Configuration of polyisoprenoids affects the permeability and thermotropic properties of phospholipid/polyisoprenoid model membranes

Article abstract of DOI:10.1016/j.chemphyslip.2011.03.004

The influence of α-cis- and α-trans-polyprenols on the structure and properties of model membranes was analyzed. The interaction of Ficaprenol-12 (α-cis-Prenol-12, α-Z-Prenol-12) and Alloprenol-12 (α-trans-Prenol-12, α-E-Prenol-12) with model membranes was compared using high performance liquid chromatography (HPLC), differential scanning calorimetry (DSC) and fluorescent methods. l-α-Phosphatidylcholine from egg yolk (EYPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as the main lipid components of unilamellar (SUVs) and multilamellar (MLVs) vesicles were used. The two-step extraction procedure (n-pentane and hexane, respectively) allowed to separately analyze the fractions of polyprenol as non-incorporated (Prenol_NonInc) and incorporated (Prenol _Inc) into liposomes. Consequently, distribution coefficients, P′, describing the equilibrium of prenol content between phospholipid (EYPC) membrane and the aqueous phase gave different log P′ for α-cis- and α-trans-Prenol-12, indicating that the configuration of the α-terminal residue significantly alters the hydrophobicity of the polyisoprenoid molecule and consequently the affinity of polyprenols for EYPC membrane. In fluorescence experiments α-trans-Pren-12 increased up to 1.7-fold the permeability of EYPC bilayer for glucose while the effect of α-cis-Pren-12 was almost negligible. Considerable changes of thermotropic behavior of DPPC membranes in the presence of both prenol isomers were observed. α-trans-Pren-12 completely abolished the pretransition while in the case of α-cis-Pren-12 it was noticeably reduced. Furthermore, for both prenol isomers, the temperature of the main phase transition (T_m) was shifted by about 1 °C to lower values and the height of the peak was significantly reduced. The DSC analysis profiles also showed a new peak at 38.7 °C, which may suggest the concomitant presence of more that one phase within the membrane. Results of these experiments and the concomitant occurrence of alloprenols and ficaprenols in plant tissues suggest that cis/trans isomerization of the α-residue of polyisoprenoid molecule might comprise a putative mechanism responsible for modulation of the permeability of cellular membranes.

Full text of DOI:10.1016/j.chemphyslip.2011.03.004

Chemistry and Physics of Lipids 164 (2011) 300–306
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Configuration of polyisoprenoids affects the permeability and thermotropic
properties of phospholipid/polyisoprenoid model membranes
Ewa Ciepichala,1,2, Malgorzata Jemiola-Rzeminskab,1,3, Jozefina Hertela,4, Ewa Swiezewskaa,∗
,
Kazimierz Strzalkab,∗∗
a Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland
b Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The influence of ␣-cis- and ␣-trans-polyprenols on the structure and properties of model membranes
was analyzed. The interaction of Ficaprenol-12 (␣-cis-Prenol-12, ␣-Z-Prenol-12) and Alloprenol-12
(␣-trans-Prenol-12, ␣-E-Prenol-12) with model membranes was compared using high performance
liquid chromatography (HPLC), differential scanning calorimetry (DSC) and fluorescent methods. l-␣-
Phosphatidylcholine from egg yolk (EYPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as
the main lipid components of unilamellar (SUVs) and multilamellar (MLVs) vesicles were used. The
two-step extraction procedure (n-pentane and hexane, respectively) allowed to separately analyze the
fractions of polyprenol as non-incorporated (PrenolNonInc) and incorporated (PrenolInc) into liposomes.
Consequently, distribution coefficients, Pꢀ, describing the equilibrium of prenol content between phos-
pholipid (EYPC) membrane and the aqueous phase gave different log Pꢀ for ␣-cis- and ␣-trans-Prenol-12,
indicating that the configuration of the ␣-terminal residue significantly alters the hydrophobicity of the
polyisoprenoid molecule and consequently the affinity of polyprenols for EYPC membrane. In fluores-
cence experiments ␣-trans-Pren-12 increased up to 1.7-fold the permeability of EYPC bilayer for glucose
while the effect of ␣-cis-Pren-12 was almost negligible. Considerable changes of thermotropic behavior
of DPPC membranes in the presence of both prenol isomers were observed. ␣-trans-Pren-12 completely
abolished the pretransition while in the case of ␣-cis-Pren-12 it was noticeably reduced. Furthermore,
for both prenol isomers, the temperature of the main phase transition (Tm) was shifted by about 1 ◦C to
lower values and the height of the peak was significantly reduced. The DSC analysis profiles also showed
a new peak at 38.7 ◦C, which may suggest the concomitant presence of more that one phase within the
membrane.
Received 14 October 2010
Received in revised form 31 January 2011
Accepted 16 March 2011
Available online 1 April 2011
Keywords:
Polyprenol
Alloprenol
cis/trans isomerization
Liposome
Membrane permeability
Membrane thermotropic properties
Results of these experiments and the concomitant occurrence of alloprenols and ficaprenols in plant
tissues suggest that cis/trans isomerization of the ␣-residue of polyisoprenoid molecule might comprise
a putative mechanism responsible for modulation of the permeability of cellular membranes.
© 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Polyprenols are a well known subgroup of polyisoprenoid alco-
hols described already in the 1960s (Stone et al., 1967). The
highest accumulation of polyprenols has been noted in plant pho-
tosynthetic tissues, but they have also been detected in wood,
seeds, flowers and in bacterial cells (reviewed in Swiezewska
and Danikiewicz, 2005). Polyisoprenoid alcohol chains are built of
5–100 and more isoprenoid units creating polymers that differ in
the chain-length and/or geometrical configuration. With respect
to the structure, the hydrocarbon chain of polyprenols is built
of an ␻-terminal isoprenoid residue followed by 2 or 3 internal
trans residues and a stretch of cis residues; typical polyprenols –
ficaprenols are finally decorated with an ␣-cis-terminal residue
(Fig. 1A). In 2007 our group described a new type of polyisoprenoid
alcohols of plant origin – alloprenols (Fig. 1B) (Ciepichal et al., 2007).
Abbreviations: Pren-12, polyprenol composed of 12 isoprene units; PrenInc,
incorporated prenol; PrenNonInc, non-incorporated prenol.
∗
Corresponding author at: Dept. of Lipid Biochemistry, Institute of Biochem-
istry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw,
Poland. Tel.: +48 22 592 35 10; fax: +48 22 592 21 90.
∗∗
Corresponding author. Tel.: +48 12 664 65 09; fax: +48 12 664 69 02.
E-mail addresses: eciepichal@ibb.waw.pl (E. Ciepichal), MJR@mol.uj.edu.pl
(M. Jemiola-Rzeminska), hertel@ibb.waw.pl (J. Hertel), ewas@ibb.waw.pl
(E. Swiezewska), strzalka@mol.uj.edu.pl (K. Strzalka).
1
These authors contributed equally to the manuscript.
Tel.: +48 22 592 35 09.
Tel.: +48 12 664 65 37.
Tel.: +48 22 592 35 01.
2
3
4
0009-3084/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2011.03.004

E. Ciepichal et al. / Chemistry and Physics of Lipids 164 (2011) 300–306
301
Fig. 1. Structure of A/␣-cis-polyprenol, B/␣-trans-polyprenol, C/dolichol; t and c indicate the number of internal trans- and cis-isoprenoid residues, respectively; ␻, ␣ and s
stand for ␻-, ␣-terminal and saturated isoprenoid residues, respectively.
In contrast to typical ␣-cis polyprenols (e.g. ficaprenols), alloprenols
posses the ␣-terminal residue in a trans configuration. Interest-
ingly, the proportion of the amounts of ␣-cis vs. ␣-trans polyprenols
is 1 to 1 in the leaves of Allophylus caudatus and eleven other plant
species (Marczewski et al., 2007). It should be also mentioned that
␣-dihydro-polyprenols (syn. dolichols, Fig. 1C) are common con-
stituents of animal and yeast cells.
trans-prenols into unilamellar phospholipid vesicles was exam-
ined using quantitative HPLC-UV analysis following the extraction
procedure. The effect of polyisoprenoid alcohols on the perme-
ability of model lipid bilayers for glucose, as an example of a
low-molecular-weight electrically neutral metabolite, and for pro-
tons was monitored by using of fluorescence probes while the
influence of polyprenols on thermotropic properties of phospho-
lipid bilayers was followed using differential scanning calorimetry
(DSC). Our results indicate that configuration of the polyisoprenoid
chain affects the behavior of polyprenols in the bilayer. The occur-
rence of the additional ␣-trans double bond results in a decreased
degree of incorporation of polyprenols into the membrane and at
the certain membrane concentration it also results in its enhanced
permeability. Interestingly, such a configurational change seems
not to be crucial for the formation of lipid domains within the
membrane.
In contrast to polyisoprenoid phosphates, functioning as cofac-
tors in the biosynthesis of bacterial peptidoglycan and eukaryotic
glycoproteins and substrates for protein prenylation (Gutkowska
et al., 2004), the role of free polyisoprenoid alcohols is still
uncertain. High hydrophobicity of polyisoprenoids causes theirs
localization in cellular membranes, e.g. mitochondria, chloroplast
envelopes, Golgi membranes (Swiezewska et al., 1993). However,
explanation of the presence of polyisoprenoid molecules within
the biological membranes has remained for many years a ques-
tion of debate due to their length which exceeds the thickness of
the bilayer. The first molecular model of a dolichol (Murgolo et al.,
2. Materials and methods
˚
1989) suggested the dimensions of Dolichol-19 (Dol-19) as 53.07 A
˚
(length) × 30.94 A (width), and the molecule was proposed to con-
2.1. Chemicals
sist of three geometrical regions, a central coiled segment and two
flanking regions. According to the recent model the dimensions
l-␣-Phosphatidylcholine from egg yolk (EYPC) and 1,2-
dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased
from Sigma–Aldrich (St. Louis, MO, USA). Prenol-12 – natural
␣-cis-isomer isolated from the leaves of Magnolia kobus was
from the Collection of Polyprenols (Institute of Biochemistry and
Biophysics, Polish Academy of Sciences, Warsaw, Poland). All sol-
vents of spectral purity and chromatographic materials were from
Merck (Darmstadt, Germany). 6-Carboxyfluorescein and valino-
mycin were from Sigma–Aldrich and pyranine was from Molecular
Probes (Eugene, USA). All other chemicals were purchased from
Sigma–Aldrich and were of p.a. grade.
˚
˚
of Dol-19 were decreased to 31.87 A (length) × 15.41 A (width).
These findings also revealed that the 3D conformations of Dol-19,
Dol-19P (Dolichyl-19-Monophosphate), and Pren-11P (Prenyl-11-
Monophosphate) were nearly identical with their three coiled,
helical domains (Zhou and Troy, 2003) arranged as a central seg-
ment and two flanking arms.
Such a model explains well the possible orientation of the
mainly-cis polyisoprenoid alcohols in model membranes (Zhou
and Troy, 2005). It is also in line with the earlier observations
showing that polyprenols, dolichols and their phosphorylated
derivatives alter the structure of the phospholipid bilayer by pro-
moting the formation of a nonlamellar (inverted hexagonal, Hex
II) structure. These structural changes explained well the effect of
polyisoprenoids on the increase of the fluidity, permeability and
fusiogenicity of the membranes observed earlier (Chojnacki and
Dallner, 1988 and references therein). It has been reported (voltam-
metric methods) that incorporation of polyprenols into a model
lipid membrane (dioleoylphosphatidylcholine, DOPC) decreased
the activation energy and increased membrane conductance and
membrane permeability coefficient (Janas et al., 2000). However, in
all the studies only typical polyisoprenoids with di-trans-poly-cis or
tri-trans-poly-cis structure were taken into consideration and the
effect of discrete structural changes (configuration of the ␣-trans-
residue within the prenol molecule) has not been studied yet. This
question seems intriguing in the light of the undisclosed biological
function of alloprenols.
2.2. Synthesis of ˛-trans-Pren-12 (Alloprenol-12)
Aldehyde of Prenol-12 was prepared as earlier (Ciepichal et al.,
2007) with some modifications. Briefly: 60 mg of Polyprenol-12 in
1 ml of dichloromethane was stirred with 150 mg of pyridinium
chlorochromate for 20 min at room temperature. Aldehyde was
isolated by column chromatography (Florisil, dichloromethane).
Fractions containing aldehyde (55 mg, mixture of ␣-cis/trans alde-
hydes, 9:1 ratio) were pooled, evaporated under nitrogen (30 ◦C)
and dissolved in 3 ml of methanol. Enhanced isomerization of the
␣-unit of aldehyde was achieved by the incubation of Prenal-12 in
the presence of sodium carbonate (molar ratio of aldehyde:sodium
carbonate, 1:15) at room temperature for 16 h (60% conversion).
Further reduction of Prenal-12 (mixture of ␣-cis/trans aldehydes)
with sodium borohydride (molar ratio of aldehyde:sodium borohy-
dride, 2:1) in the same solution (40 min, room temperature) yielded
a mixture of ␣-cis- and ␣-trans-Pren-12 (99% conversion). Mix-
ture of ␣-cis- and ␣-trans-Pren-12 was extracted (hexane:water;
1:1), concentrated and products were separated by column chro-
matography (silica gel 60 column, linear gradient of diethyl ether
in hexane from 0 to 10%). Each step of preparation of ␣-trans-Pren-
12 was followed by TLC (silica gel plates, ethyl acetate:toluene, 1:9
In this work the influence of ␣-cis-Prenol-12 and ␣-trans-
Prenol-12 (Alloprenol-12), occurring concomitantly in plant cell
membranes, on the properties of the phospholipid bilayer was ana-
lyzed. As model membranes unilamellar (SUVs) and multilamellar
(MLVs) vesicles from natural (l-␣-phosphatidylcholine, EYPC)
or synthetic (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC)
lecithin, were prepared. Efficiency of incorporation of ␣-cis- and

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E. Ciepichal et al. / Chemistry and Physics of Lipids 164 (2011) 300–306
Table 1
Distribution of ␣-cis-Pren-12 and ␣-trans-Pren-12 between unilamellar EYPC liposomes and aqueous phase. The distribution coefficient Pꢀ = mPrenInc/mPrenNonInc. Pren-12
content in the initial lipid mixture was within the range from 10 to 100 mol%, which corresponds to polyprenol:EYPC molar ratio in the range from 1:10 to 1:1. The
quantitative evaluation of the amount of prenols, PrenInc and PrenNonInc, was assayed by means of HPLC analysis on the basis of integrated peak areas from HPLC elution
profiles. Data are means of three independent experiments SD. Abbreviation: PrenInc, incorporated prenol; PrenNonInc, non-incorporated prenol.
Pren-12 content mol%
10
20
30
log Pꢀ
40
50
100
␣-cis-Pren-12
␣-trans-Pren-12
0.16 0.02
−0.66 0.03
0.31 0.03
−0.79 0.01
0.19 0.02
−0.68 0.03
−0.01 0.03
−0.61 0.08
0.00 0.02
−0.72 0.04
0.20 0.09
−0.75 0.02
(v/v), stained with iodine vapors). The purity of ␣-trans-Pren-12
was finally confirmed using HPLC/Photodiode Array detector with
a dual pump apparatus (Waters Ass., USA) using a Hypersil ODS
reversed-phase column (Agilent, USA) (4.6 mm × 60 mm, particle
size 3 ␮m) as described earlier (Skorupinska-Tudek et al., 2003).
The structure of thus obtained ␣-trans-Pren-12 was confirmed by
HPLC/ESI-MS (Pren-12 with a molecular formula C60H98O gave a
pseudomolecular ion peak m/z 857.7 ([M+Na]+)) and by means of
1H and 13C NMR as previously described (Ciepichal et al., 2007).
or ␣-trans-Pren-12 was within the range of 1–25 mol%. The sample
was vortexed for 5–10 min and the vesicles were subjected to five
cycles of the freeze–thaw procedure (Mayer et al., 1985). Obtained
multilamellar vesicles were converted to unilamellar vesicles by
a high pressure extrusion technique (Olson et al., 1979). Each
sample was extruded seven times through a double polycarbon-
ate filter, pore size 200 nm, using a Liposofast Basic Equipment
(Avestin, Ottawa, Canada). To eliminate proton gradient, valino-
mycin (1.3 mM) was added and the vesicles were finally incubated
overnight at 4 ◦C (Clement and Gould, 1981). Gel exclusion chro-
matography on a G-75 Sephadex column (0.5 cm2 × 15 cm) was
performed in order to separate unilamellar vesicles from non-
trapped fluorescence dyes. The liposomal preparation was divided
into three fractions of 1 ml each and these aliquots were subse-
quently used for the permeability experiments.
Proton permeability was measured upon addition of 66 ␮l 0.1 M
HCl to the vesicle suspension, with instant mixing. The permeation
of protons across the membrane was recorded as a decrease in
the fluorescence of pyranine monitored at 508 nm under 460 nm
excitation.
Glucose permeability was measured upon addition of 1 ml 1.6 M
glucose to 1 ml vesicle suspension. The increase in fluorescence was
monitored at 515 nm under 494 nm excitation.
2.3. HPLC analysis of the capacity of liposomes for ˛-cis- and
˛-trans-Pren-12
Small unilamellar vesicles (SUVs) were prepared at room tem-
perature by the injection method (Kruk et al., 1997). The evaporated
lipid mixture of egg yolk phosphatidylcholine supplemented with
either ␣-cis- or ˛-trans-Pren-12 was kept under vacuum for 2–3 h,
dissolved in ethanol and injected under continuous stirring into
10 mM Hepes buffer (pH 7.7) containing 50 mM KCl and 1 mM
EDTA. The final EYPC concentration was 0.5 mM, ethanol concen-
tration of 1.25% and polyprenol content in the lipid mixture in the
range of 2–100 mol%, which corresponds to the polyprenol:EYPC
molar ratio in the range of 1:50–1:1.
Carboxyfluorescein was used in the glucose permeability exper-
iments as a fluorescence marker due to its self-quenching effect at
high concentrations. Upon addition of glucose to the vesicle sus-
pension an immediate shrinkage of the vesicles takes place as a
result of water efflux. Rapid increase in the concentration of car-
boxyfluorescein inside the vesicles is the reason of quenching and
decrease in the fluorescence intensity. When glucose gradually per-
meates through the lipid bilayer, concomitant with water, vesicles
regain their size due to the water influx and fluorescence is grad-
ually increased. The recorded constant value of the fluorescence
intensity is reached when vesicles recapture their original volume.
Relative permeability of the vesicles for glucose and protons was
calculated as described previously (Berglund et al., 2000; Berglund
et al., 2004), permeability coefficient for control (pure EYPC vesi-
cles) was set to 1.0.
Subsequently, a two-step extraction procedure was performed.
Non-incorporated polyisoprenoids (PrenolNonInc) were removed
with a brief n-pentane washing as described earlier (Degli Esposti
et al., 1981). The aqueous phase was then subjected to fur-
ther extraction according to the published method (Kröger, 1978)
with the subsequent modification (Jemioła-Rzemin´ ska et al.,
2001) aimed to solubilize liposomes and extract polyisoprenoid
alcohols localized in the membrane (PrenolInc). The resulting
organic fractions (n-pentane and hexane) were collected and
evaporated separately, the obtained residues were dissolved in
chloroform:methanol (2:1, v/v). HPLC analysis was performed as
described above. All these experimental steps were performed at
21 ◦C. Estimated amounts of Pren-12 were used for calculation of
the distribution coefficient Pꢀ = mPrenInc/mPrenNonInc thus higher neg-
ative values of log Pꢀ (Table 1) indicate elevated accessibility of
Pren-12 to pentane.
Fluorescence measurements were performed in a 1 cm quartz
cuvette at 25 ◦C using a Perkin Elmer LS 50B spectrofluorometer.
Measurements were performed in triplicate, values were calcu-
lated as mean SD.
Measurements were performed in triplicate, values were calcu-
lated as mean SD.
2.4. Permeability experiments – parallel assays for protons and
glucose
2.5. Differential scanning calorimetry (DSC) analysis
Glucose and proton permeability measurements were per-
formed in parallel using aliquots of the same batch of vesicles
according to the method published earlier (Berglund et al., 2004).
Previous experiments confirmed that there was no interference
between carboxyfluorescein and pyranine when present concur-
rently inside the vesicles (Berglund et al., 2002). In a test tube, to the
mixture of EYPC and either ␣-cis- or ␣-trans-Pren-12 dried under
N2, 0.5 ml of 10 mM HEPES–NaOH buffer (pH 7.7) containing 1 mM
EDTA, 50 mM KCl, 1 mM carboxyfluorescein and 2 mM pyranine
was added. The lecithin content was 5 mg and the content of ␣-cis-
The multilamellar vesicles (MLVs) were prepared from DPPC and
either ␣-cis- or ˛-trans-Pren-12 as described previously (Jemiola-
Rzeminska et al., 2002). The final DPPC concentration was 1 mM
and ␣-cis- or ˛-trans-Pren-12 content in the samples was in the
range of 1–50 mol%.
DSC experiments were performed using a CSC Model 6100 Nano
II differential scanning calorimeter (Calorimetry Sciences Corpora-
tion, USA). The sample cell was filled with about 400 ␮l of MLV
suspension and an equal volume of distilled water was used as a
reference. The data were collected in the range of 25–55 ◦C at the

E. Ciepichal et al. / Chemistry and Physics of Lipids 164 (2011) 300–306
303
Fig. 2. Incorporation of A/␣-cis-Pren-12 and B/˛-trans-Pren-12 into EYPC unilamellar vesicles. The quantitative evaluation of the amount of polyprenols was performed on
the basis of the integrated peak areas from HPLC elution profiles. Means of three independent experiments SD are shown.
brane and the aqueous phase, calculated as Pꢀ = mPrenInc/mPrenNonInc
(where m represents the amount (g) of prenol recovered from each
phase) gave the log Pꢀ mean value −0.70 0.06 for ␣-trans-Prenol-
12 (Table 1) indicating that the distribution of ␣-trans-Prenol-12 is
independent on the prenol concentration in the initial lipid mix-
ture. In contrast, the log Pꢀ values calculated for incorporation of
␣-cis-Prenol-12 into EYPC vesicles presented a complicated profile
with three distinct regions. Namely, for the lowest ␣-cis-Pren-12
concentration (below 30 mol%) almost linear increase of its con-
tent in the two phases was observed while for the middle values of
the ␣-cis-Pren-12 concentration (above 35 and below 50 mol%) its
amounts were almost equal in both phases. Surprisingly, strong
increase of incorporation of ␣-cis-Pren-12 into the lipid mem-
brane was observed at its highest concentrations (75 and 100 mol%)
(Table 1 and Fig. 2).
scan rate of 1 ◦C min−1 both for heating and cooling. Measurements
were performed in triplicate, representative graphs are presented.
3. Results
3.1. Incorporation of polyprenols into lipid bilayers
Concomitant occurrence of equal amounts of typical ␣-cis-
together with newly described ␣-trans-polyprenols in leaves of
some plants raised the question of the effect these latter poly-
isoprenoids wielded on lipid membranes. Because of the limited
availability of natural ␣-trans-Prenol-12, a suitable semi-synthetic
method was established, as described in Section 2. This approach
was based on the fact that alkaline treatment of the molecule
containing conjugated double bonds system (–C C–C O in the
aldehyde, Prenal-12) results in cis to trans isomerization.
The effect of the configuration of the ␣-isoprenoid residue on
the incorporation efficiency of polyprenols into the unilamellar
lecithin vesicles was analyzed. The two-step extraction procedure
followed by the HPLC analysis enabled us to compare the amount of
polyprenols incorporated into the lipid bilayer with that not accom-
modated within the membrane interior during vesicle formation.
As shown in Fig. 2A, the amount of both, ␣-cis- and ␣-trans-Pren-
12 incorporated into the liposomes increased with the increasing
Pren-12 to EYPC molar ratio. The incorporation of prenol towards
membranes upon such experimental conditions might be dis-
cussed as distribution between the phospholipid (EYPC) membrane
and the aqueous phase. Distribution coefficients describing the
equilibrium of prenol content between phospholipid (EYPC) mem-
3.2. Effect of polyprenols on the permeability of model
membranes
Literature data on the effect of dolichols and polyprenols on
induction of pore formation resulting in the increased permeabil-
ity of model membranes prompted us to measure the influence
of polyprenols on the permeability of the egg yolk lecithin mem-
brane using the fluorescence-based method (Berglund et al., 2002).
Simultaneous incorporation into the liposomal structure of two
fluorescent markers: carboxyfluorescein, due to its selfquenching
effect at high concentrations (Weinstein et al., 1981), and pyranine,
which is a pH dependent fluorescent probe, enabled parallel mea-
surements of vesicle permeability for glucose and protons. Control

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E. Ciepichal et al. / Chemistry and Physics of Lipids 164 (2011) 300–306
Fig. 3. The influence of ␣-cis-Pren-12 and ␣–trans-Pren-12 on the permeability of
model EYPC membranes for glucose. The data represent the means of three inde-
pendent experiments SD.
experiments revealed that incorporation of polyprenols into the
egg yolk lecithin bilayer did not change fluorescence parameters
such as the excitation or the emission wavelengths neither of car-
boxyfluorescein not pyranine. What is even more important, no
fluorescence quenching was observed in such system (not shown).
In Fig. 3 the effect of Pren-12 in ␣-cis- and ␣-trans- configura-
tion on the permeability of EYPC bilayer for glucose is presented.
While ␣-cis-Pren-12 induced almost no changes, the influence of
␣-trans-Pren-12 was strongly dependent on its content. At concen-
trations lower than 5 mol% it caused strong fluctuations and even
the decrease in permeability for glucose, and this effect was even
more pronounced for the highest concentrations (15 and 25 mol%).
Interestingly, for vesicles containing 7 mol% of ␣-trans-Pren-12 a
significant increase in the permeability, reaching 170% of the con-
trol value determined for EYPC model membrane was observed.
Pilot measurements of the permeability of EYPC bilayer for pro-
tons revealed increased values for liposomes containing higher
concentrations (7–25 mol%) of polyprenols (180% of control for
liposomes containing 7 mol% of ␣-trans-Pren-12). However, signif-
icant conclusions cannot be drawn from these preliminary results
because of the relatively broad dispersion of the measured val-
ues. Similarly, modest increase of permeability for protons resulted
from the incorporation of either all-trans-Pren-9 or ␣-cis-mainly-
trans-Pren-9 into EYPC liposomes (data not shown).
Fig. 4. DSC heating profiles obtained for multilamellar DPPC liposomes containing
variable amounts of A/␣-cis-Pren-12 and B/␣-trans-Pren-12. The arrow indicates a
new signal appearing at 38.7 ◦C. Scans were obtained at a heating rate of 1 ◦C min−1
and are representatives of the three independent runs.
The thermotropic behavior of the DPPC liposomes containing
polyprenols observed during the successive cooling scans (after
10 min of equilibration at 60 ◦C) in the temperature range from 60 to
3.3. The effect of Prenol-12 isomers on the thermotropic behavior
of DPPC membranes
20 ◦C showed the peak characteristic of the L␣ → P␤ transition (data
ꢀ
The high-sensitivity DSC heating profiles obtained for DPPC
dispersions in excess of water with increasing concentrations of
polyprenols are shown in Fig. 4. In the thermal range of 25–50 ◦C for
the pure DPPC multilamellar vesicles we found two thermotropic
phase transitions: a highly cooperative gel-to-liquid-crystal transi-
not shown). However, the transition pathways in heating and cool-
ing runs displayed significant hysteresis. The slightly lower excess
heat capacity values recorded during cooling may indicate that
molecular rearrangements have a substantially longer response
time. The pattern observed for cooling of the DPPC/polyprenol sys-
tems was essentially similar to the corresponding heating one with
a progressive broadening of main transition peak, its shift to the
lower temperatures as well as the occurrence of the additional
low-temperature peak at high polyprenol concentrations.
◦
−1
ꢀ
tion (PB → L␣) at 42.2 C with an enthalpy change of 8.5 kcal mol
and the so called pretransition at 34.6 ◦C with ꢀH of 0.9 kcal mol−1
ꢀ
ꢀ
(LB → PB ), which is consistent with the literature data (Koynova
and Caffrey, 1998). The presence of polyprenols significantly
altered the thermotropic behavior of DPPC. With the content as
low as 1 mol% of ␣-cis-Pren-12, the pretransition was considerably
reduced, while in the case of ␣-trans-Pren-12 it was completely
abolished and no longer detected for any higher polyprenol con-
centration.
4. Discussion
Polyprenols and dolichols are ubiquitous components of all liv-
ing cells. Their widespread distribution in biomembranes raises the
question of their physiological role as modulators of membrane
properties. The apparent increase in content of polyisoprenoid alco-
hols upon tissue senescence and during plant resistance response
to biotic stress (Bajda et al., 2009) prompted us to reinvestigate
the basis of the interaction between polyisoprenoid alcohols and
the lipid matrix. The way in which model membranes are per-
turbed by introduced polyprenols can be a useful indication of how
they interact with the lipid bilayer with a subsequent influence
on the membrane properties and relevance for a variety of pro-
Moreover, the temperature of the main phase transition (Tm
)
was shifted by about 1 ◦C to lower values and the height of the
peak was reduced significantly, the effects being more pronounced
for the highest (15 and 50 mol%) polyprenol concentrations. Upon
analysis of the DSC profiles depicted in Fig. 4, it is also clearly seen
that concurrently with a significant broadening of the main phase
transition peak and the disturbance of its symmetry, a new peak
appeared at 38.7 ◦C (for Pren-12 content higher than 5 mol%), which
may suggest the concomitant presence of more that one phase
within the membrane.

E. Ciepichal et al. / Chemistry and Physics of Lipids 164 (2011) 300–306
305
cesses which occur in biological systems. Increase of membrane
permeability seems crucial for cellular response and adaptation to
adverse conditions since environmental stress causes an enhanced
exchange of metabolites through cellular compartments. Alter-
ation of membrane fluidity and permeability might be achieved
by the adjustment of the content of polyisoprenoid alcohols. In
the current report the effect of polyprenols on the permeability
and thermotropic properties of membranes was elucidated focus-
ing on the recently discovered new isomeric forms of polyprenols
(␣-trans-polyprenols).
between the lipid acyl chains. Together with the small but signif-
icant decrease of the temperature, this effect is undoubtedly an
indication that polyprenols enter the hydrocarbon region of DPPC
multibilayers. Strong influence of polyprenols on the pretransition
may be explained in terms of greater distortion of the DPPC pack-
ing in the gel state. With high ␣-trans-Pren-12 contents, apart from
the modifications observed in the shape of the main phase tran-
sition peak, an additional peak appeared, which may suggest the
existence of spatial non-homogeneity (microdomains), or phase
separation. It seems plausible that this observation is related with
an intriguing increase of the membrane permeability for glucose
and protons observed at 7 mol% content of Pren in the bilayer.
Hovewer, more in-depth studies are required to prove it. Such an
effect has never been observed for dolichol-doped PC liposomes.
A previous report described only a minor decrease of Tm (Vigo
et al., 1984) or even no effect of dolichols: Dol-20 (Valtersson et al.,
1985) and Dol-19 (Wang et al., 2008) on phospholipid bilayers.
However, observations recorded for dolichyl-P showed that it dra-
matically perturbed DMPC thermotropic behavior leading to the
abolition of the transition from the gel to the liquid crystalline
phase.
It is also worth mentioning that the literature data reveal a
strong relationship between the influence exerted by dolichols
on phosphatidylethanolamine (PE) liposomes and the structure
of the fatty acyl chains of PE. In the case of DPPE a progressive
increase in the temperature of gel to liquid-crystal phase transition
was observed upon incorporation of increasing amounts of Dol-19
(Wang et al., 2008) while destabilization of the PE bilayer accompa-
nied by promoted formation of inverted hexagonal phase (HII) was
reported for unsaturated PE (Valtersson et al., 1985; Wang et al.,
2008).
Dissimilar effects of Pren-12 and dolichols on PC membranes
might be explained on the one hand by the difference in their
length which affects the amphipatic balance and makes it more
or less favorable for hydrophilic affinity. For prenyllipids such as
plastoquinones and ubiquinones (Jemiola-Rzeminska et al., 2002;
Skowronek et al., 1996; Jemiola-Rzeminska et al., 1996) as well as
for dolichol derivatives (Valtersson et al., 1985) the magnitude of
bilayer destabilization was shown to depend on the number of iso-
prene residues. Interestingly, while an increase in the number of
isoprene units caused enhanced transversal penetration, at con-
centrations higher than 1–2 mol%, the gradually increasing fraction
of long-chain ubiquinone-10 (UQ-10, coenzyme Q) was found in
an aggregated state in the bilayer midplane region. On the other
hand, the difference in the geometry of the ␣-isoprene residue
might perhaps also have an impact on the final interaction between
polyisoprenoid and fatty acid chains.
It seems reasonable to discuss our DSC and permeability data
on the basis of location of the polyisoprenoid molecules within the
lipid bilayer which, however, is still being debated. Earlier studies,
based on EPR and NMR data, suggested a model in which dolichol
(Dol-19) was sandwiched between the two faces of a phospholipid
matrix with both ends of the polyisoprenoid molecule localized in
the bilayer interior (Valtersson et al., 1985; McCloskey and Troy,
1980; de Ropp and Troy, 1985). A model published recently pre-
dicts that polyisoprenoid chains (Dol-19 and Dol-19P) are in close
apposition with the acyl chains of the host phospholipids (Zhou
and Troy, 2005). The preferred orientation of Dol-19 is primarily
parallel to the plane of the bilayer, whereas Dol-19P is preferen-
tially arranged more perpendicular to the plane of the bilayer. Data
reported here, i.e. the observed effect of Pren-12 isomers on the
pre- and main phase transition of DPPC are consistent with this
model and point to the fact that Pren-12 and Dol-19 alike interact
with the phospholipids mainly through hydrophobic interactions
between the coil region of the polyisoprenoid chain and the fatty
acyl chains of the phospholipids.
Three types of experiments were performed. At first the effi-
ciency of incorporation of polyprenols into the unilamellar EYPC
vesicles was analyzed. There are several lines of evidence proving
that polyprenols and dolichols are incorporated into the phospho-
lipid bilayer (Zhou and Troy, 2003, 2005; Valtersson et al., 1985;
Wang et al., 2008). However, to the best of our knowledge this is
the first quantitative analysis of this phenomenon. The two-step
extraction method applied in this study was aimed at differen-
tiation between the pool of Pren-12 molecules incorporated into
the membrane and those which were associated with the liposo-
mal surface. Quantification of the Pren-12 fraction easily removed
by the mild pentane washing vs. total content of Pren-12 permit-
ted comparison of the incorporation of both isomers of Pren-12
into the EYPC bilayer. Interestingly, different values of the distri-
bution coefficient found for the Pren-12 isomers, log Pꢀ, reveal that
the configuration of the ␣-terminal residue significantly alters the
hydrophobicity of the polyisoprenoid molecule and consequently
the affinity of polyprenols for the EYPC membrane. Observed dif-
ferences indicate much higher partition of the ␣-cis- than that of
␣-trans-Pren-12 between thephospholipid region andwaterphase,
however the mode of interaction of former isomer with phos-
pholipids seems to be concentration-dependent. Especially strong
increase of the ␣-cis-Pren-12 membrane incorporation observed
at its high concentrations might suggest structural changes of the
vesicles however direct experimental data are required to explain
this observation.
In the second set of experiments it was shown that ␣-trans-
Pren-12 noticeably modified the permeability of unilamellar egg
yolk phosphatidylcholine liposomes for glucose (an increase up
to 1.7-fold for 7 mol%) while the effect of ␣-cis-Pren-12 was
almost negligible. According to the preliminary results both iso-
mers increased the permeability of liposomes for protons (at 7 mol%
content of Pren-12). These data are in agreement with earlier stud-
ies based on the electrophysiological technique, which showed
that Pren-11 (undecaprenol) increased the permeability coeffi-
cients of macrovesicular dioleoylphosphatidilcholine membranes
(hemispheric bilayers) for Na+ and Cl− ions. It was concluded that
undecaprenol substantially decreased the energy barrier for ion
migration through membranes resulting in increased ionic con-
ductance (Janas et al., 1994). Similarly, an increase of ion (Cl−)
permeability measured in the presence of transmembrane poten-
tial, was observed for Pren-16 incorporated into DOPC bilayers
(Janas et al., 2000). However, similar experiments performed for
Pren-16 diphosphate revealed the increase of the activation energy
of ion migration (up to approx. 1.8-fold) in DOPC bilayer (Janas and
Walin´ ska, 2000) and Pren-11P decreased DOPC membrane conduc-
tance and ionic permeability (Janas et al., 1994).
Increased permeability of the membrane most probably results
from structural perturbations which occur in the membrane due
to the incorporation of polyisoprenoid alcohols. This aspect was
studied within the third set of experiments where thermotropic
behavior of the dipalmitoylphosphatidylcholine multilamellar
vesicles supplemented with various amounts of ␣-cis- or ␣-trans-
Pren-12 was analyzed using the DSC technique. Incorporation of
both Pren-12 isomers resulted in a broadening of the phospholipid
main transition, which may reflect a reduction of the cooperativity

306
E. Ciepichal et al. / Chemistry and Physics of Lipids 164 (2011) 300–306
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