J. Biochem. Biophys. Methods 65 (2005) 81–87

www.elsevier.com/locate/jbbm

Short note

Conformation study of the membrane models by

the Maxwell displacement current technique and

oxidative stress

M. Weis a, M. Kopani b,*, P. Michalka b, Cs. Biro b, P. Celec c,

L’. Danisovic d, J. Jakubovsky b

a Slovak University of Technology, Faculty of Electrical Engineering and Information Technology,

Department of Physics, Bratislava, Slovakiab Comenius University, School of Medicine, Institute of Pathology, Bratislava, Slovakia

c Comenius University, School of Medicine, Institute of Pathophysiology, Bratislava, Slovakiad Comenius University, School of Medicine, Institute of Medical Biology and Genetics, Bratislava, Slovakia

Received 5 September 2005; received in revised form 23 October 2005; accepted 23 October 2005

Abstract

The role of biological membranes as a target in biological radiation damage is still unclear. Recently

much attention has been paid to the dynamic behaviour of the cell membrane. Maxwell displacement

current technique (MDC) provides new possibility of conformation study of the membrane models.

Oxidative stress can impair macromolecules in the cell on a molecular level. MDC technique enables to

study the changes in molecular orientations and/or conformations of cell membranes. The combination of

different methods in structural biology can clarify membrane chemical and physical properties.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Maxwell displacement current; Oxidative stress; Free radical; Conformation; Membrane

1. Introduction

A free radical is a cluster of atoms where one atom contains an unpaired electron. This

molecule is an extremely unstable configuration. Radicals quickly react with other molecules or

radicals to achieve the stable configuration. Accumulation of these molecules can cause cell

0165-022X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.jbbm.2005.10.005

* Corresponding author. Tel.: +421 2 59357454.

E-mail address: martin.kopani@fmed.inuba.sk (M. Kopani).

M. Weis et al. / J. Biochem. Biophys. Methods 65 (2005) 81–8782

damage or death. An imbalance between free radical production and antioxidant mechanisms is

called oxidative stress. On a molecular and cellular level oxidative stress impairs macro-

molecules in the cell—proteins, DNA and lipids. Nevertheless, oxidative stress has been shown

to be a major pathophysiological factor participating in the pathogenesis of many important

diseases.

The membranes, bilayers of the amphiphilic molecules, represent the basic structural

component of all biological systems. They participate at substance exchanges as well as have

influence at the metabolism processes. The simple model of the membrane is provided by well-

controlled lipid monolayers which assemble spontaneously from solvated molecules at the air–

water interface [1,2]. These insoluble monolayers of surfactants at the air–liquid interface

(Langmuir films) exhibit very interesting physical properties attributed to low-dimensional

systems as well as provide promising applications as models of biological membranes for

studying lipid–protein interactions [3–5]. Artificial membranes provide new possibilities for

study of the processes on a molecular level (e.g. lock-and-key mechanism) and enable to design

a phantom membrane with defined area densities of function proteins.

Shah and Schulman [6] measured surface pressures and surface potentials of mixed

monolayers of dicetylphosphate–cholesterol, dipalmitoyl lecithin–cholesterol, egg lecithin–

cholesterol, and phosphatidic acid–cholesterol. They investigated the interaction between

elements of compounds. They found that there is no interaction between lecithin and

cholesterol, but that there is ion–dipole interaction between dicetylphosphate and

cholesterol, as well as between phosphatidic acid and cholesterol. The surface potential

is shown to be a more reliable parameter for the study of interactions in monolayers than

the surface pressure.

Taneva and Keough [7] investigated the interaction of three surfactant proteins with

monolayers of surfactant components preformed at the air–water interface by means of surface

pressure techniques. Interaction of divalent ions of Ca with monolayers components was also

examined. Calcium ions did not affect the intrinsic surface activity of surfactant protein but

reduced the surface pressure. The results revealed that lipid-induced changes in the

conformations of the proteins might have modulated the interactions of three surfactant

proteins. The membrane phospholipid molecules create a spherical lipid bilayer shell around the

cell. Due to their thermodynamic properties they spontaneously form a double layer.

The bilipid layer is variably permeable. Some molecules are allowed to diffuse through the

membrane. The layer is extremely permeable to water molecules. The breaks or ruptures of the

cell membrane are spontaneously repaired. Lipid peroxidation is reaction of lipid with free

radicals. Peroxidation of lipids in cell membranes can damage cell membranes by disrupting

fluidity and permeability [8,9]. Lipid peroxidation can also adversely affect the function of

membrane bound proteins such as enzymes and receptors [10–12]. Several lipid molecules

containing double bonds can be peroxidized. The mechanisms inducing lipid peroxidation are

complex.

The role of biological membranes as a target in biological radiation damage is still unclear.

Benderitter et al. [13] irradiated human erythrocyte membranes and measured biochemical and

biophysical properties by measurements of MDA and the lipid peroxidation index. The source of

radiation used low doses gamma rays of 60Co. They found that the lipid compartment of the

membrane became more fluid. The lipid–protein membrane interface became more rigid. The

exposure to chemical substances like ethanol can cause tissue specific damage. The membrane

alterations can be seen in light and electron microscope as shown by Celec et al. [14]. The

structure and organization of membranes can be investigated by various methods. Brzustowicz et

M. Weis et al. / J. Biochem. Biophys. Methods 65 (2005) 81–87 83

al. [15] used nuclear magnetic resonance and grazing X-ray diffraction to study the molecular

organization of cholesterol in phospholipid bilayers. From the results, it can be drawn that

cholesterol has low affinity to polyunsaturated fatty acids and lateral phase separation of

membrane constituents. The combination of different methods in structural biology can clarify

membrane chemical and physical properties [16–20].

To study nonhydroxy galactocerebrosides as a part of myelin at the air–water interface of a

Langmuir-Blodgett trough fluorescence and atomic force microscopy was used. The

investigation revealed that galactocerebrosides form domain consisting of nanotubes with a

diameter of 30 nm [21].

2. The Maxwell displacement current method

Recently much attention has been paid to the dynamic behaviour of the cell membrane—

change of molecular orientation or conformation [22–24]. Many various experimental

techniques have been designed to the order parameter investigated in organic monolayers on

the water surface [25,26]. Only a few of them are suitable for the study of time-dependent

orientation properties. For detection of changes in charge states of the molecules and relation

with structural and/or conformational changes, a contactless method has been progressed which

is based on measuring Maxwell’s displacement currents (MDC), which was originally

introduced by Iwamoto and Majima [27,28]. The displacement current flows in a circuit

containing a metal/air/Langmuir monolayer/metal structure during lateral compression of the

monolayer, which results in the change in surface concentration of the monolayer constituting

molecules and as well as in the orientation change of the direction of the molecular electric

dipoles [29 30].

The basic components of the MDC experimental setup are attached to the commercially

available computer-controlled Langmuir trough (Fig. 1, top). The top electrode is suspended in

air, parallel to the interface, without a direct mechanical contact with a floating monolayer on the

water surface. The air gap between the top electrode and the monolayer is adjusted to certain

spacing (approx. 1 mm) by monitoring the capacitance of the electrode system. The

displacement current is detected with a sensitive electrometer. The sensitivity of measuring

the current is 0.1 fA, the background noise is suppressed by a multiple electrical shielding to 2

fA, in a laminar-flow box on an antivibrating stand.

As the surfactants the model phospholipids like dipalmitoyl-phosphatidylcholine (DPPC),

dimyristoyl-phosphatidylcholine (DMPC) or dilauroyl-phosphatidylcholine (DLPC) [31], as

well as functional membrane proteins, e.g. transport protein (e.g. biotin), ion channels

(gramicidin), or receptor proteins (mostly chemoreceptors and photoreceptors [32]) can be

used. Thus the phospholipids-proteins blend provides excellent opportunity to study selected

interactions. Molecules are usually spread on the subphase (bidistilled deionized water) from

chloroform solution and allowed 15 min before compressions to solvent evaporation.

Due to dynamic processes in the monolayer associated with the change in charge distribution

caused by its compression, the induced charge in the top electrode varies with time and this

generates a current flowing through the outer circuit via the electrometer.

Generally, under lateral compression of the monolayer molecular tilt can be changed

continually or suddenly, which is coupled with phase transition of the Langmuir film. Thus for

any monolayer of straight chain materials a phase diagram can be constructed. In Fig. 1 (bottom)

representative measurement and calculated molecular dipole moment projection to the normal of

phospholipids monolayer are shown (details about the dipole moment calculations are in next

Fig. 1. (Top view) Schematic view of the experimental setup for displacement current measurement. Rod-like polar

molecules execute precessional motion on the air–water interface with maximal tilt angle HA (A and l stand for the area

per molecule and the dipole moment of molecule, respectively). Electrical shielding of the top electrode is not drawn.

(Bottom left) Record of the displacement current–area isotherm measurement. (Bottom right) Dipole moment projection

to the normal calculated from MDC measurement. Rapid growth of dipole moment projection at 110 A2 indicates

ordering of molecules during the phase gas–liquid transition.

M. Weis et al. / J. Biochem. Biophys. Methods 65 (2005) 81–8784

chapter). The position of peak maxima in the current–area isotherms (Fig. 1, bottom left) is in

accordance with the values of the area of the phase transition. This indicates that the rate of the

ordering process is highest at the 2D gas–liquid transition of the phospholipid monolayer.

Position of the Langmuir film phase transition determines separation of surfactants and is in

accordance with intermolecular forces [33]. Hence, influence of the external factors (e.g.

adsorption from bulk of subphase to surface layer) as well as internal processes (e.g. slow

collapse process) can be simply recognized.

The level of the displacement current detected is very low and therefore the problem of

background should be carefully considered. The water used in the experiment was bidistilled

with subsequent deionization. Therefore, the amount of extrinsic ions was minimized. The effect

of intrinsic ion (H+, OH�) motion is negligible because no external potential is applied between

the electrodes and, furthermore, the air gap between the top electrode and the water surface is a

good electrical insulator and there is no leakage current in the circuit. These predictions were

always verified by measuring MDC on water subphase before spreading the organic monolayer.

Here the background signal is at the level of few fA, the value being at least one order of

magnitude less than the signal detected after forming the monolayer. The displacement current

technique is sensitive only to dynamic charge processes, which in this arrangement are caused by

lateral compression of the monolayer. Therefore any time-independent charge (e.g. polarized

water surface or additional substances in subphase) distributed at the interface has no effect. This

is an advantage in comparison with conventional electrical measurements, e.g. the Kelvin probe

method.

M. Weis et al. / J. Biochem. Biophys. Methods 65 (2005) 81–87 85

3. Analysis

The complicated behaviour of the Langmuir monolayer is the result of interplay between

different degrees of freedom of amphiphilic molecules. Commonly used simplified molecular

models assigned for analytical solutions take into account only some of them, with the aim of

giving qualitative explanation of particular aspects of the behaviour of the system.

The analysis is based on the assumption that each molecule behaves like a weak dipole

moment with a negative pole bound to the water surface. The influence of the polar water

molecules on a final measured signal was neglected. Individual molecules have random

directions within a certain solid angle and execute a random precessional motion with a maximal

possible tilt HA from the vertical axis. Generally, we consider the molecule as a rod-like rigid

body without a possibility of bending.

If we consider the organic film as a system of electric dipole moments then it is possible to

calculate the induced charge on the upper electrode with the method of images

Qi ¼ hlziNG ¼ lhcosHiNG: ð1Þ

where l is the dipole moment of one molecule (lz is projection of l to the normal), N is the

number of molecules under the top electrode and G is the geometrical factor depending only on

the distance between the top electrode and the top plane of the monolayer and on the shape and

area of the upper electrode. The hcosHi stands for the statistical mean value cosH where H is

the angle between the vector of dipole moment and the normal. Detailed analysis of dipole

moment projection of simple fatty acid was described in [34].

As we show in our previous studies [34] the current flowing in the outer circuit can be

expressed as a time change of the induced charge

I ¼ BQi

Bt¼ lNG

BhcosHiBt

þ lhcosHiG BN

Bt: ð2Þ

By integrating the displacement current with respect to time, the induced charge Q can be

obtained and in this way we also evaluated the vertical component of the molecular dipole

moment. Thus, the dipole moment projection to the normal lz should be calculated as

lz ¼ lhcosHi ¼ 1

GN

ZIdt ð3Þ

The numerical integration result is shown in Fig. 1 (bottom right).

4. Conclusion

Langmuir film created by phospholipids is a well-known approach to design the biological

membrane model with controlled composition and external conditions. MDC technique provides

new possibility of conformation study of the membrane models. This technique is sensitive only

to the order parameter as well as to the dipole moment of the membrane molecules. Thus,

influence of the subphase polarization is negligible. It enables to study the changes in molecular

orientations and/or conformations at different subphases recording only the signal of the

membrane model. Presented method is very sensitive to time deviations of the signal and

therefore appropriate for the study of the time-dependent processes. In association with other

techniques (electron microscopy, atomic force microscopy, diffraction, fluorescence microscopy)

more information about membrane structure and function can be achieved.

M. Weis et al. / J. Biochem. Biophys. Methods 65 (2005) 81–8786

Acknowledgements

This work was supported by grant of Science and Technology Assistance Agency No. APVT-

20-003104 and Scientific Grant Agency of the Ministry of Education of Slovak Republic and the

Slovak Academy of Sciences No. VEGA-1/1158/04.

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