Electrochemical properties of bilayer lipid membranes modified with metal porphyrins

93

Bioelectrochemistry and Bioenergetics, 23 (1990) 93-100 A section of J. Elecrroanal. Chem., and constituting Vol. 298 (1990) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

Short communication

Electrochemical properties of bilayer lipid membranes with metal porphyrins

modified

Paw& Krysihski Laboratory (Poland) (Received

of Electrochemistry

Dept.

of Chemistry,

18 May 1989; in revised form 12 September

Warsaw

University,

Pasteura

I, 02-093

Warsaw

1989)

INTRODUCTION

In recent years there has been an increasing interest in the application of electroanalytical techniques to problems of biological importance. Biochemists are now rather successful in their electroanalytical studies of biomolecules (cf. ref. l), while electrochemists are involved in the development of fuel cells based on the properties of biological membranes and interfaces. It is an attractive notion, also for our Laboratory, that a combination of techniques from electrochemistry be applied to the study of properties of cell membranes [2-41 and biomolecules in their native or pseudonative state. The planar, bilayer lipid membrane (BLM) is one of the most suitable models for studies of the electrical behaviour of membrane bound molecules found in various types of biomembranes. The membrane, which is formed in a controlled manner, can function as the sole conductor of charges between the two compartments partitioned by this membrane. This feature makes the BLM very useful for studies of electrochemical phenomena associated with the membrane itself and the membrane bound molecules [5-81. Most investigations in this area, however, ignore the possible screening effect of the fixed charges of the membrane surface against the interfacial redox reaction of membrane bound molecules placed in the redox gradient. The aim of the present work is to demonstrate this effect for the case of Fe(III)and Mn(III)-tetraphenyl porphyrin chloride (Fe(III)TPP-Cl or Mn(III)TPP-Cl) modified BLMS made from neutral or negatively charged phospholipids. EXPERIMENTAL

All reagents and porphyrins were obtained commercially and were of the highest quality available. The BLMs were formed from the negatively charged or neutral phospholipids using the micropipette technique according to the published methods 0302-4598/90/$03.50

0 1990 Elsevier Sequoia

S.A

94

(cf. refs. 5 and 6). The BLM forming solutions were made from bovine brain phosphatidylserine (PS) - for the negatively charged BLMs - or from synthetic dipalmitoyl phosphatidylcholine with natural cholesterol (PC/Chol) - for the neutral ones - all saturated in n-decane. To endow the BLM with the desired properties, the membrane forming solution was saturated with Fe(III)TPP-Cl or Mn(III)TPP-Cl. The BLM was formed in the aperture (1 mm) in the Teflon septum of the two Teflon chambers of equal volume (5 ml) filled with 0.1 M KC1 acidified to pH 5.5 and considered as the supporting electrolyte. The formation of the bilayer lipid membrane was monitored electrically by applying a small (10 mV amplitude) square potential pulse from the pulse generator to the BLM and monitoring on the oscilloscope the course of the membrane capacitance charging/discharging current from the input standard resistor. The membrane was considered “black” or bilayer formation, small if its capacitance was larger than 0.6 PF cme2. After membrane amounts (50 ~1) of 0.1 M K,Fe(CN), or FeSO, in 0.1 M KC1 were added to the one side of the membrane (donor side), whereas equal amounts (50 ~1) of 0.1 M K,Fe(CN), or Fe,(SO,), in 0.1 M KC1 were added to the other side (acceptor side). The BLM system was studied at room temperature by means of cyclic voltammetry. The conventional system for voltammetry was used (Fig. l), slightly altered to

n

generator

I

. Potent

i0Stat

i C

-05Ci

controller

I I

Teflon

0.1 __

M -

KCI -

p-T_

cell

,

J Fig. 1. Schematic diagram of BLM measuring setup. E,, E,, E,: saturated calomel electrodes (SCEs) of low resistance, respectively.

auxiliary, reference

and working

95

make possible the capacitance measurements and to detect the current response within the range 0.5 nA-10 PA. In the experiments, the typical three-electrode system was used as follows: one low resistance, saturated calomel electrode (SCE) was placed on the donor side of the BLM, whereas the other two SCEs (treated as the reference and auxiliary electrodes) were on the acceptor side of the membrane. This provided a potentiostatically controlled polarization of one side of the BLM (the solution iR drop is negligible due to the BLM resistance of about 1014 a). The voltammograms were recorded for applied voltage changing linearly in the range * 150 mV of a scan rate of 50 mV/s.

RESULTS

AND DISCUSSION

Negatively charged redox couple - K, Fe(CN), / K_,Fe(CN), The obtained voltammograms are shown in Figs. 2a and b. They were recorded for: (1) unmodified PS (Fig. 2a) and PC/Chol (Fig. 2b) BLMs in the absence of a redox couple in the bathing solution; the presence of a redox couple in the bathing solution did not affect the shape of the voltammogram; (2) Fe(III)TPP-Cl or Mn(III)TPP-Cl modified BLMs in the absence of a redox couple; the very minute transient current changes probably due to structural disturbances of the BLM caused by the porphyrin molecules, should be noted; (3) the neutral, Fe(III)TPP-Cl modified BLM placed in the redox gradient. The BLM modified with Mn(III)TPP-Cl, regardless of the lipid, was unaffected by the presence of a redox couple. The latter curve is of particular interest, since it presents the typical behaviour of a BLM with an ionic type of conductance [5]. Moreover, it is almost symmetrical (only up to 20 mV of open circuit potential, donor side positive), suggesting that the observed current increase is neither due to the diffusion of aqueous phase components nor the result of conjugated interfacial redox reactions of the BLM working as a “bipolar” electrode, as the membrane is highly rigid due to the presence of sterol and saturated fatty acid chains. These results suggest the situation outlined in Fig. 3, where the interfacial reduction of Fe(III)TPP-Cl to Fe(II)TPP by Fe(CN)zpresent in the bathing solution is shown, producing an excess of Cl- anions within the membrane phase due to the reaction: Fe(III)TPP-Cl + Fe(CN):+ Fe(II)TPP + Fe(CN)i+ Cll. These additional charges, moving in the external electric field, can decrease the membrane resistance. As is evident from Fig. 2a, this interfacial reaction is inhibited by the electrical screening effect if the BLM is negatively charged (PS). In order to verify whether or not this effect can account for the observations, the diffuse double layer (Gouy-Chapman) theory was used to evaluate the ion depletion in the vicinity of the membrane. This theory can describe adequately the potential adjacent to a phospholipid membrane [9]. For the case of “solvent free”, negatively charged PS membranes, the value of the electrostatic potential at the surface in 0.1 M NaCl was reported to be - 78 mV [lo], corresponding to an area of 2 X lO_” m2 of the PS molecule. Now the Poisson-Boltzmann equation gives the

96

(bl

Fig. 2. (a) Negatively charged phosphatidylserine BLMs. (1) Unmodified, in the presence or absence of negatively charged redox couple in bathing solution; (2) modified with Fe(III)TPP-Cl or Mn(III)TPP-Cl in the absence of negatively charged redox couple; the presence of negatively charged redox couple did not affect the BLM behaviour. (b) Neutral phosphatidylcholine/choIesteroI BLMs. (1) Unmodified, in the presence or absence of negatively charged redox couple in bathing solution; (2) Modified with Fe(III)TPP-Cl in the absence of negatively charged redox couple, or Mn(III)TPP-Cl in the absence or presence of negatively charged redox couple; (3) modified with Fe(III)TPP-Cl in the presence of negatively charged redox couple in bathing solution.

97

MEMBRC7NE PHClSE

WOTER PH?lSE

Fe(II

Fe(

Fig. 3. The possible mechanism of the effect of K,Fe(CN), Fe(III)TPP-Cl.

concentration potential:

on the neutral BLM modified with

of counterions of valence z at the surface of the membrane for this

c(0) = c, exp( - zF+ (O)/RT)

(1)

where c(0) is the concentration of counterions at the surface, c, the concentration of these ions in the bulk, F the Faraday constant, q(O) the potential at the surface, and R and T are the gas constant and temperature (K), respectively. Using this equation one can evaluate that the concentration at the membrane surface for the case of Fe(CN)z- is about 700 times and for the case of Fe(CN)z-, 2 x lo5 times lower than that in the bulk solution. Note, that when the concentration of negative groups at the PS-BLM is for some reason only half the cited value [lo], the electrostatic potential at the membrane surface becomes - 50 mV, giving concentrations about 350 and 2500 times lower than that in the bulk for Fe(CN)iand Fe(CN)z-, respectively. This result strongly supports the hypothesis that ion depletion in the vicinity of the membrane can account for the observed effects. Positively charged redox couple - FeSO,/ Fe,(SO,),, The screening effect against the negatively charged substrates should not affect, but rather assist, the redox reaction for positively charged substrates such as

Fe,(SO,),/FeSO,. The Gouy-Chapman theory and Poisson-Boltzmann distribution equation (eqn. 1) predict an about 450 and 9 X 10’ times excess of Fe2+ and respectively, in the vicinity of the membrane as compared to the bulk Fe”,

I /nA

(al

3.

-2.5 2.

-_-E/mu

t

-2.5

(b)

100

-E/mV

5.0 Fig. 4. (a) Negatively charged BLM. (1) Unmodified in the presence or absence of redox couple in bathing solution; (2) Fe(III)TPP-Cl modified BLM in the absence of redox couple; (3) same as (2), but in the presence of positively charged redox couple. (b) Fe(III)TPP-Cl modified, negatively charged BLM in the presence of positively charged redox couple, after a short potential pulse.

99

concentration. In fact, one can observe even a hindrance of bilayer formation, as the ferric and ferrous cations adsorb strongly at the membrane surface. With these cations present on both sides of the negatively charged (PS) BLM, doped with Fe(III)TPP-Cl, two types of voltammograms can be observed (Figs. 4a and b). The first one, shown in Fig. 4a is, to some extent, similar to that obtained for the neutral, doped BLM in the presence of K,Fe(CN),/K,Fe(CN),, and may correspond qualitatively to the proposed mechanism (Fig. 3). The observed smaller current response may be due to the electrostatic barrier originating from the negative surface charges impeding free movement of the Cll ions across the interface. The second type of voltammogram, observed only after a short, - 500 mV potential pulse application, is shown in Fig. 4b. The voltammograms shown here exhibit diode-like behavior within the range of applied voltage of + 150 mV. This observed asymmetry was reported to be apparently due to the asymmetry of the system in which electrons pass through the BLM only from the donor side to the acceptor side [6]. Such behavior is usually described by the “electrodic model” of the membrane, with the BLM acting as a bipolar electrode [5,6,11]. Figure 4b also presents a kind of “switch” effect, since the diode-like shape was “switched” from the curve shown in Fig. 4a by the potential pulse. The next short potential pulse of even smaller amplitude, caused an additional tenfold increase of the current within the same potential scan range ( f 150 mV).

Fig. 5. Optical (vis) spectra of: (a) () Mn(III)TPP-Cl n-decane + K,Fe(CN), in water. (b) () Fe(III)TPP-Cl n-decane+ K,Fe(CN), in water. See text for details.

in n-decane, (- - -) Mn(III)TPP-Cl in n-decane, (- - -)Fe(III)TPP-Cl

in in

100

The Interface between Two Immiscible Electrolyte Solutions (ITIES) system as verification of the proposed BLM behavior The question remained why such an effect was not observed for the case of Mn(III)TPP-Cl modified BLMs. For this purpose, as well as to verify the possibility of interfacial redox reactions in the investigated BLM systems, experiments with an ITIES as BLM model system were carried out. The ITIES system was created with a water phase containing K,Fe(CN), as the reductor in contact with an n-decane phase containing Fe(III)or Mn(III)TPP-Cl as the substrate. The system was shaken vigorously. By means of spectral analysis the course of the interfacial redox reaction was followed (Figs. 5a and b). As was expected on the basis of the BLM experiments, only the optical spectrum of Fe(III)TPP-Cl was changed. The spectrum of Mn(III)TPP-Cl remained unchanged, probably due to the more negative value of its formal potential [12]. The reduction of Fe(III)TPP-Cl was reversible. If, after reduction, the ITIES system was left in contact with the atmosphere, the spectral signal of Fe(II)TPP disappeared. ACKNOWLEDGEMENTS

This work was started at the Universitat Tiibingen (F.R.G.), Institut fiir Chemische Pflanzenphysiologie, headed by Prof. Dr. H. Metzner, within the framework of an exchange program between our Universities. The author is greatly indebted to Prof. S. Mint for his guidance and for the idea to apply electrochemical methods to studies of biological systems. This work was supported by a grant from the Polish Academy of Sciences. REFERENCES 1 E. Pungor (Ed.), Bioelectroanalysis 1, Proceedings of 1st Bioelectroanalytical Symposium, Matrafiired, 1986, Akademiai Kiado, Budapest, 1987. 2 P. Krysihski and S. Mint, Bioelectrochem. Bioenerg., 10 (1983) 261. 3 P. Krysihski, E. Herzyk and S. Mint, Bioelectrochem. Bioenerg., 12 (1984) 367. 4 E. Herzyk, Bioelectrochem. Bioenerg., 17 (1987) 231. 5 H.T. Tien, Prog. Surf. Sci., 19 (1985) 169. 6 P. Krysihski and H.T. Tien, Prog. Surf. Sci., 23 (1986) 317. 7 J. Barber (Ed.), Topics in Photosynthesis, Vol. 3, Elsevier, Amsterdam, 1979, p. 115. 8 R. Antolini, A. Gliozzi and A. Gorio (Eds.), Transport in Membranes: Model Systems and Reconstitution, Raven Press, New York, 1982. 9 S. McLaughlin, Annu. Rev. Biophys. Biophys. Chem., 18 (1989) 113. 10 A.P. Winiski, A.C. McLaughlin, R.V. McDaniel, M. Eisenberg and S. McLaughlin, Biochemistry, 25 (1986) 8206. 11 M.A. Habib and J. O’M. Bockris, J. Bioelectr., 3 (1984) 247. 12 J.-H. Fuhrhop. Struct. Bonding, 18 (1974) 1.