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ORIGINAL PAPER

The Interfacial Tension of the Lipid Membrane Formed

from Lipid–Amino Acid Systems

Aneta D. Petelska•Monika Naumowicz•

Zbigniew A. Figaszewski

Published online: 13 May 2011

? The Author(s) 2011. This article is published with open access at Springerlink.com

Abstract

posed of phosphatidylcholine (lecithin, PC)–valine (Val),

phosphatidylcholine–isoleucine (Ile), phosphatidylcholine–

tyrosine(Tyr),andphosphatidylcholine–phenylalanine(Phe)

has been studied. The membrane components formed 1:1

complexes. The interfacial tension measurements were used

to determine the membrane surface concentration A3

membrane interfacial tension c3, and the stability constant K.

The interfacial tension of lipid membranes com-

-1, the

Keywords

Phosphatidylcholine ? Valine ? Isoleucine ? Tyrosine ?

Phenylalanine ? Complex 1:1

Interfacial tension ? Bilayer membrane ?

Introduction

Natural cell membranes have been studied by numerous

techniques including physicochemical ones. An important

property of a cell membrane is its interfacial tension, which

determines its rigidity and, as a result, affects its stability.

A cell membrane is a very complex system, and it contains

various structural components that can influence its inter-

facial tension. Therefore, it is easier to study the effect of

various factors; e.g., amino acid–lipid interaction using

artificial phospholipid bilayer model membranes. The

properties of the artificial membrane should be well known

and generally similar to the properties of the membranes of

living cells. Lipid monolayers, lipid bilayers, collodion,

cellophane, millipore, ion-exchanger, or other membranes

have been used as artificial membranes [1], but the inter-

facial tension of a cell membrane is best measured by

means of a spherical bilayer lipid model membrane.

Aminoacidsareubiquitouslyfoundinalllivingcells[2,3].

Therefore, although they are usually only present in low

concentrations in cells, amino acids represent interesting

model substances to examination the interaction between

amino acids and bilayer lipid membranes. These interactions

have been examined used numerous experimental studies

[4–6].MacCallumetal.[7,8]havecalculatedthedistribution

in a lipid bilayer of small molecules mimicking 17 natural

amino acids in atomistic detail by molecular dynamics sim-

ulation.Theresultsgivedetailedinsightinthemolecularbasis

of the preferred location and orientation of each side chain as

well as the preferred charge state for ionizable residues.

To understand complex biological systems it is valuable

to analyze simpler model systems. It would be useful, for

example, to be able to determine the influence of individual

amino acids on the interactions of peptides with a cell

membrane. Jacobs and White used a variety of techniques

to examine the thermodynamics and binding of a general

class of tripeptides, to small phosphatidylcholine vesicles

[9]. These experiments revealed peptide induced alterations

of the lipid order and modulations of the lipid acyl chain

motion. Later work related these thermodynamic parame-

ters with structural information from neutron diffraction

experiments [10]. These experiments were used to deter-

mine the general location of the peptide in the bilayer, if

the peptide inserted in hydrocarbon region or was confined

to the water–hydrocarbon interface. Specific information

concerning the structure of the lipid–peptide complex

A. D. Petelska (&) ? M. Naumowicz ? Z. A. Figaszewski

Institute of Chemistry, University in Bialystok, Al.

J. Pilsudskiego 11/4, 15-443 Bialystok, Poland

e-mail: aneta@uwb.edu.pl; elchem@uwb.edu.pl

Z. A. Figaszewski

Laboratory of Electrochemical Power Sources, Faculty of

Chemistry, University of Warsaw, Pasteur St. 1, 02-093 Warsaw,

Poland

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Cell Biochem Biophys (2011) 61:289–296

DOI 10.1007/s12013-011-9207-3

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would be extremely useful in further understanding the role

of individual amino acids in peptide–lipid interactions.

It is well known the importance of investigations of inter-

actions between proteins and biological membranes. We

decided to give a deeper sight to this problem by the determi-

nationofwaysofinteractionsbetweenmainstructuralelements

of proteins and membranes: amino acid molecules and

phospholipid bilayers correspondingly, using interfacial ten-

sion method. By means of interfacial tension method,

remarkableprogressisbeingmadeintheunderstandingoflipid

bilayer behavior influence on the peptide–lipid interaction.

The effects of membrane composition on interfacial

tension were previously described in phosphatidylcholine–

other lipid [11], phosphatidylcholine–fatty acid, and

phosphatidylcholine–amine systems [12]. This work is a

continuation of our studies concerning the interaction of

lipid bilayers with molecules of increasing complexity

using membranes composed of phosphatidylcholine–amino

acid system. We describe the dependence of interfacial

tension on membrane composition in phosphatidylcholine

(PC)–valine (Val), PC–isoleucine (Ile), PC–tyrosine (Tyr),

and PC–phenylalanine (Phe) over a possible range of

compositions, and present a comparison of the stability

constants of the complexes and the surface areas occupied

by the membrane components.

Theory

In the cases where the membrane components do not form

chemical compounds, their interaction can be described by

the following set of equations [13, 14]:

c1m1A1þ c2m2A2¼ c

m1

m1þ m2

x1þ x2¼ 1

1;A?1

2

¼ x1

ð1Þ

where A?1

components 1 and 2; m1;m2(mol m-2) are the quantities of

components 1 and 2 per unit area of the membrane; c1;c2

(N m-1) are the interfacial tensions of membranes assem-

bled from pure components 1 and 2; c (N m-1) is the

measured interfacial tension of the membrane; and x1;x2

are the solution mole fractions of components 1 and 2.

The elimination of m1and m2yields the linear equation:

Þx1¼A2

A1

(mol m-2) are the surface concentration of

c ? c1

ð

c2? c

ðÞx2

ð2Þ

Membranesmayalsobeassembledfromtwocomponents

capable of forming a complex. The stoichiometry of the

complex may vary, but because the first stability constant in

these complexes is usually the largest [15], we assumed that

the complexes are primarily of 1:1 stoichiometry.

In cases where the membrane components form a 1:1

complex, interactions in the membrane may be described

by a previously published set of equations [14].

The equilibrium between the individual components and

the complex is represented by:

AðComponent1Þ þ BðComponent2Þ , ABðComplex)

and the basic equation describing the interaction between

components 1 and 2 can be written as [11, 14]:

c ? c1

þ c3? c2

¼ KA?1

þ c3? c

where B1¼ A3=A1and B2¼ A3=A2:

Equation 3 may be simplified by taking into account the

high stability constant of the complex. Applying this sim-

plification results in linear behavior for small (x2\x1) and

large (x2[x1) x2values [11, 14].

ðÞB2x1þ c ? c2

ÞB1x2þ c1? c2

3B1B2 c ? c1

ÞB1x2? c ? c2

ðÞB1x2

½? c3? c1

Þ x1? x2

Þ x2? x1

Þ x1? x2

ðÞB2x1

½

ðððÞ?

ððÞ½

ðððÞ þ c3? c

ðÞB2x1

½?

ð3Þ

c1? c

ðÞx1? x2

x2

Þx2? x1

x1

¼ ?B1c3þ B1c

ð4Þ

c2? c

ð¼ ?B2c3þ B2c

ð5Þ

When calculating the stability constant for the complex,

Eq. 3 can be simplified to x1¼ x2[11].

K A?1

123

ð

¼ c2A?1

? c2A?1

??2A?1

1þ c1A?1

??2A?1

???1c ? c3

2? c A?1

Þ2

1þ c1A?1

2? c A?1

1þ A?1

1þ A?1

2

??

2

??

c2A?1

?

1þ c1A?1

A?1

2

?c3

??

???

1þ A?1

2

?

ð6Þ

The parameters describing the complex may be used to

calculate theoretical points using the equation presented

below (agreement between the theoretical and experimental

values implies that the system is well described by the

above equations):

KA?1

þ?KA?1

? KA?1

þ KA?1

? KA?1

where

1A?1

2

1A?1

1A?1

1A?1

1A?1

a1þ a2

ð

c2a1þ c3a2

2a3c3c3a2þ c1a2

2a1c1a1c2þ a2c3

ðÞ a3? a1

c1a1? c3a3

ðÞc2

Þ a1þ a2

Þ a3? a1

Þ

Þ ? a4A?1

2

ðÞ

2

ððÞ þ a4A?1

3

a3þ a2

ðÞ?c

ð

ð

3

c2a3þ c1a2

ðÞ ¼ 0

ð7Þ

a1¼ A?1

a2¼ A?1

a3¼ A?1

a4¼ A?1

þ c2? c3

3

x2? x1

2x1

ðÞ

1x2

3

c1? c2

ðÞ x2? x1

Þx2A?1

ðÞ þ c1? c3

ðÞx1A?1

2

?

ð

1

?

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For systems containing two lipid components, 1:1

complex formation was assumed to be the explanation for

deviation from the additivity rule. Model curves were

constructed usingcalculated parameters such as equilibrium

constants, molecular areas of the complexes, and interfacial

tension of molecules and complexes. The accuracy of the

models was verified by comparison to experimental results.

Experimental

Measuring Apparatus and Measuring Procedures

The interfacial tension method is based on Young and

Laplace’s equation:

2c ¼ RDp:

The interfacial tension, c, in a lipid bilayer sample is

determined by measuring the radius of curvature of the

convex surface, R, formed when a pressure difference, Dp

is applied across the bilayer [16].

The apparatus and measurement method were described

in previous papers [13, 17]. The measurement vessel con-

sists of two glass chambers separated by a mount holding a

1.5 mm diameter circular Teflon element axially pierced

by a small orifice. Spherical membranes were formed by

the Mueller–Rudin method [18] on the flat end of the

Teflon element. Both chambers were filled with an elec-

trolyte solution. The membrane-forming solution was

introduced to the flat wall of the Teflon element using a

micropipette, and pressure was applied to the left chamber

using a manometer (VEB).

The convexity of the spherical cap was measured by

means of a microscope with an objective equipped with a

scale with 0.1-mm-interval scale marks. Therefore, the

instrument readings of the lipid spherical cap were made

with 0.05 mm precision. The convexity of the lipid mem-

brane of the spherical cap, together with the Teflon element

diameter corresponding to the lipid spherical cap diameter,

yielded the radius of curvature. The measurement of the

spherical cap was difficult because the spherical cap is

hardly visible. While using yellow light its visibility gets

better.

The radius of curvature, R, was determined using this

value and the diameter of the Teflon element, corre-

sponding to the diameter of the lipid cap and the convexity

of the lipid membrane, which was presented below in

drawing a and b:

where R radius of curvature, r radius of the Teflon cap,

h convexity of the lipid membrane.

Radius of curvature was calculated from equation:

R ¼ r2þ h2?2h:

membrane and overpressure provoking the membrane

convexity. Then we calculated the interfacial tension val-

ues from radius of curvature and pressure difference values

according to Young and Laplace’s equation.

The interfacial tension was measured on a freshly cre-

ated lipid bilayer membrane 12–15 times. For each mem-

brane about 10 instrument readings of the lipid spherical

cap diameter, formed by pressure difference applied on

both sides, were made. These measurements were made

within the whole range, from the very low values of

the lipid spherical cap diameter to those almost equal to the

Teflon element radius. From all of instrument readings the

arithmetic mean and standard deviation were enumerated.

Measurements with preparation of the electrolyte solution

were made 2–3 times in order to test the repeatability of

these determinations. The experimental results are pre-

sented with error bars in the figures.

We measured radius of curvature of bilayer lipid

Reagents

The following reagents were used for the preparation of the

membrane-forming solution:

1.Phosphatidylcholine (99%, Fluka) (fatty acid compo-

sition: 16:0 *33%, 18:0 *4%, 18:1 *30%, 18:2

*14%, 20:4 *4%).

Cell Biochem Biophys (2011) 61:289–296 291

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2.

3.

4.

5.

L-Valine (99.5%, Fluka);

L-Isoleucine (99.5%, Fluka);

L-Tyrosine (99.5%, Fluka);

Phenylalanine (99.5%, Fluka).

The molecular weights of the lecithin, valine, isoleucine,

tyrosine, and phenylalanine were approximately 752.08,

117.15, 131.17, 181.19, and 165.19 g mol-1, respectively.

The as-received phosphatidylcholine was purified by

dissolving in chloroform and evaporating the solvent under

argon. The stock membrane-forming solutions consisted of

20 mg cm-3of the desired substances (PC, Val, Ile, Tyr, or

Phe) in 20:1 n-decane:butanol. The solution containing the

membrane components was not saturated and could

therefore contain the components in any proportion. During

membrane formation, the solvent was removed, leaving a

membrane composed of lipids in the same ratio as the stock

solution. Bilayer membranes were obtained as bubbles at

the Teflon cap constituting a measuring vessel component.

The use of n-decane as the solvent allows one to obtain

membranes of thickness and capacity values similar to

those of membranes formed of monolayers [19, 20]; there

is almost no solvent retained in the bilayer. A small

quantity of butanol added has a negligible effect on the

interface tension values of the bilayers created; however, it

considerably accelerates the formation of the membranes.

The formation of the bilayers was monitored visually and

electrically by measuring the membrane capacitance at low

frequency (1 Hz). Capacity of the membranes increased

with time after bilayers formation until a steady-state value

was reached some 10–20 min later. The measurements

were begun only after the low frequency capacitance was

stable, increasing by, 1%/h. When the capacitance had

stabilized, it was assumed that diffusion of solvent out of

the bilayer was complete, although some n-decane mole-

cules would remain dissolved in the membrane interior.

The bilayers area was determined with a microscope with a

micrometer scale built into the lens.

The electrolyte solution contained 0.1 M potassium

chloride and was prepared using triple-distilled water and

KCl produced by POCh (Poland). The KCl was calcined to

remove any organic impurities.

All solvents were chromatographic standard grade. The

n-decane was purchased from Merck and the chloroform

and butanol were obtained from Aldrich.

All experiments were carried out at 293 ± 2 K.

Results and Discussion

The effect of the presence of amino acids on interfacial

tension of the membranes formed from PC was stud-

ied. The dependence of interfacial tension of the lipid

membrane as a function of composition was studied at

room temperature (293 ± 2 K) in all the feasible concen-

tration range. The interfacial tension values reported in this

paper refer to the two sides of bilayer membrane surface

area unit.

Figure 1 contains a graph of c ? c1

for the four systems PC–Val (Fig. 1a), PC–Ile (Fig. 1b),

PC–Tyr (Fig. 1c), and PC–Phe (Fig. 1d). According to

Eq. 2, when the membrane components do not interact

these functions should yield straight lines. This is clearly

not the case, which suggests that a complex or other

structure exists in PC–Val, PC–Ile, PC–Tyr, and PC–Phe

bilayers. Because the use of Eq. 3 presupposes the exis-

tence of 1:1 complexes, our initial assumption was that the

complexes formed were 1:1. The interfacial tension of the

lipid membrane was studied over a wide range of lipid

compositions.

ðÞx1versus c2

ð?cÞx2

Phosphatidylcholine–valine complex

Equation 2 predicts that in the absence of interactions, the

plot of Fig. 1a should yield a straight line. The nonlinear

natureoftheplotindicatessomeformofinteractionbetween

phosphatidylcholine and valine. Such interactions in phos-

phatidylcholine–amino acid systems in monolayer can be

explained in terms of complexes [21]. The 1:1 complex is

formed in the initial stage of complexation, followed by

other compositions in subsequent stages. In our case, an

equation derived to describe the equilibrium of 1:1 complex

formation was sufficient for the entire concentration range.

Figure 2a depicts the interfacial tension of a PC–Val

membrane as a function of valine mole fraction. The

dependences of interfacial tension of lipid membranes

formed from the PC–Val system was executed in the

function of the composition to 60% of the valine contents,

because only to such contents of component 2 (valine) with

lecithin were the forming bilayer membrane.

The interfacial tension value of pure lecithin membrane

(component 1), c1was measured directly and presented

earlier [13],whichisequal1.62 9 10-3N m-1.There isno

accurate literature data on interfacial tension values for the

pure amino acid (valine, isoleucine, tyrosine, and phenyl-

alanine), because these components are not creating the

bilayer membrane. However, in order to characterize the

course of the experimental curves, the c2value for the pure

components are necessary, which will be used for calcula-

tion. In this case, the interfacial tension hypothetical values

for membranes built from amino acids were determined

adjusting the experimental curve with the polynomial of the

other mark extrapolating the x2= 1 value. An example of

this extrapolation for valine membrane is presented in

Fig. 3.Theinterfacialtensionvaluesobtainedinthiswayfor

pure valine, isoleucine, tyrosine, and phenylalanine are

292Cell Biochem Biophys (2011) 61:289–296

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equal to 7.0 9 10-4, -2.7 9 10-3, -3.5 9 10-3, and

5.3 9 10-3N m-1, respectively. Negative values of inter-

facial tension for membrane built from pure isoleucine and

tyrosinearepointingtothefactthatitisnotpossibletocreate

the bilayer membrane from pure amino acid. Thermody-

namic potential forthis bilayer would have a negative value,

i.e., the bilayer is not forming.

The valine and phenylalanine membrane interfacial

tension value is positive. However, it is not possible to

create a bilayer lipid membrane from the pure component

because the forming solution above 60% of valine or 25%

of phenylalanine was granulated in the solution.

Based on the literature, an assumption was made that

a 1:1 complex (termed compound 3) formed the most

prevalent structure and was characterized by a maxi-

mal stability constant K [15]. Given this assumption,

the dependence of interfacial tension on the composition of

the membrane-forming solution is described by Eq. 3. The

interfacial tensions of membranes formed from the pure

components were experimentally or theoretically deter-

mined. The constants B1;B2 and c3were determined

assuming that the value of the stability constant for the PC–

Val complex was sufficient with respect to the simplified

Eq. 3 to Eqs. 4 and 5.

(a)

0,0E+00

1,0E-03

2,0E-03

3,0E-03

4,0E-03

5,0E-03

(γ2-γ)x2

(γ-γ1)x1

(b)

0,0E+00

1,0E-03

2,0E-03

3,0E-03

4,0E-03

5,0E-03

6,0E-03

(γ2-γ)x2

(γ-γ1)x1

x2

x2

(c)

0,0E+00

1,0E-03

2,0E-03

3,0E-03

4,0E-03

5,0E-03

(γ2-γ)x2

(γ-γ1)x1

(d)

0,0E+00

1,0E-03

2,0E-03

3,0E-03

4,0E-03

5,0E-03

00,050,10,150,20,25

0

0,050,10,150,20,25

00,050,10,150,20,25

00,050,1

(γ2-γ)x2

0,150,20,25

(γ-γ1)x1

x2

x2

Fig. 1 Graph of Eq. 2 for

phosphatidylcholine–valine (a),

phosphatidylcholine–isoleucine

(b), phosphatidylcholine–

tyrosine (c), and

phosphatidylcholine–

phenylalanine (d), where x2is

the mole fraction of component

2 (valine, isoleucine, tyrosine,

phenylalanine, respectively)

(a)

0,0E+00

1,0E-03

2,0E-03

3,0E-03

4,0E-03

5,0E-03

6,0E-03

x2

γ, N m-1

(b)

-4,0E-03

-2,0E-03

0,0E+00

2,0E-03

4,0E-03

6,0E-03

x2

γ , N m-1

(c)

-4,0E-03

-2,0E-03

0,0E+00

2,0E-03

4,0E-03

6,0E-03

x2

γ , N m-1

(d)

0,0E+00

1,0E-03

2,0E-03

3,0E-03

4,0E-03

5,0E-03

6,0E-03

00,20,40,60,81

00,20,40,60,81

00,20,40,60,81

00,20,40,60,81

x2

γ, N m-1

Fig. 2 The interfacial tension c

of phosphatidylcholine–valine

(a), phosphatidylcholine–

isoleucine (b),

phosphatidylcholine–tyrosine

(c), and phosphatidylcholine–

phenylalanine (d), as a function

of mole fraction x2of

component 2 (the experimental

values are marked by points and

the theoretical values are

indicated by the curve)

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