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from Aldrich Chemicals (Steinheim, Germany) or from Fluka
Chemie AG (Buchs, Switzerland). Cholesterol and myristic
acid were obtained from Sigma Chemicals (Deisenhofen,
Germany) and GPC, sn-glycero-3-phosphatidylcholine, from
Lukas Meyer (Hamburg, Germany). Octadec-14-ynoic acid,
octadec-9-ynoic acid and octadec-14-ynoic acid, deuterated at
position C-13, have been synthesized, as described earlier.17
The methyl ester of octadec-14-ynoic acid was obtained by the
reaction of the corresponding acid with CH N , according to
temperature spectra was measured twice for each sample. The
data analysis is thus based on an average of six measurements.
IR data analysis
For the analysis of the CH wagging band region the Bruker
2
IFS 66 spectrometer software (Opus 3.0) was used. In the case
of the phospholipid model membranes the spectral region of
interest (1315 to 1400 cm~1) was Ðrst Ñattened by a linear
baseline. Afterwards, the spectra were iterated using Ðve vibra-
tion bands with Gaussian and Lorentzian contributions of
variable amount. Their initial positions were 1378 cm~1
(symmetric methyl deformation mode), 1368 cm~1 (kink and
gtg sequences), 1354 cm~1 (double gauche sequences), 1342
cm~1 (end gauche sequences) and 1330 cm~1. During the
least-squares Ðt analysis the band intensities and widths were
varied independently. Variation of the band positions on the
other hand was restricted to ^ 2 cm~1 of the values given
2
2
standard procedures.
For the preparation of the phospholipids 1,2-dioctadec-(14-
ynoyl)-sn-glycero-3-phosphatidylcholine [DO(14-yne)PC] and
1,2-dioctadec-(9-ynoyl)-sn-glycero-3-phosphatidylcholine
[DO(9-yne)PC] a previously published preparation method
was used.15,16 Thus, the desired octadecynoic acid (1 g, 3.57
mmol), dissolved in 40 ml of dry chloroform, was Ðrst trans-
formed into its imidazolide by addition of N,N-carbon-
yldiimidazole (630 mg, 3.89 mmol) and stirring at room
temperature for about 45 min. Afterwards, carefully dried sn-
glycero-3-phosphatidylcholine (335 mg, 1.3 mmol) and 1,8-
diaza-bicyclo[5.4.0]undec-7-ene (543 mg, 3.57 mmol) were co-
added. After further stirring at room temperature for at least
24 h, the solvent was removed. DO(14-yne)PC and DO(9-yne)
PC of high purity were obtained from the residue by rec-
rystallization in acetone. 1,2-Dimyristoyl-sn-glycero-3-phos-
phatidylcholine (DMPC) was obtained in an analogous way
by using myristic acid instead of octadecynoic acid.
above. The integrated intensities of the CH wagging bands
2
were furthermore normalized with respect to the methyl defor-
mation band. The amount of speciÐc gauche sequences were
calculated according to the procedure given by Senak et al.11
that is based on reference measurements on n-alkanes and a
theoretical approach using the rotational isomeric state (RIS)
model.20 Care was taken over the fact that the phospholipids
contain only one methyl group per fatty acid chain in contrast
to two methyl groups in n-alkanes. The total number of
gauche conformers per chain is obtained by taking into
account that two gauche bonds are necessary to build up one
kink/gtg or double gauche sequence. The estimated uncer-
tainty for the various conformers is 10 to 15%.
To obtain the corresponding phosphatidyl acid, 1,2-
dioctadec-(14-ynoyl)-sn-glycero-3-phosphatidyl acid [DO(14-
yne)PA], the choline head group was split o† by the enzyme
phospholipase D (PLDP), employing the following procedure:
300 mg (0.38 mmol) DO(14-yne)PC were dissolved in 10 ml
chloroform and heated to 45 ¡C. Subsequently, 5 ml of a buf-
The experimental data from the measurements on octadec-
14-ynoic acid, dissolved in
a small amount of tetra-
chloromethane, were treated slightly di†erent. At Ðrst, the
vibrational bands arising from the solvent were eliminated by
fered solution (0.1 M NaAc, 0.1 M CaCl , 0.01 M EDTA at
2
pH 5.6) containing the enzyme were co-added. The biphasic
the subtraction of a pure CCl spectrum recorded under the
reaction mixture was stirred vigorously for about 2È3 h. By
4
same experimental conditions. This was followed by a further
addition of 50 ml of Folch solution (CHCl /MeOH \ 2 : 1)
3
linear baseline correction. The Ðnal determination of the band
intensities was done in the same way as described above.
the reaction was stopped. The product was separated from the
organic phase and further recrystallized in acetone.
For the analysis of the CH wagging progressions,8,9 sub-
2
traction of the underlying PO ~ antisymmetric stretching
2
Sample preparation
band near 1230 cm~1 was required. For this reason, an IR
Multilamellar dispersions (steroid-free samples) were prepared
by the hydration of 10 mg of dry lipid in about 15 ml
deuterium-depleted water. Homogenization was obtained by
repeated freezeÈthawing, centrifuging and vortexing of the
sample. The construction of the IR cell excluded a loss of
water and thus incomplete hydration. The cholesterol contain-
ing samples were prepared by dissolving the appropriate
phospholipid and cholesterol (3 : 2 molar ratio) in a small
amount of freshly distilled chloroform. The solvent was par-
tially removed by a stream of nitrogen gas, and the residual
solvent was evaporated under vacuum for a few hours. After
this procedure the dry mixture was dispersed in deuterium-
depleted water in the same way, as described above. Liquid
alkynes were used without any further treatment and octadec-
14-ynoic acid was dissolved in a small amount of tetra-
chloromethane.
spectrum at the highest available temperature in the liquid
crystalline phase (typically at 353 K) was recorded and sub-
tracted from the spectrum acquired at the other temperatures.
Afterwards, a Ñattened baseline was generated in the spectral
region of interest by selecting appropriate spectral minima. All
progression intensitiesÈa sum of the k \ 1 to k \ 4
componentsÈwere further normalized with respect to the
progression band intensity obtained for the pure
phospholipid/water dispersions at 278 K. Di†erences in the
PO ~ antisymmetric stretching band region caused by the
2
addition of cholesterol have been taken into account by com-
parison with the IR spectrum at 353 K. Further details about
the actual procedures can be found in refs. 8 and 9.
The frequencies of the CH symmetric stretching vibrations
2
were determined from the interpolated zero crossings in the
Ðrst derivative spectra.
IR measurements
Di†erential scanning calorimetry (DSC)
Both phospholipid dispersions and reference alkynes were
The phase behaviour of the various samples was established
by di†erential scanning calorimetry. Typically a temperature
range of 200 to 350 K at heating rates of 2 K min~1 or 5 K
min~1 was examined using a Netzsch DSC 204 calorimeter
(Selb, Germany).
placed in 25 or 50 lm thick infrared cells with CaF windows.
2
The IR cell was thermostatted with a variable temperature
unit from LOT/Oriel (Langenberg, Germany). All IR spectra
were recorded with a Bruker IFS 66 FT IR spectrometer
(Karlsruhe, Germany). Typically 32 to 128 interferograms
were collected, apodized with a triangular function and
Fourier transformed with one level of zero-Ðlling. The spectral
resolution was 2 cm~1. For each system three samples of the
same composition were prepared. The whole series of variable
Results and discussion
In the following we present the results from a variable tem-
perature FTIR study of model membranes based on the phos-
4834
Phys. Chem. Chem. Phys., 1999, 1, 4833È4841
Wolfangel, Peter
