Interactions of Melatonin with Anionic Phospholipid Model Membrane: An
FTIR Study
Ipek Sahin1, Nadide Kazanci1, Feride Severcan2
1
Department of Physics, Faculty of Science, Ege University, 35100 Bornova-İzmir,TURKEY
2
Department of Biology, Middle East Tecnical University, 06531 Ankara, TURKEY
Abstract: Interactions of melatonin with anionic dipalmitoyl phosphotidylglycerol (DPPG) multilamellar liposomes
(MLVs) were investigated as a function of temperature and different melatonin concentration by using Fourier transform
infrared (FTIR) spectroscopy. The results revealed that low concentration of melatonin (1mol %) does not induce
significant change in the overall shape of thermotropic profile of DPPG membrane. In contrast, at higher concentration of
melatonin (15 mol %), the phase transition shifts to lower temperatures. Low concentration of melatonin has no
significant change in the frequency values of the CH2 stretching mode implying a negligible effect on the order of the
system, whilst high concentration of melatonin disorders the system. It also increases the bandwidth of the CH 2
asymmetric stretching modes .It also makes strong hydrogen bonding with the C=O stretching and PO 2 antisymmetric
double bond stretching band of DPPG or with the water molecules around both in the gel and liquid crystalline phases.
Keywords: Melatonin, Dipalmitoyl phosphatidylglycerol, Membrane, Fourier Transform Infrared, lipid order, membrane
fluidity.
1 Introduction
Melatonin, N-acetyl-5-methoxytryptamine, is a hormonal
product of pineal gland. Its synthesis is higher at night than
during the day in all vertebrates including man. Once
melatonin is produced in the pineal gland it is quickly
released into vascular system. The rapid release of
melatonin is generally believed to relate to its high
lipophilicity which allows it to readily pass through the
membrane of pinealocytes [1]. Melatonin’s action as a free
radical scavenger is well established [2], being effective in
protecting DNA, membrane lipids and some cytosolic
proteins. The effects of melatonin in several diseases at
clinical level are reported in recent reviews. Some of these
diseases are cardiovascular disorders [3], diabetes [4],
Alzheimer’s disease [5], Aids [6] and cancer [7]. However
the precise mechanisms underlying its effect are not well
established and studies for this purpose are in progress. It
would be possible that the membrane action of melatonin
could be one of the mechanisms responsible for its
beneficial effects. Despite its importance, a limited number
of studies are available in the literature about the
interaction of melatonin with membranes at molecular
level [8-9]. They mainly report the effect of melatonin on
membrane dynamics, which are not always consistent with
each other. In order to better understand the function of
melatonin at molecular level, it is important to study its
interaction with membrane components and specifically
with lipids. Negatively charged phospholipids are present
in all biological membranes in a fraction ranging from 5 to
1
25 molar percent. Within them, phosphatidylglycerol is
abundant in the plasma membrane of microorganisms,
choloroplast membranes in plants and to a lower extent in
mammalian cells. Several structural and physicochemical
properties of vesicles formed by charged phospholipids are
strongly dependent on ionic composition of suspension
medium [10].
In the present study, we have investigated in detail
the interaction of melatonin with dipalmitoyl
phosphatidylglycerol (DPPG), which contains an anionic
(phosphate) head group, model membrane using Fourier
transform
infrared
(FTIR)
spectroscopy.
FTIR
spectroscopy was used to monitor subtle changes in the
structure and function of the lipid assemblies by analyzing
the frequency, the bandwidth changes of the different
vibrational modes representing the acyl chains, interfacial
region and the head group region of lipid molecules. For
this reason in addition to membrane dynamics, this
teqnique allowed us to obtain other physical properties of
binary mixtures of melatonin and phospholipid
membranes. We investigated the effects of melatonin on
lipid phase transition, membrane acyl chain order,
hydration state of head group and glycerol backbone
region.
2 Materials and Methods
Melatonin and DPPG were purchased from Sigma (St.
Louis, MO, USA) and used without further purification.
number of gauche conformers [11-12]. Furthermore the
bandwidths of the CH2 stretching bands give dynamic
information about the system [11, 13].
Figure 1. shows the temperature depence of the frequency
of the CH2 antisymmetric stretching bands of DPPG MLVs
in the presence and absence of low ( 1 mol % ) and high (
15 mol % ) concentration of melatonin. In the curve of
DPPG MLVs the frequency values at temperatures below
32 0C are characteristic of conformatially highly ordered
acyl chains with a high content of trans isomers as found
in solid hydrocarbons, whereas, the values at temperatures
above 45 0C are characteristic of conformatially disordered
acyl chains. with a high content of gauche conformers as
those found in liquid hydrocarbons. The pretransition
occurs around 35 0C and the abrupt shift in the peak
frequency of the CH2 stretching modes of DPPG, which
takes place during the main endothermic phase transition
CH2 ANTISYMMETRIC STRETCHING
2925
WAVENUMBER (cm -1)
For the infrared measurements, pure phospholipid
MLVs were prepared according to the procedure, reported
by Toyran and Severcan (2003) [11]. To prepare DPPG
MLVs, 5 mg of phospholipid were dissolved in chloroform
in a round-bottomed flask. A dried lipid film was obtained
by evaporating it with a nitrogen flux and then pumping it
for at least 2 h under vacuum by using Heto spin vac. The
film was hydrated by adding 25  l of 10 mM phosphate
buffer, pH 7.4. Liposomes were formed by vortexing the
mixture at a temperature above the gel-to-fluid phase
transition for 20 min. In order to prepare melatonin
containing liposomes, appropriate amount of melatonin
was taken from the stock solution, in which melatonin was
dissolved in ethanol, and put in a round-bottomed flask.
The excess ethanol was evaporated by nitrogen stream and
then 5 mg of DPPG were added and dissolved in the same
round-bottomed flask by chloroform. The same procedure
for the preparation of pure DPPG liposomes was then
followed. Sample suspensions of 20  l were placed
between CaF2 windows with the cell thickness of 12  m.
Infrared spectra were obtained using a Spectrum 1 Perkin
Elmer FTIR spectrometer equipped with a DTGS detector.
Interferograms were averaged for 50 scans at 2 cm-1
resolution. Temperature was regulated by a Graseby
Specac digital temperature controller unit. The samples
were incubated for 10 min at each temperature before data
acquisition. Samples were scanned between 25 and 47 0C
with 2 0C intervals, and between 50 and 70 0C with 5 0C
intervals.
2924
2923
2922
2921
dppg
% 1 mol mel.
%15 mol mel.
2920
2919
2918
2917
25
3 Results and Discussion
The infrared spectra of DPPG MLVs, both pure and
containing different concentration of melatonin (1 mol %
and 15 mol %), were investigated as a function of
temperature. The C-H stretching modes at 2800-3000 cm-1,
C=O stretching mode at 1735 cm-1 and PO2
antisymmetric stretching double bands at 1220-1240 cm-1
were considered. All experiments were repeated three
times and similar trend were observed at each repeat.
Various kinds of information can be derived from
these bands. Frequency shifts in different regions or
changes in the widths of corresponding peaks can be used
to extract information about various physicochemical
processes taking place in the systems. For example, the
frequencies of the CH2 stretching bands of acyl chains
depend on the degree of conformational disorder and
hence the frequency values can be used to monitor the
average trans/gauche isomerization in the systems. The
shifts to higher wavenumbers correspond to an increase in
2
30
35
40
45
50
55
60
65
0
TEMPERATURE ( C)
Fig. 1. Temperature-dependent variation in the frequency
of the CH2 antisymmetric stretching mode of DPPG MLVs
in the presence and absence of melatonin.
(~ 41 0C), has been associated with the change from all
trans to gauche conformers [14]. As seen from the figure,
with the addition of 1 mol % concentration of melatonin
into the DPPG MLVs, the shape of phase transition curve
does not change and no significant shift for the midpoint
temperature of phase transition curve is observed. The
effect of high concentration (15 mol %) of melatonin on
the thermotropic phase transition is different than lower
melatonin concentration. The main phase transition
temperature shifts to lower values without affecting the
general shape of transition profile. At temperature ranges
corresponding to the gel phase (< Tm), no significant
change is observed in the frequency of the CH2 stretching
band with the addition of 1 mol % melatonin. This
70
-1
BANDWIDTH (cm )
14
CH2 ANTISYMMETRIC STRETCHING
C=O STRETCHING
dppg
1736,5
-1
WAVENUMBER (cm )
indicates that, low melatonin concentration has a
negligible effect on the order of DPPG MLVs both in the
gel and liquid crystalline phase. However, inclusion of 15
mol % melatonin increases the frequency in the liquid
crystalline phase which indicates an increase in the number
of gauche conformers. The increase in the number of
gauche conformers implies a decrease in the order of
bilayer [12, 13]. Similar effects were also observed for the
CH2 symmetric stretching band (not shown).
13
% 1 mol mel.
1736
% 15 mol mel.
1735,5
1735
1734,5
1734
1733,5
1733
25
12
11
dppg
% 1 mol mel.
% 15 mol mel.
10
9
8
7
6
25
30
35
40
45
50
55
60
65
70
0
TEMPERATURE ( C)
40
45
50
55
60
65
70
TEMPERATURE ( C)
Fig. 3. Temperature dependence of the frequency of the
C=O stretching mode of DPPG MLVs in the presence and
absence of melatonin.
which indicates that melatonin causes a strong hydrogen
bonding. The hydrogen bonding occurs in between the
C=O groups of DPPG and either with the hydroxyl group
of melatonin or with water molecules in the environment
[12].
PO2- ANTISYMMETRIC STRETCHING
WAVENUMBER (cm )
1222
-1
3
35
0
Fig. 2. Temperature dependence of the bandwidth of the
CH2 antisymmetric stretching mode of DPPG MLVs in the
presence and absence of melatonin.
Figure 2. shows the temperature dependence of the
bandwidth of the CH2 antisymmetric stretching band of
DPPG MLVs in the absence and 1 mol % and 15 mol %
melatonin concentrations. Bandwidth was measured at
0.75 × peak height position. As seen from the figure, low
melatonin concentration does not induce any effect while
high concentration of melatonin slightly increases the
dynamics.
One of the most useful infrared band for probing the
polar part of the membrane is that of band due to the ester
group vibrations at 1730 cm-1 (C=O stretching).
Temperature dependence of the frequency of the C=O
stretching modes of DPPG multibilayers in the absence
and presence melatonin is shown in Figure 3. As seen from
the figure, a dramatic decrease in the frequency, in
comparison to that of DPPG, is observed in the presence of
melatonin, both in the gel and liquid crystalline phase,
30
1221
dppg
1220
% 1 mol mel.
1219
% 15 mol mel.
1218
1217
1216
1215
25
30
35
40
45
50
55
60
65
70
0
TEMPERATURE ( C)
Fig. 4. Temperature dependence of the PO 2 antisymmetric
double stretching mode frequencies of DPPG MLVs in the
presence and absence of melatonin.
The other band for probing directly the head group of
DPPG is PO2 antisymmetric double stretching band
which is located at 1260 cm-1. As seen from Figure 4. the
frequency of this band also shifts to lower values with the
addition of melatonin into DPPG MLVs which indicates
hydrogen bonding of the phosphate group either with
melatonin or water molecules.
In the current study we found that addition of
melatonin into the DPPG membrane system eliminates
pretransition, and shifts the main transition (T m) to lower
temperatures. Previously Saija et al. [15] and Severcan et
al. [16] also reported a decrease in Tm in the presence of
melatonin, in zwitterionic model membranes. Studies on
the effect of melatonin on membrane dynamics are very
limited and these studies were not always consistent with
each other [2, 8, 9, 15, 16].These spin label ESR,
fluorescence, UV, FTIR spectroscopic and DSC
calorimetric studies used rat microsomal membranes and
different type of model membranes composed of
dimyristoyl
phosphatidylcholine
(DMPC)
and
dipalymitoyl phosphatidylcholine(DPPC) in the form of
multilamellar vesicles, unilamellar vesicles, reversed
micelles. In the current study we observed an increase in
membrane dynamics as melatonin concentration was
increased. This is in agreement with our previous study
where we studied melatonin-zwitterionic model membrane
interactions. In that referred study we also observed a
significant increase in membrane dynamics in the presence
of melatonin [15]. The result of the current study related to
membrane dynamics is also in agreement with previous
DSC study [15]. However the results of present study
shows that the effect of melatonin is seen to be less
profound in DPPG in comparison to DPPC membranes.
In addition to the order and dynamics studies of
the acyl chains, we also investigated the interfacial region
and polar head group of the DPPG MLVs. At 15 mol %
melatonin concentration (Figs. 3, 4), the C=O and PO2
functional groups of the lipid ester groups shift to a lower
frequency values. This result indicates a greater hydration
of the carbonyl groups resulting in an increase in the
number of H-bonded carbonyls. It may also reflect an
increase in H-bonding between the carbonyl groups and
hydroxyl groups of the melatonin. The strong hydrogen
bonding induced melatonin at the carbonyl and phosphate
groups in DPPG membranes suggests that melatonin
positions itself in the bilayer with a prefential location in
the interfacial region. However, due to the interaction of
strong hydrogen bonding, it may also significantly change
lipid acyl chain flexibility and lipid dynamics, as reported
previously for melatonin/ neutral phospholipid systems
[16].
4 Conclusion
In the present study we have investigated for the first time
the effect of melatonin on the phase transitions profile,
4
lipid order and dynamics and hydration states of the head
group and the region near the head group of anionic DPPG
MLVs as a function of temperature and at low and high
melatonin concentration. The results revealed that
melatonin alters physical properties of DPPG membranes.
However its effect is seen to be less profound in
comparison to melatonin-DPPC interactions [16].
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