Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 61-64, 2005

AcceptedEvaluation of the Associate Constant of a Phospholipid – Ca2+ Complex Usinga Phospholipid Monolayer Adsorbed at an Aqueous | 1,2-dichloroethane Interface
Yumi Yoshida,*, a Kohji Maeda,a and Osamu ShiraibaDepartment of Chemistry, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan
Division of Nuclear Engineering Science, Research Reactor Institute, Kyoto University, Kumatori, Osaka590-0494, Japanb

Received: November 15, 2004; In Final Form: April 21, 2005
The complex formation of Ca2+ with a phosphatidylcholine adsorbed at the interface between water and 1,2dichloroethane, W | DCE, was evaluated to elucidate the adsorption of ions on biomembrane surfaces. An electrochemical method combined with interfacial tension measurement was adopted for evaluation of the complex
formation of the phospholipid and the ion. The association constant for the complex formation of Ca2+ with thelipid was three orders of magnitude higher than those of monovalent cations with the lipid. The present result
suggests that the strong complex formation of highly hydrophilic metal cations with a lipid membrane results in ahigher affinity to biomembrane surfaces.

1. Introduction
The evaluation for binding of an ion to a phospholipid
membrane surface is very important in understanding theadsorption behavior of an ion on a biomembrane surface or abacterial surface. 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 and so on.1 Results obtained by these techniques
reflect not only the complex formation of an ion with the polar
head group of a phospholipid but also the hydrophobic interaction caused by breaking the water structure. For example, thebinding strength of anion with a BLM corresponded to thehydrophobicity of the anions;1 e.g. tetraphenylborate > ClO4− >I− > SCN− > NO3− > Br− > Cl− > SO42−.
An electrochemical method combined with interfacial
tension measurement has been proposed to evaluate thecomplex formation of a phospholipid with an ion. 2,3 In themeasurement, the associate constant of a phospholipid with anion is estimated from the interfacial potential for desorption of
the phospholipid monolayer at the aqueous | organic interface
created by the transfer of the ion associated with the phospholipid. The electrochemical method has the advantage of
avoiding the effect of the hydrophobic interaction.3 We haveestimated the association constants of a phospholipid withvarious monovalent cations.3
In the present work, we attempted to apply the method toevaluate the complex formation of a phospholipid with a morehydrophilic cation such as Ca2+.
2. Experimental
Chemicals. The phospholipid employed was L-α-phosphatidylcholine dioleoyl, PC, of analytical grade (SIGMA, LotNo. 32k5202). Bis(triphenylphosphoranylidene)ammonium
tetrakis [3,5-bis(trifluoromethyl)phenyl] borate, BTPPA+TFPB–,
was obtained as a precipitate by mixing a methanol solution ofBTPPA+Cl– with a methanol solution of Na+TFPB–, and theprecipitate was purified by recrystallization based on the*Corresponding author. E-mail: yyoshida@kit.jp. FAX: +81-75724-7518.

temperature dependence of the solubility of the salt in ethanol.
Na+TFPB– was synthesized according to the procedure described
previously.4, 5 Tetraphenylarsonium sulfate, (TPhAs+)2SO42 –,was prepared by the titration of TPhAs + Cl – (Aldrich) with
Ag2SO4 in an aqueous solution.6 The salt of tetraphenylborate
with TPhAs+, TPhAs+TPhB–, was prepared according to themethod of previous studies.6 The 1,2-dichloroethane, DCE, waspurified according to the method described in the literature7
and then saturated with distilled water.
All other chemicals used were of reagent grade quality.
Measurement of the interfacial tension at a polarized
W|DCE interface. The interfacial tension, γ, at the polarized
W|DCE interface was measured by the Wilhelmy vertical platetechnique8 applying the potential difference, E, at the W|DCEinterface.3 Figure 1 shows the cell used for the measurement ofγ. The W|DCE interface was formed in a glass beaker (35 mmdiameter). A glass plate (width l = 23.85 ± 0.05 mm, thickness
d = 0.3 ± 0.01 mm, Kyowa Interface Science Co., Ltd.) silanizedElectrobalance4537612Figure 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) an 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, (7) asilanized glass plate (From Ref. 3)© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

Yoshida

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

with dimethyldichlorosilane was placed on the W|DCE interface,and connected to an electrobalance.
The γ value was estimated from the detaching force of theglass plate from the W|DCE interface:(1)

where ∆W is the weight difference between the glass plate incontact with the interface and the same plate detached from theinterface.
Ca2+ was added to W as a chloride salt. The pH of W was notcontrolled by a buffer and was confirmed to be in the range 6–7.
0.01 M (M = mol dm−3) BTPPA+TFPB–, which is a supporting
electrolyte, was added with PC to the DCE.
The interfacial potential difference between the W and DCE,
E, was applied as the potential of W with reference to 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 netin DCE. The configuration of TPhAsE is given by eq 2.
5 × 10–4 M

10–3 M

LiCl(TPhAs+)2SO42–

TPhAs+TPhB–

(W)

(W)

(DCE)

Ag AgCl 0.1 M

30(2)

25

In the presence of PC, the E was held at 0.2 V until thedecrease in γ stopped before starting the potential sweep. Theadsorption equilibrium of PC was achieved at 0.2 V, wherestrong adsorption of PC was observed.3 The potential dependence of γ was measured by changing E from a negative to a
positive potential.
E was related to the Galvani potential difference at theW|DCE interface, ∆φ, as,E = ∆φ + Eref ,γ / mN m -1

γ = ∆W/(2l + 2d),dependence of γ showed the ideal electrocapillary curve of aquadratic function, which indicates the formation of an electrical double layer near the interface. Therefore, E was applied
exactly at the W|DCE interface. When PC was added to DCE,
the γ value decreased to approximately 7 mN m−1 at a ∆φ of−0.15 V, and remained constant at ∆φ values between −0.3 and0.05 V. In this potential range, the difference in γ between thepresence and absence of PC was significant. When the ∆φ wasshifted to more a positive potential than 0.05 V, the γ value
increased steeply, and the difference in γ between the presence
and absence of PC became insignificant. These results indicate that the adsorbed PC was released from the interface at amore positive potential than 0.05 V as described in the literature.2, 3, 12-17 The threshold of ∆φ for lipid desorption, 0.05 V, isdefined as ∆φdesorb hereafter.
Figure 3 shows a plot of γ at −0.15 V as a function of thebulk concentration of PC in DCE. When the concentration ofPC was in the range between 2 × 10 −6 and 2 × 10 −5 M, γdecreased at the constant rate of 14 mN m−1 with the concentration of PC, indicating that the adsorption of PC at the W|DCE

where Eref is the potential of the reference electrodes employed.
The Eref of TPhAsE was 0.350 V with reference to the standard
potential3 where the Gibbs free energy for the transfer of anion from W to DCE, ∆Gtr, is recognized to be zero based on
Parker’s extrathermodynamic assumption.9 Using eq 3, experimental data for E was converted to ∆φ and is shown as thus
hereafter.
Voltammetry for the Ca2+ transfer at a micro W|DCE interface. The Ca2+ transfer at the W|DCE interface was measured
as a voltammogram by scanning E applied at a micro W|DCE
interface and measuring the current for the ion transfer.10, 11
The cell consisted of W and DCE separated by a polyester film
16 µm thick with a micro hole 50 µm in diameter. 11 The
W|DCE interface was formed at the micro hole. The supporting
electrolyte added to DCE was BTPPA+TFPB–.
All electrochemical measurements were carried out at 25 ±0.5 ˚C.
Apparatus. The potentiostat/galvanostat for voltammetryat the micro W | DCE interfaces and that for the γ measurement
were Model HECS-318 (Huso Electro Chemical System) andModel HA 1010MIS (Hokuto Denko Co.), respectively. Thefunction generator and X-Y recorder used were Model HB-111(Hokuto Denko Co.) and Type 3086 (Yokogawa ElectricWorks, Ltd.), respectively. The electrobalance and the electrobalance lift were HD-60 (A&D Company, Ltd. ) and SYLB7K (Sanyu Tech., Ltd.).
3. Results and Discussion
Adsorption of a PC monolayer at the W|DCE interface.
Figure 2 shows the γ value at the W|DCE interface measured
as a function of ∆φ. In the absence of PC in DCE, the potential

2015105(3)0-0.4∆ desorb-0.2

0.0

0.2

0.40.6∆ φ / V vs. TPhE
Figure 2. The potential dependence of the interfacial tension in thepresence (●) or absence ( ) of 10–5 M PC at the interface between
DCE containing 0.01 M BTPPA + TFPB – and W containing 0.1 M
CaCl2.

·2015γ / mN m-1

62

1050-6.0-5.5-5.0-4.5-4.0-3.5

log CPC / M
Figure 3. The interfacial tension at −0.15 V as a function of the logarithmic concentration of PC, log CPC, in DCE.

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

Evaluation of the Associate Constantinterface was saturated. The surface excess concentration relative to both solvents, ΓPC, was calculated to be 2.45 µmol m−2
based on the slope, and was consistent with the reported value of
2.31 µmol m−2 for a phospholipid monolayer in the presence ofNaCl in W.3 The γ decreased at lower rate when the concentration of PC was higher than 5 × 10−5 M, indicating that PC
was present the critical aggregation concentration.
Voltammograms for the transfer of Ca2+ facilitated by a PCat the W|DCE interface. Previous studies have suggested
that the desorption of PC at ∆φ > ∆φ desorb is caused by thecomplex formation of a cation with PC followed by transfer
across the interface.2, 3, 13, 18 In order to confirm the complex
formation of Ca2+ with PC, voltammograms for the transfer ofCa2+ were measured at the micro W|DCE interface.
Figure 4 shows voltammograms for the transfer of Ca2+ inthe presence of PC in DCE. In the absence of PC in DCE, theCa2+ transfer from W to DCE was observed as the positive
current at 0.45 V in the voltammogram (line 1). When PC wasadded to the DCE, a new positive current appeared in thevoltammograms at negative potentials greater than 0.45 V (line2− 5). The new positive current increased with the increase of
the concentration of PC in DCE and shifted by about −0.060 V
for a tenfold increase in the Ca2+ concentration. Considering theprevious study of ion transfer facilitated by a neutral ligand,19
the new positive current is found to be due to the Ca2+ transfer
facilitated by the addition of PC to DCE. The facilitation of theCa2+ transfer by the addition of PC results from the complex
formation of PC with Ca2+.
The potential range allowing the Ca2+ transfer facilitated by
PC was greater than 0.05 V. The potential range agreed withthat observable for desorption of the PC monolayer from theinterface, ∆φ > 0.05 V (∆φdesorb), as shown in Figure 2. Therefore,

2.0

541.0I / nA

3201632+

the Ca transfer facilitated by the complex formation with PC
causes desorption of the PC monolayer at ∆φ > ∆φdesorb. The
process of the adsorption-desorption behavior of PC in Figure
2 is illustrated in Figure 5.
An association constant of Ca 2+ with a PC monolayer
adsorbed at the W|DCE interface. The adsorption-desorption behavior of PC follows from transfer of the complex of PC
with a hydrophilic cation, Mz. The behavior can be expressedas eq 4.2, 3, 18zzz
PC(O) ←→ PC(ads) + M ←→ MPC (ads) ←→ MPC (O)(4)

Here, the subscripts (O) and (ads) denote the species in thebulk organic phase and the adsorbed species at the interface,respectively. Samec et al. proposed a theory for the potential
dependence of γ by assuming the reaction in eq 4 and using
two Frumkin isotherms for adsorption of PC and MPCz.2 The
theory suggests that the onset of the increase in interfacial
tension such as ∆φdesorb is determined mainly by the transfer
potential for Mz, ∆φ M0’, the bulk concentration of hydrophilic
salts, MX, in W, C 0MX, and the association constant in O, Kass =CMPC(O) / CPC(O)CM(O).

∆φdesorb ≈ ∆φ 0'M − (RT/zF) ln (KassC 0MXDMPC1/2DPC−1/2 ), (5)where D is the diffusion coefficient.
The Kass value was evaluated from ∆φdesorb in Figure 2. Byusing eq 5, the log Kass of Ca2+ with PC was calculated to be
19.3 mol −1 dm 3 . In calculation, the following points wereassumed: the lipid/ion stoichiometry of the complex was 1:1,and D of CaPC2+ was equal to that of PC. The value of ∆φ 0 for
Ca2+ was estimated to be 0.59 V based on ∆φ 0 for Mg2+,20 thepotential difference between the positive current for thetransfer of Ca2+ and that for the transfer of Mg2+ in the voltammogram recorded in the absence of PC.
Figure 6 shows the Kass of PC with cations estimated from∆φdesorb. The values of Kass for monovalent cations other thanCa2+ are the values reported in our previous work3 which wasestimated according to a procedure similar to the present work
The log Kass of PC with Ca2+ was higher by 3 times than thoseof PC with monovalent cations. It has been reported that theassociation of BLM with a metal cation is stronger with theincrease of the surface charge density of the metal cation.1 Thecomplex formation between cations and the PC monolayer
adsorbed at the interface demonstrates a trend similar to thatreported in the study of BLM.
25-1.00.2

0.40.6∆ φ / V vs. TPhE
Figure 4. Voltammograms measured at micro interfaces between Wcontaining 0.1 M CaCl2 and DCE containing 0.01 M BTPPA+TFPB–and (1) 0, (2) 10−4, (3) 2 × 10−4, (4) 5 × 10−4, and (5) 10−3 M PC. Scanrate was 1 mV s−1.

∆ϕ < ∆ϕdesorb
(W)

Ca2+

∆ϕ > ∆ϕdesorb

Ca2+log Kass / mol-1 dm30-0.220151050+

Li(DCE)

Ca2+Figure 5. Potential dependence of the PC adsorption at the W | DCEinterface.

+Na+++++++++K Cs H4 H3 H2 NH )4N rg a2A CN 3)N )2N 3)3 H3
H H3 CH (CC(((CFigure 6. The association constants, Kass, of PC with the cations inDCE evaluated from the onset of the increase in the potential dependence of γ, ∆φdesorb. The Kass of cations other than Ca2+ were calculatedin Reference 3. Arg+ indicates the arginine cation. The value of Kassfor (CH3)4N+ was too small to determine.

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J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

4. Conclusion
An associate constant for the complex formation of a phospholipid with Ca2+ was evaluated from the interfacial potential
for desorption of the phospholipid monolayer from the WDCEinterface. Based on this method, the log Kass of Ca2+ with PC
was calculated to be 19.3 mol−1 dm3: higher by 3 times thanthose of PC with monovalent cations. The affinity of the PC
monolayer adsorbed at the WDCE interface to a metal cationis almost similar to that of BLM. Therefore, localization ofmetal cations on the BLM surface may be responsible for thecomplex formation of the cation with PC.
Acknowledgements. This study was partly supported byGrants-in-Aid for Scientific Research (No. 16350043 and No.
16550072) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
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