STUDIA UNIVERSITATIS BABEŞ-BOLYAI, PHYSICA, SPECIAL ISSUE, 2003
PHOSPHOLIPID MODEL-MEMBRANES STUDIED BY FTIR
SPECTROSCOPY
Feride Severcan1 and Dana Dorohoi2
1
Middle East University of Ankara, Biology Department,
Ankara, Turkey,
E-mail <feride@metu.edu.tr>
2
“Al.I.Cuza” University, Faculty of Physics, 6600 Iasi,
Romania,
E-mail <ddorohoi@uaic.ro>
Local fluidity of the acyl chains in model-membrane of the type
dipalmitoylphosphatidyl glycerol (DPPG) and DPPG/GS containing
peptide gramicidin S (GS) in different amounts is revealed by FTIR CH 2
symmetric and asymmetric stretching modes of phospholipid acyl chains.
The influence of temperature as well as of the peptide content on
GS/DPPC and GS/DPPG model-membrane fluidity, described by order
parameter, has been studied.
A mathematical model was developed to describe the thermotropic main
transition of the model membrane. Experimental data were fitted by
functions obtained in this model.
1. Introduction
The inner compartment of the cells and organelles is isolated from the
outer media by the biological membranes consisting in principal from
phospholipids
such
as
dipalmitoylphosphatidylcholine
(DPPC)
and
dipalmitoylphosphatidylglycerol (DPPG).
The biological functions of membranes are assured by a large variety of
processes. The fluidity of the lipid constituents is one of the most important factors
associated with morphological changes in the biological membranes. DPPG is
usually considered as model membrane in trials to evidence the perturbative action
of temperature and/or of drugs inducing the lipid fluidization [1,2].
As well as DPPC [3-5], DPPG is an amphionic phospholipid with two
hydrophobic fatty acid chains separated from a charged head group by a glycerol
backbone. This phospholipid forms bilayers separated by water with the interior
fatty acid chains oriented in parallel to each other and the phospholipid head faced
out, in contact with water. The stability of the phospholipid membranes is assured
by minimization of hydrophobic interactions and by maximization of the
hydrophilic ones.
The nature of phospholipid interactions influences the mechanism of
membrane penetration by some peptides, like antibiotics. When these interactions
have an electrostatic nature [6], such as in the case of DPPG and Gramicidin S
FERIDE SEVERCAN AND DANA DOROHOI
(GS) [7,8], membrane destabilization can occur by pores formation or by detergent
like mechanism [9,10], favoring the leakage of the cellular content [11].
A variety of physical methods has demonstrated the presence of defects in
the packing of lipid molecules in the solid bilayer. A pretransition and a main
phase transition were evidenced [12,13]. At the main phase transition the defects
are the sites of initial melting of the lipids forming small pools of fluid lipid
molecules. Therefore both fluid and solid domains coexist at the main phase
transition temperature. As temperature is raised beyond, the remaining solid lipids
will rapidly and cooperatively melt in a first order process into an all fluid phase.
When temperature of fluid lipid is gradually lowered, the pathway from solid to
fluid phase does not exactly retrace the pathway from solid to fluid phase, because
of the difference in the free energies of the solid and fluid domains formed in one
direction from those formed in the other. DPPC model membrane hysteresys of
main phase has been evidenced by M. Geith [13].
The transition between the gel phase and the liquid crystalline phase of the
model membrane is essentially induced by temperature [13-18], by the defects in
the bilayer structure, or by the chemical reagents [18,19].
There is a main phase transition temperature at which the number of the
systems in the gel phase equalizes the number of the systems in the liquid
crystalline phase. Close to the main phase transition the membrane permeability is
the greatest.
The studies on drug influence on the model membranes are beneficial for
the understanding of the mechanisms of the human erythrocites lysis [20] under
antibiotic action.
2. Theoretical notions
Let us consider a system consisting from N subsystems that can have only
two thermodynamic phases gel and liquid crystalline ones. Let suppose that the
transition between these phases is a reversible thermodynamic transformation:
Gel phase  Liquid crystalline phase
(1)
Relation (1) suggests that by the increasing temperature of the system, subsystems
can pass in the liquid crystalline phase and, by the system cooling, the subsystems
can return in the gel phase.
Let be N g the number of the subsystems in the gel phase and N l the
number of the subsystems in the liquid crystalline phase. These numbers are
dependent on temperature and satisfy the equation:
N g  Nl  N
(2)
At low temperatures, when the system is in the gel phase, we can consider that N g
tends to N, while at the temperature higher than the melting point Ng is near zero,
because the system passed in its liquid crystalline phase and Nl tends to N.
Such a condition is satisfied by phospholipid acyl chains from the system
DPPG/GS with various concentrations of GS. DPPG/GS systems support a
PHOSPHOLIPID MODEL-MEMBRANES STUDIED BY FTIR SPECTROSCOPY
reversible thermodynamic transformation of the type (1). For a fixed value of the
GS molar ratio, N has a fixed value. By temperature increasing, N l increases and
Ng decreases as relation (1) predicts. Cooling the samples to lower temperatures,
Ng increases and Nl decreases, to assure the returning of the system at its gel phase.
The reversibility of the transformation (1) has been experimentally
demonstrated [13] by usıng wavenumber modıfıcatıon of CH2 stretchıng mode
measured in the DPPC/GS systems. So, by temperature increasing the
wavenumbers of the CH2 stretching mode increased, showing a sharp modification
at 41.5 0C, the melting point of DPPC, and then, at the sample cooling, they
decreased in the same way to the values of the wavenumbers corresponding to the
gell phase.
For the sample in the gel phase, supposing that all the subsystems are
ordered, one can write:
(3)
N ( gf   gi )  h  c ( g  0 )
where  gf and  gi are the interaction energy of a pair of subsystems in the final (f)
and initial (i) states of the spectral transition, when the molecules are in the gel
phase (g):  g is the wavenumber measured in the gel phase of the system and  0 is
the wavenumber measured in the gas phase of the same system.
A similar relation can be written for the liquid crystalline phase of the system:
N ( l f   li )  h  c ( l   0 )
(4)
by using the interaction energies,  l f and  li , between two molecule from the
liquid crystalline phase in the vibration states (f and i) participating to the IR
transition and the wavenumbers  l and  0 corresponding to the system in its
liquid crystalline phase and in gas phase.
For the system at a given temperature T, different from T m, one can define
the ratios of the molecules from the gel and liquid crystalline phases, by:
pg 
Ng
N
and pl 
Nl
N
(5)
The ratios p g and p l satisfy the relation:
p g  pl  1
(6)
One the other hand, for the system at a given temperature T, having Ng
subsystems in the gel phase and Nl subsystems in liquid crystalline phase, one
obtains:
(7)
N g ( gf   ig )  N l ( lf   il )  h  c (  0 )
 is the wavenumber measured for the system at temperature T. From equations
(7), (3), (4) and (5) one obtains:
pg 
 l 
 l  g
(8)
FERIDE SEVERCAN AND DANA DOROHOI
pl 
  g
 l  g
(9)
It results that the ratios of the subsystems in the gel phase (pg) and in the liquid
crystalline phase (pl) from the system at temperature T can be estimated by the
wavenumbers of the IR bands. The measurements reffer to the system in the gel
phase ( g ), in the liquid crystalline phase ( l ) as well as in an intermediate phase
( ). The intermediate phase is represented by a mixture of these phases.
Wavenumber  increases with the temperature increasing. So, the equations (8)
and (9) are indicators of the pl increasing and of the p g decreasing by temperature
increasing.
In equations (8) and (9) wavenumber  depends both on temperature and
on molar ratios of GS from the studied systems. It results that the variation of the
wavenumber  when these parameters are modified determines the manner in
which the number of the molecules varies from the gel and liquid crystalline states.
Relations (8) and (9) show that at the increasing temperature, if the
wavenumber  increases, the relative number of the subsystems in the liquid
crystalline phase must increase in the same way with the decreasing of the relative
number of the subsystems in the gel phase.
It also results that the derivatives versus temperature of the wavenumbers
in the intermediate state could be considered as indicators of the variation of the
relative numbers of the subsystems in the gel and in the liquid state at one fixed
temperature.
The derivatives of the numbers p g and p l versus temperature are linear
functions of the wavenumber derivative versus temperature:
p g
T

1 
 l   g T
pl
1 

T  l   g T
(10)
(11)
From equations (10) and (11) it results that, the manner in which the number of the
subsystems from the studied system varies can be monitored by the wavenumber
derivatives versus temperature in the points from the understudied temperature
range.
The higher are the wavenumber derivative values, the faster are the thermodynamic
phase transitions of the type (1).
2. Materials and methods.
DPPG and GS were purchased from Sigma Chemical Co St. Luis Mo,. They
were used without purification. Multilamellar vesicles DPPG and DPPG/GS were
obtained from DPPG dried films, phosphate buffer and stocks of drug-ethanol
PHOSPHOLIPID MODEL-MEMBRANES STUDIED BY FTIR SPECTROSCOPY
solutions, using the procedure proposed by F. Severcan et al [4]. DPPG/GS
vesicles with a drug concentration of 1-10 mol% were used in this study.
The FTIR spectra of multilamellar DPPG and DPPG/GS systems were
registered with a FTIR Bomeme BM 157 spectrometer, using CaCl2 cells. A
Unicam Specac Temperature Controller was used for temperature modifying. The
FTIR spectra were averaged from 100 scans. The water vaporous influence was
eliminated by subtracting the FTIR spectra of buffer solution from the model
membrane spectrum, at each studied temperature. Experimental data were obtained
in a large interval of temperature [27.1-70] 0C.
4. Results and Discussion
In order to estimate derivatives (10) and (11) for the DPPG/GS systems,
we used the wavenumbers of the CH2 stretching vibrations that appear near 2850
cm-1 (asymmetric stretching mode) and near 2917 cm-1 (symmetric stretching
mode) in the gel phase of the samples.
With temperature increasing, the corresponding bands slowly shift to the
higher frequencies. Significant changes in the values of the wavenumbers of these
bands appear at the melting point temperature. Then wavenumbers increase very
slowly with the temperature increasing. Fig. 1 illustrates the shapes of the
modifications of the wavenumbers of the CH2 symmetric mode versus temperature.
Fig.1 Wavenumber of the symmetric stretching band vs temperature
Fig.1 shows us that DPPG and DPPG/GS model membranes have two
distinct thermodynamic phases. One phase at low temperatures, named gel phase,
in which the system is characterized by the smallest value of the wavenumber and
the other phase, at high temperatures, named liquid crystalline phase and
characterized by the highest value of the wavenumber. Thermodynamic transition
(1) takes place between these phases.
FERIDE SEVERCAN AND DANA DOROHOI
These experimental data permit us to use (8) and (9) in order to estimate
the relative number of the subsystems from the gel pg and liquid crystalline pl
phases at the fixed temperature.
Table 1
Wavenumber derivatives for symmetric stretching vibration of CH 2 groups of DPPG
Nr.
T(K)
Gramicidin content (mol%)
0
1
3
6
9
1
301.5
0
0
0.05
0.05
0
2
303.5
0.05
0
0
0.05
0
3
304.5
0.1
0
0.1
0
0.2
4
306
0.07
0.07
0
0.07
0
5
307.1
0.09
0.09
0.09
0
0.18
6
308.7
0.06
0
0
0
0
7
310.7
0
0.1
0.05
0.15
0
8
312.5
0.11
0.28
0.28
1.72
1.67
9
314.3
0.61
0.61
2.61
2.33
2.56
10
314.7
0.25
0.25
0.25
025
0.25
11
317.3
0.11
0.11
0.12
0
0.08
12
319.8
0.04
0.04
0.08
0.08
0.04
13
322.9
0.06
0.03
0
0.07
0.1
14
327
0.02
0.02
0.02
0.05
0
15
329.9
0.03
0
0
0
0.1
16
334.1
0.02
0.02
0.07
0.02
0
The values of the derivatives contained in Table 1 show us that:
In the ranges (26.5-39.5) 0C and (41.7-61.1) 0C, the derivatives have small positive
values, showing no significant changes are in the system from point of view of the
numerical values Ng and Nl.
Pl values rapidly increase beginning to 39.5 0C and become constant at
0
41.7 C. The values of the derivatives in 41.7 0C are indeed small. It results that the
thermodynamic transition (1) between the two phases of the system gives rise in
the domain (39.5-41.7) 0C for all the studied systems.
The values of the wavenumbers derivatives are significantly high at 41.3
0
C, for GS concentrations 0, 1, 3 mol % indicating a fast modification of the ratios
pg and pl at this temperature. Thus, for the molar ratios 0, 1 and 3 mol%, the main
phase transition happens at 41.3 0C. It results that for the molar concentrations in
the range (0-3] mol%, GS do not modify the melting point of the DPPG/GS
systems.
Contrarly, for 6 and 9 mol% GS in the systems DPPG/GS, the values of the
wavenumber derivatives are significant at 39.5 0C and decrease at 41.3 0C. For
these samples, the melting point can be considered as being at 39.5 0C, decreased
with approximately 1.8 0C compared with pure DPPG.
In Fig.2 are plotted the derivatives of the wavenumbers versus the temperature for
asymmetric stretching vibration mode of CH2 acyl chains that offer the same
information such as Table 1. From these graphs it results that for concentrations 0,1
and 3 mol% GS, the number of the subsystems from the gel phase rapidly modifies
at 41.3 0C, while for the molar concentrations 6 and 9 mol%, the number of the
PHOSPHOLIPID MODEL-MEMBRANES STUDIED BY FTIR SPECTROSCOPY
subsystems from the gel phase modifies rapidly at 39.50C. One can affirm that the
big concentration such as 6 and 9mol% of GS in the DPPG/GS mixtures
determines the decreasing of the main point temperature.
Fig.2 Wavenumber derivatives for asymmetric stretching vibration of CH 2 group
Numerical values of the wavenumber derivatives show that the main point
temperature decreases with about 2 0C for the DPPG/GS systems with high molar
concentrations of peptide.The concentration role is evidenced only for the big
molar ratios of GS in membrane model and only in the temperature range (39.541.7) 0C. Our results are in agreement with those obtained in [18].
The hypothesis that GS increase permeability of the membrane by the
increase of the curvature stress in the bilayers of DPPG, is also supported by the
derivative values of the bandwidths expressed in the temperature range (39.5-41.7)
0
C, near the mean point of the thermodynamic transition of the type (1).
Using the derivatives of the ratios pg and pl versus temperature we
calculated the number of the subsystems from the gel and from the liquid
crystalline phases. The values of Ng for the studied DPPG/GS systems, computed
for a system composed from one hundred subsystems, are plotted in Fig.3.
One can estimate the main point of transition (1) for pure DPPG as being at
41.5 0C. For DPPG/GS systems with molar ratios of peptide smaller that 3 mol%
the main phase temperature is lowered at 41 0C and, finally DPPG/GS systems with
molar ratios of peptide higher that 6 mol% pass in liquid crystalline phase at 38.8
0
C.
FERIDE SEVERCAN AND DANA DOROHOI
Figure 3 Number of the subsystems from the gel state vs temperature for CH 2 asymmetric
stretching mode.
5. Conclusions
The proposed model gives results in accordance with the previous ones and
permits to obtain quant.itative information about the thermodynamics of the
lipid/peptide systems. The modifications in the model membrane fluidity are
expressed as functions on the wavenumbers of CH2 vivrations in DPPG acyl
chains, directly dependent on the order degree in the local bilayer.
This study shows that the compromise between order and disorder that
characterises model membranes is extremely vulnerable to the antibiotics.
Acknowledgement
Dana Dorohoi gratefully acknowledges partial financial support of this study by
Tubitak Committee of Turkey in a NATO PC-B Programme
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