The interfacial tension of the lipid membrane formed from lipid-amino acid systems.

Abstract

The interfacial tension of lipid membranes composed of phosphatidylcholine (lecithin, PC)-valine (Val), phosphatidylcholine-isoleucine (Ile), phosphatidylcholine-tyrosine (Tyr), and phosphatidylcholine-phenylalanine (Phe) has been studied. The membrane components formed 1:1 complexes. The interfacial tension measurements were used to determine the membrane surface concentration A (3)(-1), the membrane interfacial tension γ(3), and the stability constant K.

Full text

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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
123
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%).
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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
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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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