex formation of alkali metal ions with a PC monolayer adsorbed at the W|DCE interface Fig. 6(a) is the potential dependence of c in the presence of an alkali metal cation such as Li+, Na+, K+ or Cs+ in W. The PC desorption in Range B was also observed in every case of the alkali metal cations. Fig. 6(b) shows the voltammograms for the transfer of the alkali metal cation in the presence of 5 · 104 M PC in DCE. In the case of Li+, Na+or K+, a positive current for the facilitated ion transfer appeared in Range B in the voltammogram. For Cs+, a positive current for the facilitated transfer of Cs+ was not observed clearly, but a large current appeared near the final rise for the transfer of Cs+ when the potential sweep was fast, e.g., 20 mV s1. Desorption of PC causes convection near the interface and accelerates diffusion of PC to the interface. The large current might be due to a Cs+ transfer facilitated by complex formation with PC. These results indicate that not only Na+ but also Li+, K+ and Cs+ form a complex with PC. The Kass values calculated from D/desorb are summarized in Table 1. 3.6. The complex formation of anions with a PC monolayer adsorbed at the W|DCE interface
D/desorb D/1=2 0
21
ð7Þ
where D/0M is the transfer potential for M+ ion, Kass = cMPC(O)/cPC(O)cM(O) the association constant in O, D the diffusion coefficient, and c0MX the bulk concentration of hydrophilic salts, MX, in W. The lipid/ion stoichiometry of the complex is assumed to be 1:1. Assuming the theory reported by Samec et al., the Kass was calculated from D/desorb in Fig. 3. The Kass of
The effects of anions on the PC monolayer were investigated based on the potential dependence of c and voltammograms in the presence of Na+X ðX ¼ SO2 in 4 ; CH3 COO ; Cl ; Br ; NO3 ; I ; ClO4 Þ W. The potential dependence of c in Fig. 7(a) shows that adsorption of the PC layer was not influenced by the transfer of any anions. Fig. 7(b) indicates that negative
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Fig. 6. The potential dependences of the interfacial tension (a) in the presence of 105 M PC + 0.01 M BTPPA+TFPB in DCE and 0.1 M MCl in W and the voltammograms (b) measured at the micro interface between W containing 0.1 M MCl and DCE containing 5 · 104 M PC + 0.01 M BTPPA+TFPB. (- - - -) in (b) indicate the voltammograms in the absence of PC in DCE. Scan rate was 1 mV s1: M+ = Li+ (d, 1), Na+ (s, 2), K+ (., 3) and Cs+ ($, 4).
Fig. 7. The potential dependences of the interfacial tension (a) in the presence of 105 M PC + 0.01 M BTPPA+TFPB in DCE and 0.1 M NaX in W and the voltammograms (b) measured at the micro interface between W containing 0.1 M NaX and DCE containing 5 · 104 M PC + 0.01 M BTPPA+TFPB. (- - - -) in (b) indicate the voltammograms in the absence of PC in DCE. Scan rate was 1 mV s1: X ¼ SO2 4 (d,1), CH3COO (s, 2), Cl (., 3), Br ($, 4), NO3 (j, 5), I (h, 6) and ClO4 (r, 7).
currents for the facilitated transfers of the anions were not observed. These results suggest that no anions form a complex with PC, irrespective of their hydrophobicity. The negative current with respect to the transfer of an anion often shifted to a negative potential in the presence of a PC monolayer. The PC monolayer seems to interfere with the transfer of anions. The reason cannot be elucidated yet, because quantitative results have not been obtained. A potential shift was also caused by adding dilute PC to the conventional interface. The interference of the PC monolayer was observed only for the ion transfer with no complex formation of PC. For example, (CH3)4N+ did not form a complex with PC as described in the following Section 3.7. The transfer of (CH3)4N+ also shifted to a more positive potential in the presence of PC as Fig. 8(b). Grandell et al. have reported that the transfer of picrate shifted to a more negative potential in the presence of PC [23]. 3.7. The complex formation of the ammonium ions with a PC monolayer adsorbed at the W|DCE interface Fig. 8 shows the potential dependences of c and the voltammograms at the micro interface in the presence þ þ þ of NHþ or 4 ; CH3 NH3 ; ðCH3 Þ2 NH2 ; ðCH3 Þ3 NH
Fig. 8. The potential dependences of the interfacial tension (a) in the presence of 105 M PC + 0.01 M BTPPA+TFPB in DCE and 0.1 M MCl in W and the voltammograms (b) measured at the micro interface between W containing 0.1 M MCl and DCE containing 5 · 104 M PC + 0.01 M BTPPA+TFPB. Scan rate was 1 mV s1. (- - - -) in (b) indicate the voltammograms in the absence of PC in DCE.: + þ þ Mþ ¼ NHþ 4 (d, 1), CH3 NH3 (s, 2), ðCH3 Þ2 NH2 (., 3), (CH3)3NH ($, 4) and (CH3)4N+ (j, 5).
Y. Yoshida et al. / Journal of Electroanalytical Chemistry 578 (2005) 17–24
(CH3)4N+ in W. The pH of W was in the range of 6–7 and was not controlled by any buffer. In a neutral pH solution, the ammonium ions are monovalent. The desorption of PC in Range B was observed in the case þ þ þ of NHþ but 4 ; CH3 NH3 ; ðCH3 Þ2 NH2 or ðCH3 Þ3 NH + not in the case of (CH3)4N . A positive current for the facilitated transfer depending on the concentration of PC was obtained in the case of every ammonium ion except for (CH3)4N+. Two mechanisms are expected for the effects of PC such as desorption of PC in Range B and the positive current depending on the concentration of PC; (1) the transfer of the PC protonated with H+ of the ammonium ions, or (2) the transfer of the ammonium ion complexed with PC. The values of pKa of the phosphodiester of PC and the ammonium ion in W are 1.7 [2] and more than 9 [24], respectively, indicating that protonation of PC in mechanism (1) is impossible in neutral W. Assuming mechanism (1), the positive current should occur at a more positive potential with the increasing number of methyl groups in the ammonium molecule since pKa in W of an ammonium ion increases with an increase in the number of methyl groups [24]; e.g., pKa = 9.5 for þ NHþ 4 and 10.7 for ðCH3 ÞNH3 . But the experimental results show an opposite tendency. Therefore, desorption of PC in Range B and the positive current occurred according to mechanism (2). The association constants calculated from D/desorb are summarized in Table 1. 3.8. Complex formation of arginine cation with a PC monolayer adsorbed at the W|DCE interface Arginine, Arg, in neutral W is a monovalent cation having one negative charge and two positive charges because the pKa values of Arg are 2.7 (–COOH), 9.04 þ ðNHþ 3 Þ, 12.48 ðANHCð@NH2 ÞNH2 Þ. Fig. 9 shows the potential dependence of c and the voltammogram in the presence of Arg+Cl in W. The PC desorption was observed at a potential more positive than 0.2 V. A positive current depending on the concentration of PC appeared in the voltammogram. Therefore, PC forms a complex with Arg+. The association constant of the complex with Arg+ is indicated in Table 1. 3.9. Selectivity of the complex formation of a PC monolayer with ions Table 1 shows the Kass of PC with cations estimated from D/desorb based on Eq. (7). It has been reported in previous studies [2] that the binding of a BLM with a metal cation is stronger with an increase of the surface charge density of the metal cation. In the present work, the complex formation of PC with an alkali metal ion indicates a tendency similar to the reported results. We consider that the binding of BLM with a metal cation
23
Fig. 9. The potential dependence of the interfacial tension (a) in the presence of 105 M PC + 0.01 M BTPPA+TFPB in DCE and 0.1 M ArgCl in W and the voltammograms (b) measured at the micro interface between W containing 0.1 M ArgCl and DCE containing (1) 104, (2) 2 · 104, (3) 5 · 104 or (4) 103 M PC + 0.01 M BTPPA+TFPB. ( ) in (a) and (- - - -) in (b) indicate the interfacial tension and the voltammograms, respectively, in the absence of PC in DCE. Scan rate was 1 mV s1.
is determined mainly by the complex formation of the polar head group of PC with the metal cation. In the studies using the BLM, the binding of an anion to the BLM has been reported. The more hydrophobic the anion, the stronger is the binding to the BLM [2]. However, we could not observe the complex formation of PC with any anion, including hydrophobic anions such as ClO 4 . The binding of BLM with an anion might be determined not by a complex formation with the polar head group of PC but by other interactions such as a hydrophobic interaction caused by breaking of the water structure. The binding strength of a BLM with NHþ 4 has been recognized to be weak compared with metal cations in previous studies [2]. Present results, on the contrary, reveal the complex formation of PC not only with NHþ 4 þ þ but also with CH3 NHþ 3 ; ðCH3 Þ2 NH2 or ðCH3 Þ3 NH . The Kass of the PC complex with the ammonium ion decreased with the increase in the number of methyl groups in the ammonium ion, indicating that the complex formation is due to hydrogen bonding between H of the ammonium ion and the phosphodiester in the polar head group of PC. The Kass of the PC complex with Arg+ is larger than that of other ammonium ions. It is considered that the large Kass of Arg+ is caused by the complex formation of PC with each of two amino groups in Arg+. The result of Arg+ suggests that the
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more amino groups in a molecule, the stronger is the complex formation with PC.
4. Conclusion The selectivity of complex formation of PC with an ion was evaluated from the desorption behavior of PC layer at the W|DCE interface based on the theory reported by Samec et al. for the potential dependence of the interfacial tension in the presence of PC and confirmed by measuring the transfer of an ion at the W|DCE interface facilitated by the complex formation with PC. The ions investigated, such as alkali metal ions, NHþ 4 and alkylammonium ions had been shown to have little ability of complex formation with PC in previous work [2]. Hence, the effects of these ions on the orientation of a polypeptide on the biomembrane surface were often discussed based on other interactions such as a long-range electrostatic force [1,2]. In the present work, a neutral phospholipid such as phosphatidylcholine, however, shows remarkable complex formation with these ions and appreciable selectivity for the complex formation. Especially, the complex formation of lipids with the amino group should be noted to understand the strong binding of the basic polypeptide to the BLM. For example, melittin5+ is a basic polypeptide with three Lys and two Arg in 26 residues and disrupts the bilayer structure of the biomembrane by strong binding [26]. The binding of melittin5+ with the biomembrane has been explained mainly by the long-range electrostatic force between the positive charges on melittin5+ and the negative charge on the biomembrane [2]. However, we consider that the complex formation of amino groups in melittine5+ with PC is also important because the strong binding to the BLM has been observed using neutral lipids [26].
Acknowledgements This study was partly supported by a Grants-in-Aid for Scientific Research (Nos. 16350043 and 16550072) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References [1] R. Guidelli, G. Aloisi, L. Becucci, A. Dolfi, M.R. Moncelli, F.T. Buoninsegni, J. Electroanal. Chem. 504 (2001) 1, and references cited therein. [2] S.A. Tatulian, in: G. Cevc (Ed.), Phospholipids Handbook, Marcel Dekker, Inc., New York, 1993 (Chapter 14) and references cited therein. [3] Z. Samec, A. Troja´nek, H.H. Girault, Electrochem. Comm. 5 (2003) 98. [4] S.G. Chesniuk, S.A. Dassie, L.M. Yudi, A.M. Baruzzi, Electrochim. Acta 43 (1998) 2175. [5] Y. Yoshida, H. Yoshinaga, N. Ichieda, A. Uehara, M. Kasuno, K. Banu, K. Maeda, S. Kihara, Anal. Sci. 17 (suppl.) (2001) i1037. [6] H.H. Girault, D.J. Shiffrin, J. Electroanal. Chem. 179 (1984) 277. [7] T. Kakiuchi, M. Nakanishi, M. Senda, Bull. Chem. Soc. Jpn. 61 (1988) 1845. [8] V. Marecˇek, A. Lhotsky´, H. Ja¨nchenova´, J. Phys. Chem. B 107 (2003) 4573. [9] T. Kakiuchi, T. Kondo, M. Senda, Bull. Chem. Soc. Jpn. 63 (1990) 3270. [10] D. Grandell, L. Murtoma¨ki, K. Kontturi, G. Sundholm, J. Electroanal. Chem. 463 (1999) 242. [11] H. Nishida, N. Takada, M. Yoshimura, T. Sonoda, H. Kobayashi, Bull. Chem. Soc. Jpn. 57 (1984) 2600. [12] S.R. Bahr, P. Boudjouk, J. Org. Chem. 57 (1992) 5545. [13] S. Kihara, M. Suzuki, K. Maeda, K. Ogura, S. Umetani, M. Matsui, Z. Yoshida, Anal. Chem. 58 (1986) 2954. [14] K.M. Kadish, J.E. Anderson, Pure Appl. Chem. 59 (1987) 703. [15] L. Wilhelmy, Ann. Physik. 119 (1863) 177. [16] Y. Yoshida, Z. Yoshida, H. Aoyagi, Y. Kitatsuji, A. Uehara, S. Kihara, Anal. Chim. Acta 452 (2002) 149. [17] J. Parker, Chem. Rev. 69 (1969) 1. [18] G. Taylor, H.H.J. Girault, J. Electroanal. Chem. 208 (1986) 179. [19] H. Ohde, A. Uehara, Y. Yoshida, K. Maeda, S. Kihara, J. Electroanal. Chem. 496 (2001) 110. [20] H.H. Girault, D.J. Schiffrin, in: A.J. Bard (Ed.), Electroanalytical Chemistry, vol. 15, Marcel Dekker Inc., New York, 1989, p. 1, and references cited therein. [21] J. Li, R. Miller, H. Mo¨hwald, Coll. Surf. A, Physicochem. Eng. Aspects 144 (1996) 113. [22] H.J. Lee, C. Beriet, H.H. Girault, J. Electroanal. Chem. 453 (1998) 211. [23] D. Grandell, L. Murtoma¨ki, G. Sundholm, J. Electroanal. Chem. 469 (1999) 72. [24] D.D. Perrin, IUPAC Chemical Data Series No. 22 ‘‘Organic Ligands’’, Pergamon Press, New York, 1979. [25] Y. Shao, A.A. Stewart, H.H.J. Girault, J. Chem. Soc. Faraday Trans. 87 (1991) 2593. [26] A.M. Batenburg, J.H. van Esch, B. de Kruijff, Biochemistry 27 (1988) 2324, and references cited therein.
Electroanalytical Chemistry Journal of Electroanalytical Chemistry 578 (2005) 17–24 www.elsevier.com/locate/jelechem
The complex formation of ions with a phospholipid monolayer adsorbed at an aqueous|1,2-dichloroethane interface Yumi Yoshida
a,*
, Kohji Maeda a, Osamu Shirai
b
a
b
Department of Chemistry, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan Division of Nuclear Engineering Science, Research Reactor Institute, Kyoto University, Kumatori, Osaka 590-0494, Japan Received 21 September 2004; received in revised form 7 December 2004; accepted 7 December 2004 Available online 8 February 2005
Abstract The complex formation of phosphatidylcholine adsorbed at a water|1,2-dichloroethane, W|DCE, interface with ions was evaluated based on the desorption behavior of lipids at the interface accompanied by an ion transfer and was confirmed by measuring voltammograms for the ion transfer facilitated by the complex formation with lipids. The desorption behavior of lipids was measured according to the Wilhelmy vertical plate method, the cell of which was improved to achieve polarization of the W|DCE interface. Voltammograms for the ion transfer at the W|DCE interface facilitated by the complex formation with lipids were recorded at a micro W|DCE interface in order to avoid the influence of convection caused by the desorption of lipids at the interface. Lipids þ adsorbed at the W|DCE interface showed complex formation with alkali metal ions, NHþ 4 ; ðCH3 ÞNH3 ; ðCH3 Þ2 2 þ þ þ NH2 or ðCH3 Þ3 NH , but not with ðCH3 Þ4 N or anions such as SO4 , CH3COO , Cl , Br , NO3 , I or ClO4 . Lipids also showed strong complex formation with a basic amino acid such as an arginine cation, which has two amino groups in the molecule. The þ þ þ þ strength of the complex formation with lipids was Argþ > Liþ ; Naþ > Kþ > NHþ 4 > CH3 NH3 > ðCH3 Þ2 NH2 > Cs > ðCH3 Þ3 NH . 2005 Elsevier B.V. All rights reserved. Keywords: Phospholipid; Liquid|liquid interface; Association constants; Amino acid; Voltammetry; Interfacial tension
1. Introduction The evaluation of binding of an ion or an ionic functional group to the surface of a phospholipid membrane is very important to understand not only the ion transport through a biomembrane but also the orientation of a membrane protein on the lipid membrane [1]. The binding of an ion to a bilayer lipid membrane, BLM, has been investigated by NMR measurement, IR spectroscopy, electrophoresis, the ion-selective electrode method, the monolayer-radiotracer technique, neutron diffraction, etc. [2]. Results obtained by these techniques indicate that the binding strength of an ion
*
Corresponding author. Tel./fax: +81 75 724 7518. E-mail address: [email protected] (Y. Yoshida).
0022-0728/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2004.12.016
with the BLM was large for a large surface charge density of the cation, e.g., lanthanide > transition metals > alkali earth metals > alkali metals organic cation, and for a hydrophobic anion, e.g., tetra phenylborate > ClO 4 > I > SCN > NO3 > Br > 2 Cl > SO4 . The binding strength of an ion with a BLM has been discussed mainly based on the following interactions [2]; (1) the long-range electrostatic force, which is governed by charges located on an ion and phospholipids and the local dielectric constant near the water|membrane interface, (2) the complex formation of an ion with the polar head group of a phospholipid, and (3) the hydrophobic interaction caused by breaking of the water structure. In previous studies, the location of an ion or the orientation of a protein on the BLM surface has been mainly discussed based on interaction (1) in the absence of an inorganic salt irrespective of
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the objective ion. However, the inorganic salt at high concentration such as 0.1 M NaCl is present under biological conditions. Under biological conditions, the contribution of interaction (2) to the binding to the BLM is more important than interaction (1) in order to understand the location of an ion or the orientation of a protein on the BLM surface because interaction (1) is masked by a large amount of the inorganic salt. The purpose of the present work is to evaluate interaction (2) and to elucidate the selectivity of the complex formation of lipids with ions. According to the previous method using the BLM, interaction (2) is difficult to evaluate in distinguishing it from other interactions. Using a phospholipid monolayer adsorbed at the liquid|liquid interface, only interaction (2) can be estimated. When the phospholipid monolayer forms a complex with an ion, the phospholipid monolayer desorbs with the transfer of the ionic complex realized by applying an interfacial potential [3–7]. In addition, the complex formation can be confirmed by voltammetry for the ion transfer at the liquid|liquid interface facilitated by complex formation with the phospholipid [4,5,8]. Desorption of the phospholipid monolayer and the facilitation of ionic transfer at the liquid|liquid interface can be measured even in the presence of inorganic salts and is not influenced by interaction (3). In measurements using the phospholipid monolayer at a liquid|liquid interface, the strong complex formation of lipids with alkali earth metal ions [4,9,10], H+ [6,8] or Li+ [3,4,7] has been reported. In the case of other alkali metal ions or ammonium ions, complex formation has not been demonstrated in previous studies though we pointed out the possibility of complex formation with other alkali metal ions and ammonium ion [5]. In order to estimate the weak complex formation with these ions, we improved the measurement of the interfacial tension based on the Wilhelmy vertical plate method and confirmed the complex formation by voltammetry for the ion transfer at a micro water|1,2-dichloroethane, W|DCE, interface, where convection caused by desorption of lipids is small.
dence of the solubility of the salt in ethanol. Na+TFPB was synthesized according to the procedure described previously [11,12]. Tetraphenylarsonium sulfate, ðTPhAsþ Þ2 SO2 4 , was prepared by the titration of TPhAs+Cl (Aldrich) with Ag2SO4 in an aqueous solution [13]. The salt of tetraphenylborate with TPhAs+, TPhAs+TPhB, was prepared according to the previous studies [13]. The DCE used was purified according to the method described in the literature [14] and was saturated with distilled water. All other chemicals used were of reagent grade quality. 2.2. Measurements of the interfacial tension at a polarized W|DCE interface The interfacial tension, c, at a polarized W|DCE interface was measured by the Wilhelmy vertical plate technique [15] applying the potential difference, E, at the W|DCE interface. Fig. 1 shows the cell used for the measurement of c. The W|DCE interface was formed in a glass beaker (35 mm diameter). The objective cation or anion was added to W as a chloride or sodium salt. The pH of W was not controlled by any buffer and was confirmed to be almost 6–7. The DCE contained PC and 0.01 M BTPPA+TFPB as a supporting electrolyte. A glass plate (23.85 ± 0.05 mm width, 0.3 ± 0.01 mm thickness, Kyowa Interface Science Co., Ltd.) silanized with dimethyldichlorosilane was placed on the W|DCE interface, and connected to an electrobalance. The interfacial potential difference at the W|DCE interface was applied as the potential of W referred to that of DCE using two reference electrodes of an Ag|AgCl electrode in W and a tetraphenylarsonium ion-selective electrode, TPhAsE, in DCE and two counter electrodes of a Pt wire in W and a Pt net in DCE. The configuration of TPhAsE is given by Eq. (1).
2. Experimental 2.1. Chemicals The phospholipid employed was L-a-phosphatidylcholine dioleoyl, PC, of analytical grade (SIGMA, Lot No. 32k5202). Bis(triphenylphosphoranylidene)ammonium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, BTPPA+TFPB, was obtained as a precipitate by mixing a methanol solution of BTPPA+Cl with a methanol solution of Na+TFPB, and the precipitate was purified by recrystallization based on the temperature depen-
Fig. 1. Cell for the measurement of interfacial tension at a polarized W|DCE interface: (1) aqueous phase, (2) DCE phase, (3) a Pt wire used as a counter electrode in W, (4) a Ag|AgCl electrode used as a reference electrode in W, (5) TPhAsE used as a reference electrode in DCE, (6) a Pt net used as a counter electrode in DCE and (7) a silanized glass plate.
Y. Yoshida et al. / Journal of Electroanalytical Chemistry 578 (2005) 17–24
Ag AgCl 0.1 M LiCl (W)
5 × 10–4 M
19
10–3 M
(TPhAs+)2SO42– TPhAs+TPhB– (W)
(DCE) (M = mol dm-3)
ð1Þ Here, E was related to the Galvani potential difference at the W|DCE interface, D/, as, E ¼ D/ þ Eref ;
ð2Þ
where Eref is the potential of the reference electrodes employed. According to the same procedure described in a previous study [16], the Eref of TPhAsE was determined to be 0.350 V referred to the standard potential, where the Gibbs energy for the transfer of an ion from W to DCE, DGtr, is recognized to be zero based on the extrathermodynamic assumption of Parker [17]. 2.3. Voltammetry for ion transfer at a micro W|DCE interface The voltammogram for ion transfer at the interface between W and DCE was measured by scanning E applied at a micro W|DCE interface and measuring the current due to the ion transfer [18,19]. The cell consisted of W and DCE separated by a polyester film, 16 lm thick with a micro hole 50 lm in diameter [19]. The W|DCE interface was formed at the micro hole. In the measurements, the objective cation or anion was added to W as a chloride salt or sodium salt, respectively. The supporting electrolyte added to DCE was BTPPA+TFPB. All electrochemical measurements were carried out at 25 ± 0.5 C. 2.4. Apparatus The potentiostat/galvanostat for voltammetry at the micro W|DCE interface and that for c measurement were Model HECS-318 (Huso Electro Chemical System) and Model HA 1010MIS (Hokuto Denko Co.), respectively. The function generator and X–Y recorder used were Model HB-111 (Hokuto Denko Co.) and Type 3086 (Yokogawa Electric Works, Ltd.), respectively. The electrobalance and a electrobalance lift were HD-60 (A&D Company, Ltd.) and SYLB-7K (Sanyu Tech., Ltd.).
3. Results and discussion 3.1. Polarization at a W|DCE interface in the cell for the c measurement The polarization of the rather large W|DCE interface formed in the cell of Fig. 1 was confirmed by measuring
Fig. 2. Voltammograms in the presence (a) and in the absence (b) of 2 · 104 M (CH3)4N+Cl in W and the potential dependence of the interfacial tension in the absence of (CH3)4N+Cl measured at the W|DCE interface formed in the cell of Fig. 1. W and DCE contained 0.1 M NaCl and 0.01 M BTPPA+TFPB as supporting electrolytes, respectively. scan rate: 50 mV s1.
the transfer of (CH3)4N+. Curve (a) in Fig. 2 is the voltammogram for the transfer of (CH3)4N+ at the W|DCE interface formed in the cell. The middle potential between the positive and negative peaks agreed with the reported value [20]. The peak separation was 0.075 V. It was recognized that the influence of the IR drop on the polarization of the W|DCE interface in the cell was not serious. The electrocapillary curve in Fig. 2 was measured by using the cell of Fig. 1. The c values were reproducible by about ±0.3 mN m1 on repeated runs. The electrocapillary curve was expressed as a quadratic function, and the differential capacitance was 0.054 F m2. The c value of the potential of zero charge, pzc, was 28.1 mN m1, which agreed with the reported value at the W|DCE interface estimated by the quasi-elastic laser light scattering method [3]. 3.2. Adsorption of the PC monolayer at the W|DCE interface Fig. 3 shows the c value at the W|DCE interface measured as a function of D/ in the presence of PC in DCE. The D/ was held at 0.2 V until the decrease in c stopped before starting the potential sweep. The adsorption equilibrium of PC was achieved at 0.2 V, where strong adsorption of PC was observed in our preliminary results for c. The potential dependence of c was measured on changing D/ from a negative to a positive potential because the reproducibility of c became poor at more positive potentials than about 0.1 V as was pointed out by Kakiuchi et al. [7]. The difference between c in the presence and in the absence of PC was significant when D/ was more negative than around 0.1 V, indicating that the amount of adsorbed PC in this D/ range (Range A) was large, but the difference was less significant when D/ was more
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30
γ / mN m-1
25 20 15 10 5 0 -0.4
-0.2
0.0 0.2 ∆f / V
0.4
0.6 Fig. 4. The interfacial tension at 0 V in Fig. 3 as a function of logarithmic concentration of PC in DCE.
Fig. 3. The potential dependences of the interfacial tension in the presence of x M PC + 0.01 M BTPPA+TFPB in DCE and 0.1 M NaCl in W: x = 0 (- - - -), 2 · 106 (d), 5 · 106 (s), 105 (.), 2 · 105 (,), 5 · 105 (j) or 104 (h).
positive than around 0.1 V, indicating that the amount of adsorbed PC in this D/ range (Range B) was small in accordance with the literature [3–7,9,10]. The c value at the polarized W|DCE interface was expressed by the Gibbs adsorption equation taking into account adsorption of supporting electrolytes in both phases and PC species in DCE. X dc ¼ Ciz d~ liz þ CPC dlPC ; ð3Þ iz ¼ Naþ ; Cl ; BTPPAþ ; TFPB : ~a are the surface excess concentraHere, Ca, la and l tion relative to both solvents, the chemical potential and the electrochemical potential for the a species in the bulk phase. When the composition of the supporting electrolytes in W and DCE is not changed, Eq. (3) can be written as dc ¼ Q dD/ þ CPC dlPC ;
ð4Þ
where Q is the surface charge density. When D/ is constant, Eq. (5) can be derived from Eq. (4). Here, aPC is the activity of PC in DCE. 1 oc CPC ¼ : ð5Þ RT o ln aPC D/;li Fig. 4 is a plot of c at 0 V, which corresponds to the potential zero charge, as a function of the bulk concentration of PC in DCE. When the concentration of PC was in the range from 2 · 106 to 2 · 105 M, the slope was constant, indicating that the adsorption of PC at the W|DCE interface was saturated. The CPC was calculated to be 2.31 lmol m2 from the slope based on Eq. (5). The value of CPC agreed with the reported values of the phospholipid monolayer, 2.71 lmol m2 [21] and 2.5 lmol m2 [3]. When the concentration of PC was more than 5 · 105 M, the slope decreased, indicating that this concentration of PC was more than the critical aggregation concentration.
3.3. Complex formation of Na+ with a PC monolayer adsorbed at the W|DCE interface Previous literature references have suggested that the desorption of PC in Range B is caused by the transfer of the complex following complex formation of a hydrophilic cation with PC [3,5,8]. In order to confirm the complex formation of Na+ with PC, voltammograms for the transfer of Na+ were measured at the micro W|DCE interface. The reason for the measurement at the micro interface is to decrease the effect of convection of the interface due to desorption of PC. Fig. 5 shows voltammograms for the transfer of Na+ in the presence of PC at high concentration in DCE. The positive current appeared at a positive potential greater than 0.2 V. The positive current increased with the increase in the concentration of PC. The positive current, however, was not proportional to the concentration of PC since the PC concentration was higher than the critical aggregation concentration, 5 · 105 M, as described in Section 3.2. In the concentration range, the positive current might be controlled by diffusion of the dissociated PC and the aggregated PC. When a concentration
Fig. 5. The voltammograms measured at the micro interface between W containing 0.1 M NaCl and DCE containing (1) 0, (2) 104, (3) 2 · 104, (4) 5 · 104 or (5) 103 M PC + 0.01 M BTPPA+TFPB. Scan rate was 1 mV s1.
Y. Yoshida et al. / Journal of Electroanalytical Chemistry 578 (2005) 17–24
of Na+ in W increased by one order, the positive wave shifted by about 0.060 V to a more negative potential. This result indicates that the positive current is due to the facilitated transfer of Na+ by the complex formation with PC. The potential range for the positive current in the voltammogram agreed with Range B in the potential dependence of c. The PC desorption in Range B is caused by the transfer of the complex of PC with Na+. The association constant of an ion with PC should be estimated by voltammetric analysis of the positive current due to the facilitated transfer of Na+ according to previous studies for the facilitated ion transfer at a liquid|liquid interface [22]. From analysis of the present voltammograms, it is difficult to evaluate the association constant. Marecˇek et al. [8] estimated the association constant by analyzing the peak current for desorption of protonated PC in the presence of a dilute PC which was recorded based on the fast scan voltammetry. We had also tried to measure the peak current for desorption of PC accompanied by the transfer of Na+ at a conventional interface according to MarecˇekÕs procedure. The peak current for the PC desorption, however, could not be observed. The rate of the PC desorption following complex formation with Na+ might be slower than that of the protonated PC. 3.4. Association constant of PC with Na
+
We attempted to estimate the association constant of PC with a hydrophilic cation, M+, from the potential dependence of c. The mechanism of the desorption of PC preceded by complex formation of PC with M+ can be expressed as Eq. (6) [3,8]. þ PCðOÞ ¡PCðadsÞ þ Mþ ¡MPCþ ðadsÞ ¡MPCðOÞ
ð6Þ
Here, the subscripts (O) and (ads) denote the species in the bulk organic phase and the adsorbed species at the interface, respectively. Samec et al. proposed the theory for the potential dependence of c by assuming the mechanism of Eq. (6) and using two Frumkin isotherms for adsorption of PC and MPC+ [3]. The theory suggests that the onset of the increase in interfacial tension, D/desorb, is determined mainly by the half-wave potential, D/1/2, of the facilitated transfer of M+ by PC.
1=2
1=2
D/0M ðRT=zFÞ lnðK ass c0MX DMPC DPC Þ; 0
Table 1 The association constants, Kass, of PC with the cations in DCE evaluated from the onset of the increase in the potential dependence of c, D/desorb Ions +
Li Na+ K+ Cs+ NHþ 4 ðCH3 ÞNHþ 3 ðCH3 Þ2 NHþ 2 (CH3)3NH+ Arg+
D/desorba (V) 0.13 0.13 0.13 0.10 0.08 0.03 0.05 0.10 0.15
D/0i (V) c
0.58 0.58c 0.54c 0.39c 0.45d 0.36d 0.27d 0.17d 0.64d
log Kassb 8.6 8.6 7.9 5.9 7.3 6.6 6.4 5.6 9.3
In the presence of 105 M PC in DCE. Association constant in DCE defined in Section 3.4. c Ref. [25]. d The value of D/0 was calculated based on the potential difference between the final rise for the transfer of a cation and that for the transfer of Cs+ in the voltammogram recorded in the absence of PC. a
b
Na+ with PC is shown in Table 1. In the calculation, D of MPC+ is assumed to be equal to that of PC. 3.5. The complex formation of alkali metal ions with a PC monolayer adsorbed at the W|DCE interface Fig. 6(a) is the potential dependence of c in the presence of an alkali metal cation such as Li+, Na+, K+ or Cs+ in W. The PC desorption in Range B was also observed in every case of the alkali metal cations. Fig. 6(b) shows the voltammograms for the transfer of the alkali metal cation in the presence of 5 · 104 M PC in DCE. In the case of Li+, Na+or K+, a positive current for the facilitated ion transfer appeared in Range B in the voltammogram. For Cs+, a positive current for the facilitated transfer of Cs+ was not observed clearly, but a large current appeared near the final rise for the transfer of Cs+ when the potential sweep was fast, e.g., 20 mV s1. Desorption of PC causes convection near the interface and accelerates diffusion of PC to the interface. The large current might be due to a Cs+ transfer facilitated by complex formation with PC. These results indicate that not only Na+ but also Li+, K+ and Cs+ form a complex with PC. The Kass values calculated from D/desorb are summarized in Table 1. 3.6. The complex formation of anions with a PC monolayer adsorbed at the W|DCE interface
D/desorb D/1=2 0
21
ð7Þ
where D/0M is the transfer potential for M+ ion, Kass = cMPC(O)/cPC(O)cM(O) the association constant in O, D the diffusion coefficient, and c0MX the bulk concentration of hydrophilic salts, MX, in W. The lipid/ion stoichiometry of the complex is assumed to be 1:1. Assuming the theory reported by Samec et al., the Kass was calculated from D/desorb in Fig. 3. The Kass of
The effects of anions on the PC monolayer were investigated based on the potential dependence of c and voltammograms in the presence of Na+X ðX ¼ SO2 in 4 ; CH3 COO ; Cl ; Br ; NO3 ; I ; ClO4 Þ W. The potential dependence of c in Fig. 7(a) shows that adsorption of the PC layer was not influenced by the transfer of any anions. Fig. 7(b) indicates that negative
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Y. Yoshida et al. / Journal of Electroanalytical Chemistry 578 (2005) 17–24
Fig. 6. The potential dependences of the interfacial tension (a) in the presence of 105 M PC + 0.01 M BTPPA+TFPB in DCE and 0.1 M MCl in W and the voltammograms (b) measured at the micro interface between W containing 0.1 M MCl and DCE containing 5 · 104 M PC + 0.01 M BTPPA+TFPB. (- - - -) in (b) indicate the voltammograms in the absence of PC in DCE. Scan rate was 1 mV s1: M+ = Li+ (d, 1), Na+ (s, 2), K+ (., 3) and Cs+ ($, 4).
Fig. 7. The potential dependences of the interfacial tension (a) in the presence of 105 M PC + 0.01 M BTPPA+TFPB in DCE and 0.1 M NaX in W and the voltammograms (b) measured at the micro interface between W containing 0.1 M NaX and DCE containing 5 · 104 M PC + 0.01 M BTPPA+TFPB. (- - - -) in (b) indicate the voltammograms in the absence of PC in DCE. Scan rate was 1 mV s1: X ¼ SO2 4 (d,1), CH3COO (s, 2), Cl (., 3), Br ($, 4), NO3 (j, 5), I (h, 6) and ClO4 (r, 7).
currents for the facilitated transfers of the anions were not observed. These results suggest that no anions form a complex with PC, irrespective of their hydrophobicity. The negative current with respect to the transfer of an anion often shifted to a negative potential in the presence of a PC monolayer. The PC monolayer seems to interfere with the transfer of anions. The reason cannot be elucidated yet, because quantitative results have not been obtained. A potential shift was also caused by adding dilute PC to the conventional interface. The interference of the PC monolayer was observed only for the ion transfer with no complex formation of PC. For example, (CH3)4N+ did not form a complex with PC as described in the following Section 3.7. The transfer of (CH3)4N+ also shifted to a more positive potential in the presence of PC as Fig. 8(b). Grandell et al. have reported that the transfer of picrate shifted to a more negative potential in the presence of PC [23]. 3.7. The complex formation of the ammonium ions with a PC monolayer adsorbed at the W|DCE interface Fig. 8 shows the potential dependences of c and the voltammograms at the micro interface in the presence þ þ þ of NHþ or 4 ; CH3 NH3 ; ðCH3 Þ2 NH2 ; ðCH3 Þ3 NH
Fig. 8. The potential dependences of the interfacial tension (a) in the presence of 105 M PC + 0.01 M BTPPA+TFPB in DCE and 0.1 M MCl in W and the voltammograms (b) measured at the micro interface between W containing 0.1 M MCl and DCE containing 5 · 104 M PC + 0.01 M BTPPA+TFPB. Scan rate was 1 mV s1. (- - - -) in (b) indicate the voltammograms in the absence of PC in DCE.: + þ þ Mþ ¼ NHþ 4 (d, 1), CH3 NH3 (s, 2), ðCH3 Þ2 NH2 (., 3), (CH3)3NH ($, 4) and (CH3)4N+ (j, 5).
Y. Yoshida et al. / Journal of Electroanalytical Chemistry 578 (2005) 17–24
(CH3)4N+ in W. The pH of W was in the range of 6–7 and was not controlled by any buffer. In a neutral pH solution, the ammonium ions are monovalent. The desorption of PC in Range B was observed in the case þ þ þ of NHþ but 4 ; CH3 NH3 ; ðCH3 Þ2 NH2 or ðCH3 Þ3 NH + not in the case of (CH3)4N . A positive current for the facilitated transfer depending on the concentration of PC was obtained in the case of every ammonium ion except for (CH3)4N+. Two mechanisms are expected for the effects of PC such as desorption of PC in Range B and the positive current depending on the concentration of PC; (1) the transfer of the PC protonated with H+ of the ammonium ions, or (2) the transfer of the ammonium ion complexed with PC. The values of pKa of the phosphodiester of PC and the ammonium ion in W are 1.7 [2] and more than 9 [24], respectively, indicating that protonation of PC in mechanism (1) is impossible in neutral W. Assuming mechanism (1), the positive current should occur at a more positive potential with the increasing number of methyl groups in the ammonium molecule since pKa in W of an ammonium ion increases with an increase in the number of methyl groups [24]; e.g., pKa = 9.5 for þ NHþ 4 and 10.7 for ðCH3 ÞNH3 . But the experimental results show an opposite tendency. Therefore, desorption of PC in Range B and the positive current occurred according to mechanism (2). The association constants calculated from D/desorb are summarized in Table 1. 3.8. Complex formation of arginine cation with a PC monolayer adsorbed at the W|DCE interface Arginine, Arg, in neutral W is a monovalent cation having one negative charge and two positive charges because the pKa values of Arg are 2.7 (–COOH), 9.04 þ ðNHþ 3 Þ, 12.48 ðANHCð@NH2 ÞNH2 Þ. Fig. 9 shows the potential dependence of c and the voltammogram in the presence of Arg+Cl in W. The PC desorption was observed at a potential more positive than 0.2 V. A positive current depending on the concentration of PC appeared in the voltammogram. Therefore, PC forms a complex with Arg+. The association constant of the complex with Arg+ is indicated in Table 1. 3.9. Selectivity of the complex formation of a PC monolayer with ions Table 1 shows the Kass of PC with cations estimated from D/desorb based on Eq. (7). It has been reported in previous studies [2] that the binding of a BLM with a metal cation is stronger with an increase of the surface charge density of the metal cation. In the present work, the complex formation of PC with an alkali metal ion indicates a tendency similar to the reported results. We consider that the binding of BLM with a metal cation
23
Fig. 9. The potential dependence of the interfacial tension (a) in the presence of 105 M PC + 0.01 M BTPPA+TFPB in DCE and 0.1 M ArgCl in W and the voltammograms (b) measured at the micro interface between W containing 0.1 M ArgCl and DCE containing (1) 104, (2) 2 · 104, (3) 5 · 104 or (4) 103 M PC + 0.01 M BTPPA+TFPB. ( ) in (a) and (- - - -) in (b) indicate the interfacial tension and the voltammograms, respectively, in the absence of PC in DCE. Scan rate was 1 mV s1.
is determined mainly by the complex formation of the polar head group of PC with the metal cation. In the studies using the BLM, the binding of an anion to the BLM has been reported. The more hydrophobic the anion, the stronger is the binding to the BLM [2]. However, we could not observe the complex formation of PC with any anion, including hydrophobic anions such as ClO 4 . The binding of BLM with an anion might be determined not by a complex formation with the polar head group of PC but by other interactions such as a hydrophobic interaction caused by breaking of the water structure. The binding strength of a BLM with NHþ 4 has been recognized to be weak compared with metal cations in previous studies [2]. Present results, on the contrary, reveal the complex formation of PC not only with NHþ 4 þ þ but also with CH3 NHþ 3 ; ðCH3 Þ2 NH2 or ðCH3 Þ3 NH . The Kass of the PC complex with the ammonium ion decreased with the increase in the number of methyl groups in the ammonium ion, indicating that the complex formation is due to hydrogen bonding between H of the ammonium ion and the phosphodiester in the polar head group of PC. The Kass of the PC complex with Arg+ is larger than that of other ammonium ions. It is considered that the large Kass of Arg+ is caused by the complex formation of PC with each of two amino groups in Arg+. The result of Arg+ suggests that the
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more amino groups in a molecule, the stronger is the complex formation with PC.
4. Conclusion The selectivity of complex formation of PC with an ion was evaluated from the desorption behavior of PC layer at the W|DCE interface based on the theory reported by Samec et al. for the potential dependence of the interfacial tension in the presence of PC and confirmed by measuring the transfer of an ion at the W|DCE interface facilitated by the complex formation with PC. The ions investigated, such as alkali metal ions, NHþ 4 and alkylammonium ions had been shown to have little ability of complex formation with PC in previous work [2]. Hence, the effects of these ions on the orientation of a polypeptide on the biomembrane surface were often discussed based on other interactions such as a long-range electrostatic force [1,2]. In the present work, a neutral phospholipid such as phosphatidylcholine, however, shows remarkable complex formation with these ions and appreciable selectivity for the complex formation. Especially, the complex formation of lipids with the amino group should be noted to understand the strong binding of the basic polypeptide to the BLM. For example, melittin5+ is a basic polypeptide with three Lys and two Arg in 26 residues and disrupts the bilayer structure of the biomembrane by strong binding [26]. The binding of melittin5+ with the biomembrane has been explained mainly by the long-range electrostatic force between the positive charges on melittin5+ and the negative charge on the biomembrane [2]. However, we consider that the complex formation of amino groups in melittine5+ with PC is also important because the strong binding to the BLM has been observed using neutral lipids [26].
Acknowledgements This study was partly supported by a Grants-in-Aid for Scientific Research (Nos. 16350043 and 16550072) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References [1] R. Guidelli, G. Aloisi, L. Becucci, A. Dolfi, M.R. Moncelli, F.T. Buoninsegni, J. Electroanal. Chem. 504 (2001) 1, and references cited therein. [2] S.A. Tatulian, in: G. Cevc (Ed.), Phospholipids Handbook, Marcel Dekker, Inc., New York, 1993 (Chapter 14) and references cited therein. [3] Z. Samec, A. Troja´nek, H.H. Girault, Electrochem. Comm. 5 (2003) 98. [4] S.G. Chesniuk, S.A. Dassie, L.M. Yudi, A.M. Baruzzi, Electrochim. Acta 43 (1998) 2175. [5] Y. Yoshida, H. Yoshinaga, N. Ichieda, A. Uehara, M. Kasuno, K. Banu, K. Maeda, S. Kihara, Anal. Sci. 17 (suppl.) (2001) i1037. [6] H.H. Girault, D.J. Shiffrin, J. Electroanal. Chem. 179 (1984) 277. [7] T. Kakiuchi, M. Nakanishi, M. Senda, Bull. Chem. Soc. Jpn. 61 (1988) 1845. [8] V. Marecˇek, A. Lhotsky´, H. Ja¨nchenova´, J. Phys. Chem. B 107 (2003) 4573. [9] T. Kakiuchi, T. Kondo, M. Senda, Bull. Chem. Soc. Jpn. 63 (1990) 3270. [10] D. Grandell, L. Murtoma¨ki, K. Kontturi, G. Sundholm, J. Electroanal. Chem. 463 (1999) 242. [11] H. Nishida, N. Takada, M. Yoshimura, T. Sonoda, H. Kobayashi, Bull. Chem. Soc. Jpn. 57 (1984) 2600. [12] S.R. Bahr, P. Boudjouk, J. Org. Chem. 57 (1992) 5545. [13] S. Kihara, M. Suzuki, K. Maeda, K. Ogura, S. Umetani, M. Matsui, Z. Yoshida, Anal. Chem. 58 (1986) 2954. [14] K.M. Kadish, J.E. Anderson, Pure Appl. Chem. 59 (1987) 703. [15] L. Wilhelmy, Ann. Physik. 119 (1863) 177. [16] Y. Yoshida, Z. Yoshida, H. Aoyagi, Y. Kitatsuji, A. Uehara, S. Kihara, Anal. Chim. Acta 452 (2002) 149. [17] J. Parker, Chem. Rev. 69 (1969) 1. [18] G. Taylor, H.H.J. Girault, J. Electroanal. Chem. 208 (1986) 179. [19] H. Ohde, A. Uehara, Y. Yoshida, K. Maeda, S. Kihara, J. Electroanal. Chem. 496 (2001) 110. [20] H.H. Girault, D.J. Schiffrin, in: A.J. Bard (Ed.), Electroanalytical Chemistry, vol. 15, Marcel Dekker Inc., New York, 1989, p. 1, and references cited therein. [21] J. Li, R. Miller, H. Mo¨hwald, Coll. Surf. A, Physicochem. Eng. Aspects 144 (1996) 113. [22] H.J. Lee, C. Beriet, H.H. Girault, J. Electroanal. Chem. 453 (1998) 211. [23] D. Grandell, L. Murtoma¨ki, G. Sundholm, J. Electroanal. Chem. 469 (1999) 72. [24] D.D. Perrin, IUPAC Chemical Data Series No. 22 ‘‘Organic Ligands’’, Pergamon Press, New York, 1979. [25] Y. Shao, A.A. Stewart, H.H.J. Girault, J. Chem. Soc. Faraday Trans. 87 (1991) 2593. [26] A.M. Batenburg, J.H. van Esch, B. de Kruijff, Biochemistry 27 (1988) 2324, and references cited therein.