From the Department of Biochemistry and Molecular Genetics,
University of Virginia School of Medicine,
Charlottesville, Virginia 22908
We have semi-synthesized 18 species of mixed
chain phosphatidylethanolamines (PE) in which the sn-1 acyl
chain is derived from stearic, arachidic, and behenic acids, and the
sn-2 acyl chain is originated from
cis,cis-octadecadienoic and
cis,cis-eicosadienoic acids with the two
methylene-interrupted double bonds located at various positions. These
PEs constituting the bilayers in the aqueous dispersion were subjected
to differential scanning calorimetric experiments. The
Tm values associated with the gel-to-liquid crystalline phase transitions for these PEs are found to be
significantly smaller than those of the saturated counterparts.
Moreover, the magnitude of the Tm-lowering effect
of acyl chain diunsaturation depends critically on the positions of the
two methylene-interrupted cis double bonds in the
sn-2 acyl chain. Specifically, if the sn-2 acyl
chain is derived from cis,cis-octadecadienoic
acid, the Tm-lowering effect has the following
decreasing order:
9,12 >
6,9 >
12,15. For cis,cis-eicosadienoyl
acyl chain, the Tm-lowering effect is stronger in
the order of
10,13 >
11,14 >
8,11 >
5,8 >
14,17.
Finally, a refined molecular model is presented that can adequately explain the Tm-lowering effect of sn-2
acyl chain diunsaturation. Moreover, this same refined molecular model
can also be invoked to better interpret the
Tm-lowering effect observed for sn-1
saturated/sn-2 monoenoic PE.
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INTRODUCTION |
Biological membranes are often referred to as two-dimensional
sheetlike structures consisting mainly of proteins and lipids. Nevertheless, they do have a third dimension, albeit short typically in
between 5 and 10 nm, in the direction normal to the membrane surface.
In particular, lipids in the biological membrane are amphipathic
molecules, with their long hydrophobic moieties oriented roughly
parallel to the direction of the third dimension. Because of the high
heterogeneity of the hydrophobic moieties, the short third dimension of
the biological membrane is characterized by a nonuniform architecture.
For instance, the number of the cis carbon-carbon double
bond in the sn-2 acyl chain for most membrane lipids ranges
from 1 to 6, with the position of the rotationally rigid cis
double bond varying from the 4th carbon (
4) when
counting from the carboxyl end to the
3 carbon or the third carbon
when counting from the methyl end of the acyl chain. These lipid
molecules with various numbers and positions of cis double
bonds along the sn-2 acyl chain in the direction of the third dimension of the membrane must affect the lateral
lipid-lipid/lipid-protein contact interactions in the two-dimensional
plane of the membrane which, in turn, may contribute significantly to
some specific properties of biological membranes.
As an approach to understand the effects of structural variations in
the third dimension of the biological membrane on the lateral
lipid-lipid interactions in the two-dimensional plane of the sheet-like
membrane, we have recently investigated the influence of the numbers
and positions of cis C-C double bonds in the sn-2
acyl chain of phosphatidylethanolamine
(PE)1 on the gel-to-liquid
crystalline (or the L
L
) phase transition of the lipid bilayer (1-4), a highly cooperative behavior exhibited by most, if not all, lipid molecules in the two-dimensional plane of the lipid bilayer. In particular, we have applied the high
resolution differential scanning calorimetry (DSC) to follow the
L
L
phase transition exhibited by
several series of lipid species under two distinct subclasses of
phosphatidylethanolamine, viz. the sn-1
saturated/sn-2 monoenoic PE and the sn-1
saturated/sn-2 trienoic PE (1-3). Our DSC results indicated
clearly that the gel-to-liquid crystalline phase transitions exhibited
by these two subclasses of PE share two common features (1, 3). First, the introduction of a single cis double bond or three
methylene-interrupted cis double bonds into the
sn-2 acyl chain lowers the phase transition temperature,
Tm. Second, the largest
Tm-lowering effect occurs when the single or three
cis double bond is located in the middle of the linear
segment of the sn-2 acyl chain. Interestingly, the
Tm increases progressively as the single or
three cis-double bond migrates from the middle toward either
end of the acyl chain (1, 3). Based on the simulated molecular structures of these unsaturated lipids packed in the gel-state bilayer
as obtained by molecular mechanics (MM) calculations, we have
postulated a molecular model to explain qualitatively the observed
Tm-lowering effect of acyl chain unsaturation (1,
3). Explicitly, the MM-based molecular model predicts that in the plot
of Tm versus the positions of two
methylene-interrupted cis double bonds for a homologous
series of sn-1 saturated/sn-2 dienoic PE, the
Tm profile should exhibit a V-shaped characteristic
(3). In the present investigation, our main goal is to test the
validity of the predicted V-shaped Tm profile. In
order to achieve this goal, a large number of sn-1 saturated/sn-2 dienoic PE needs to be synthesized. Although
we have previously semi-synthesized 15 molecular species of
sn-1 saturated/sn-2 dienoic PE (4), the
sn-2 acyl chains of these lipids were
6 fatty acids which
can be purchased from commercial sources. Other dienoic fatty acids
with the methylene-interrupted cis double bonds at
3,
9, and
12 positions are not commercially available. In this
study, we first synthesized these various dienoic fatty acids and then
linked them via acylation to the appropriate lysolipids. Specifically,
the following five series of sn-1 saturated/sn-2 dienoic PE were synthesized:
C(18):C(18:2
n,n+3)PE,
C(20):C(18:2
n,n+3)PE,
C(18):C(20:2
n,n+3)PE,
C(20):C(20:2
n,n+3)PE, and
C(22):C(20:2
n,n+3)PE. These PEs constituting the
bilayers in the aqueous dispersions were then subjected to DSC
experiments to study the influence of the positions of two
methylene-interrupted cis double bonds on the bilayer's
L
L
phase transition. Another goal of
this investigation is to refine the MM-based molecular model based on
the Tm values of sn-1
saturated/sn-2 diunsaturated PE with the two double-bond
position ranging from
5,8 to
3. Specifically, we
attempted to obtain a simple molecular model which can adequately
explain how the Tm value varies as the
methylene-interrupted two-double bond migrates along the sn-2 acyl chain from the
5,8 position near
the bilayer/water interface to the
3 position near the bilayer
center.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Dienoic fatty acids with two
methylene-interrupted cis double bonds at the
6 positions
such as cis,cis-9,12-octadecadienoic and
cis,cis-11-14-eicosadienoic acids were obtained
commercially from Sigma. Lysophosphatidylcholines were supplied by
Avanti Polar Lipids (Alabaster, AL). Phospholipase D, type I from
cabbage, was purchased from Sigma. All routine reagents and organic
solvents were of reagent and spectroscopic grades, respectively, and
they were purchased from different commercial sources.
Semi-synthesis of sn-1 Saturated/sn-2 Dienoic PE--
With the
exception of
6 dienoic fatty acids, all other dienoic fatty acids
used as the starting materials for phospholipid semi-synthesis were
prepared in this laboratory using modified procedures of established
methods (5, 6). Basically, the synthesis of dienoic fatty acids
proceeded through the coupling of relevant
-acetylenic acid
(HC
C-(CH2)n-COOH) and appropriate
1-bromo-2-alkynes (R-C
C-CH2-Br) followed by catalytic cis-hydrogenation of the triple bond using the Lindlar
catalysis (6). Depending on the availability of the starting materials for making the
-acetylenic acids, it is possible to synthesize the
various dienoic fatty acids by two different routes. Detailed procedures will be published elsewhere; however, the outlines of the
two routes are presented in Fig. 1,
A and B, as reaction schemes I and II,
respectively. Specifically, the synthesis of cis,cis-5,8-eicosadienoic acid,
cis,cis-6,9-octadecadienoic acid, and
cis,cis-8,11-eicosadienoic acid can be
accomplished by the reaction scheme I, whereas that of
cis,cis-12,15-octadecadienoic acid and
cis,cis-14,17-eicosadienoic acid being carried
out using reaction scheme II. By using high pressure liquid
chromatography (7), the purity of the synthesized dienoic fatty acids
can be estimated to be greater than 95%.

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Fig. 1.
Two reaction schemes for the synthesis of
dienoic fatty acids. A, scheme I, a synthetic
route for compounds I, II, and III. B, scheme II,
a synthetic route for compounds IV and V.
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In this study, 18 molecular species of PE, in each of which the
sn-1 acyl chain is saturated and the sn-2 acyl
chain is derived from a dienoic fatty acid, were semi-synthesized and
purified according to our previously published method (4). Purity was checked by UV spectroscopy, TLC, and DSC (4); the lipid powder, after
lyophilization from benzene, was kept at
20 °C.
High Resolution DSC Measurements--
The lipid samples were
prepared according to established procedures (1, 4). Briefly, cold
aqueous buffer solution (50 mM NaCl, 0.25 mM
diethylene triaminepentaacetic acid, 5 mM phosphate buffer,
pH 7.4, and 0.02 mg/ml NaN3) was added to lyophilized lipid
powder to give a total lipid concentration of 2-5 mM. The exact lipid concentration was determined by phosphorus analysis. In
order to avoid possible auto-oxidation of the unsaturated lipids, the
cold aqueous buffer solution was degassed and then purged gently with
N2 gas prior to the lipid sample preparation. Furthermore, once the lipid sample was prepared, it was immediately degassed and
sealed under N2 followed by vortexing for about 5 min.
After vortexing, the sealed lipid sample was kept at 0 °C for about 30 min and then loaded into the DSC sample cell. The lipid sample was
further equilibrated in the DSC cell at a desired temperature (usually
15 °C below the Tm) for 120 min and then scanned. All DSC experiments were performed using a high resolution MC-2 differential scanning microcalorimeter (Microcal, Northampton, MA).
Each lipid sample was scanned at least three times at a constant scan
rate of 15 °C/h in the ascending temperature mode with at least
60-90 min of equilibration at low temperatures between scans. In order
to ascertain that the same thermal history pertained to all lipid
samples, only the Tm value from the second DSC
heating scan was reported in this study. The third DSC heating scan
served as an internal control to check whether the second DSC curve was
reproducible. The Tm value obtained at the
transition peak with maximal peak height was reproduced at ±0.1 °C
for each lipid sample. Details of the procedure for carrying out DSC
experiments and for determining the values of the phase transition
temperature (Tm) and enthalpy (
H) were described in our earlier publications (1-4).
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RESULTS |
Phase Transition Behavior of C(X):C(18:2
n,n+3)PE
with X = 18 and 20 and n = 6, 9, and 12--
The influence
of the positions of the two methylene-interrupted cis
carbon-carbon double bonds in the sn-2 acyl chain on the gel-to-liquid crystalline phase transition behavior of the lipid bilayer composed of phosphatidylethanolamines was studied by high resolution differential scanning calorimetry using two homologous series of PE, viz.
C(X):C(18:2
n,n+3)PE with
X = 18 and 20 and n = 6, 9 and 12. Specifically, the sn-1 acyl chains of the two series PE are
derived from stearic and arachidic acids, respectively, and the
sn-2 acyl chains are originated from
cis,cis-octadecadienoic acid with the positions of the two methylene-interrupted double bonds designated by
n,n+3, where n denotes the number of
carbon atoms from the carboxyl end and equals to 6, 9, and 12. Within
each series, there are three lipid species that are positional isomers.
Their numbers of carbon atoms and numbers of cis double
bonds are equal; however, the positions of the two
methylene-interrupted cis double bonds in the
sn-2 acyl chain are different. It should be mentioned at this point that the sn-2 acyl chain of saturated PE in the
single crystals is bent nearly 90° at the C(2) position due to the
fact that the torsional angles of C(1)-C(2) and C(2)-C(3) bonds are
119° and 65°, respectively (8). As a result, the
all-trans-linear segment of the saturated sn-2
acyl chain begins at the C(3) atom and ends at the terminal methyl
carbon. When two methylene-interrupted cis double bonds are
introduced into the sn-2 acyl chain of PE, the relative
positions of
6,9,
9,12, and
12,15 along the linear segment of the sn-2
acyl chain in each of the positions isomers can be schematically
illustrated as shown in Fig. 2,
C-E. It is
apparent that the two methylene-interrupted cis double
bonds,
9,12, of
C(X):C(18:2
9,12)PE are located nearly at the
center of the linear segment of the sn-2 acyl chain, whereas
the
6,9 double bonds of
C(X):C(18:2
6,9)PE and the
12,15 double bonds of
C(X):C(18:2
12,15)PE are located near the
carboxyl and methyl ends, respectively, in the sn-2 acyl
chains.

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Fig. 2.
Schematic diagrams indicating the chain
lengths of the long and short segments of the kinked sn-2
acyl chain of PE in the gel-state bilayer. A, the
conformation of the kinked sn-2
cis,cis-11,14-eicosadienoyl chain of PE at
T < Tm. The sharp bend occurs at
C(2), and the kinked sequence around the two methylene-interrupted
double bonds is
s s+s+ s .
Consequently, the long all-trans linear segment begins at
C(3) and ends at C(10). B, the same kinked sn-2
acyl chain as A. LS and SS denote the
long and short segments, respectively, separated by the double bonds.
The length of the long segment, 8 C-C bond lengths, is one C-C bond
length longer than the all-trans linear segment shown in
A. The chain lengths of the long and short segments of the
kinked sn-2 acyl chain each with a total number of 18 carbons are illustrated in C, D, and
E. The positions of the two methylene-interrupted
cis double bonds, 6,9, 9,12,
and 12,15, are indicated in the rectangular
box. It is evident that the two double bonds in
cis,cis-9,12-octadecadienoyl chain are located
nearly in the middle of the chain, since the long and short segments
differ by only one C-C bond length. The kinked chains with 20 carbons
are illustrated in F-J. The numeric numbers are the lengths
of the long and short segments in C-C bond length, and the positions of
the two methylene-interrupted cis double bonds are indicated
in the rectangular box. For
cis,cis-10,13-eicosadienoyl chain, the two double
bonds are located nearly in the middle of the chain, since the
difference between LS and SS is only one C-C bond length.
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Fig. 3 shows the first and second DSC
heating curves for aqueous dispersions prepared individually from each
of the lipid species within the two homologous series of
C(18):C(18:2
n,n+3)PE and
C(20):C(18:2
n,n+3)PE with n = 6, 9, and 12. All six lipid samples show a single endothermic transition
in the first DSC heating scans. Upon immediate second DSC heating
scans, four samples display a smaller and down-shifted endothermic
transition; moreover, these low temperature endotherms observed in the
second DSC heating scans are seen reproducibly upon repeated
reheatings. These thermal history-dependent phenomena are
identical to those typically exhibited by aqueous dispersions prepared
from C(12):C(12)PE and saturated mixed chain PE (9-12). Consequently,
the high and low temperature transitions observed in the initial and
subsequent DSC heating scans can be reasonably assigned as the
crystalline-to-liquid crystalline (the Lc
L
) and the gel-to-liquid crystalline (the
L
L
) phase transitions,
respectively.

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Fig. 3.
The first and second DSC heating scans of
aqueous dispersions prepared from
C(20):C(18:2 n,n+3)PE and
C(18):C(18:2 n,n+3)PE, where n = 6, 9, and 12. The Tm value for each phase
transition is indicated next to the corresponding thermogram. Clearly,
when the two cis double bonds are located at the
9,12 positions, this positional isomer exhibits the
smallest value of the phase transition temperature in each series of
the dienoic PE.
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The thermodynamic parameters (Tm,
H,
and
T1/2) associated with the L
L
phase transition for each of the six lipid samples
shown in Fig. 3 are summarized in Table
I. It should be mentioned that the
Tm value observed calorimetrically for the
C(18):C(18)PE dispersion is 74.4 °C (12). This Tm value is considerably higher than those of
C(18):C(18:2
n,n+3)PE with n = 6, 9, 12 as shown in Fig. 3 and Table I. Similarly, the
Tm value of 75.8 °C exhibited by lamellar
C(20):C(18)PE is also substantially higher than the
Tm values observed in Fig. 3 for
C(20):C(18:2
n,n+3)PE with n = 6, 9, and 12. The present study thus confirms and extends the previously
published DSC results (4), indicating that the acyl chain
diunsaturation markedly lowers the Tm value
associated with the PE bilayer's gel-to-liquid crystalline phase
transition. This Tm-lowering effect of acyl chain diunsaturation is well known for phosphatidylcholine bilayers (13).
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Table I
The thermodynamic parameters associated with the gel-to-liquid
crystalline phase transition of lipid bilayers prepared from various
dienoic PE
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For the two series of C(18):C(18:2
n,n+3)PE and
C(20):C(18:2
n,n+3)PE with n = 6, 9, 12 the Tm-lowering effect of acyl chain unsaturation depends on the positions of the two cis double
bonds. From Fig. 3, it is clear that the Tm-lowering
effect is largest at
9,12 and decreases as the two
methylene-interrupted cis double bonds migrate together
toward either the surface of the bilayer at
6,9 or the
center of the lipid bilayer at
12,15. The present study
thus demonstrates convincingly that a structural variation along the
third dimension of the membrane can result in different responses by
the lipid bilayer as a whole. In this case, the different
Tm-lowering responses have the following decreasing
order:
9,12 >
6,9
>
12,15.
In Fig. 2, the structural unit of the two methylene-interrupted
cis double bonds at
9,12 is seen to be nearly
in the middle of the linear segment of the sn-2 acyl chain.
Hence, the Tm-lowering effect of acyl chain
diunsaturation observed in Fig. 3 appears to be similar to that of acyl
chain monounsaturation (1), which has the following two
characteristics. First, the Tm profile has roughly the shape of the letter V. Second, the minimal Tm
occurs when the single cis double bond is nearly in the
middle of the linear segment of the sn-2 acyl chain.
Phase Transition Behavior of C(X):C(20:2
n,n+3)PE
with X = 18, 20, and 22 and n = 5, 8, 11, and 14--
Three
other series of diunsaturated phosphatidylethanolamines, in which the
sn-1 acyl chains are derived from stearic, arachidic, and
behenic acids, respectively, whereas the sn-2 acyl chains are originated from cis,cis-eicosadienoic acid
with the two methylene-interrupted cis double bonds located
at
5,8,
8,11,
11,14, and
14,17positions, were also studied by high resolution
DSC. The relative positions of these cis double bonds
(
5,8,
8,11,
11,14, and
14,17) in the sn-2 acyl chains of PEs are
schematically illustrated in Fig. 2, F-J. The
aqueous dispersion prepared individually from some of these dienoic PEs
exhibit calorimetrically the same thermal history-dependent
phase transition characteristics as those shown in Fig. 3. The values
of the thermodynamic parameters (Tm,
H, and
T1/2) associated with the
gel-to-liquid crystalline phase transition obtained from the second DSC
heating scans are summarized in Table I.
Fig. 4 shows the second DSC heating
curves obtained with aqueous dispersions prepared individually from 12 molecular species under the three series of
C(X):C(20:2
n,n+3)PE with
X = 18, 20, and 22 and n = 5, 8, 11, and 14. With the exception of C(22):C(20:2
5,8)PE, all
thermograms shown in Fig. 4 are characterized by single and nearly
symmetrical endothermic transitions peaked at distinct temperatures,
indicating that our synthesized dienoic fatty acids and mixed chain PEs
derived from them have high degrees of chemical and isomeric purities,
respectively. To the best of our knowledge, all lipid species shown in
Figs. 3 and 4, with the exception of
6 lipids, have not been studied
previously by DSC or any other biophysical method. This is perhaps due
to the fact that dienoic fatty acids leading to the synthesis of these
PEs are not commercially available.

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Fig. 4.
The second DSC heating scans of aqueous lipid
dispersions prepared from 12 lipid species in three different series of
dienoic PE as indicated. Within each series, all lipid species are
positional isomers. The positions of the double bonds,
n,n+3, vary from 5,8 to
14,17 along the
cis,cis-eicosadienoyl chain.
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The Tm values for aqueous dispersions of
C(18):C(20)PE, C(20):C(20)PE, and C(22):C(20)PE are 79.1, 82.5, and
84.5 °C, respectively (3, 12). The Tm values of
the 12 lipid species, sn-1 saturated/sn-2
eicosadienoic PEs, shown in Table I are considerably smaller than the
Tm values of their respective saturated
counterparts. A similar Tm-lowering effect has also
been observed earlier for sn-1 saturated/sn-2 octadecadienoic PE. Consequently, the observed
Tm-lowering effect of the sn-2 acyl chain
diunsaturation can be considered as a general feature for the phase
transition behavior of the lipid bilayer.
The Tm for each lipid sample derived from the three
series of
C(X):C(Y:2
n,n+3)PE with
X = 18, 20, and 22 and n = 5, 8, 11, and 14 can be compared in terms of the positions of two cis
double bonds,
n,n+3. The
Tm-lowering effect of acyl chain diunsaturation is
stronger in the order of
11,14 >
8,11 >
5,8 >
14,17.
The Estimated Tm Values for
C(X):C(20:2
10,13)PE with X = 18, 20, and
22--
The Tm-lowering order just described was
obtained in the absence of experimentally determined
Tm values for
C(X):C(20:2
10,13)PE. As shown in Fig.
2H, the
10,13 position in the sn-2
eicosadienoyl acyl chain is nearly in the middle of the linear
hydrocarbon segment of the sn-2 acyl chain. In order to
obtain a more precisely defined Tm-lowering order,
it is necessary to include data for PE with their double bonds near the
middle of the sn-2 acyl chain. Hence, we have estimated the
Tm values for
C(X):C(20:2
10,13)PE with X = 18, 20, and 22 as described below.
Recently, the gel-to-liquid crystalline phase transition temperatures
for saturated, monounsaturated, and
6-diunsaturated PE have been
shown to correlate with some fundamental structural parameters
underlying the PE molecule packed in the gel state bilayer;
consequently, relatively simple equations of Tm in
terms of the structural parameters can be derived (1, 4, 12). We can
apply a similar approach to derive a general Tm structural parameter equation for lipid bilayers composed of dienoic PE
in which the two methylene-interrupted cis carbon-carbon
double bonds are in the lower half of the sn-2 acyl chain.
Specifically, based on the 10 Tm values determined
for C(X):C(18:2
n,n+3)PE with
X = 18 and 20 and n = 9, 12 and
C(X):C(20:2
n,n+3)PE with X = 18, 20, 22 and n = 11 and 14 as shown in Table I, we
can derive a general Tm structural parameter
equation for
C(X):C(Y:2
n,n+3)PE with
X = 18, 20, 22, and Y = 18, 20, and
n = 9-14 for
C(X):C(18:2
n,n+3)PE or
n = 10-14 for
C(X):C(20:2
n,n+3)PE.
Three structural parameters underlying the dienoic PE are defined in
Fig. 5, A and B, in
which molecular graphic drawings of a monomeric and a dimeric
C(20):C(20:2
11,14)PE obtained with MM calculations are
illustrated. The three defined structural parameters are abbreviated as
C, SS, and N which specify the molecular structure of the
unsaturated lipid species packed in the gel-state bilayer, and they
have units of carbon-carbon bond length. The first one is
C, the effective chain length difference between the
sn-1 and sn-2 acyl chains. Because of the sharp
bend at C(2) and the presence of two cis double bonds at
n,n+3 positions, the sn-2 acyl chain
is shortened by about 3.5 C-C bond lengths in comparison with its
saturated counterpart (4), yielding
C = X
Y + 3.5. The second structural
parameter is SS, which defines the short segment of the kinked
sn-2 acyl chain, ranging from (n + 4)th carbon to
the terminal methyl carbon; it is related to Y and
n as follows: SS = Y
(n + 4). Finally, the thickness of the hydrocarbon core of the gel state
bilayer is defined as N. This structural parameter represents the
separation distance between the two carbonyl oxygens of the
sn-1 acyl chains in the transbilayer dimer (Fig.
5B), and is related to X and Y as follows (4):
n = X + Y
2.5, by
assuming the van der Waals contact distance between the two opposing
methyl groups from the sn-1 and sn-2 acyl chains
to be 3.0 C-C bond lengths. Based on simple equations of
Tm and structural parameters derived earlier for
saturated, monounsaturated, and
6 diunsaturated PE (1, 4, 12), one
can tentatively formulate a general Tm structural
parameter expression for dienoic PE as shown in Equation 1.
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(Eq. 1)
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When experimental Tm values and the
computational data (N,
C, and SS) obtained with the 10 respective lipid species shown in Table
II are substituted individually into
Equation 1, the resulting 10 simultaneous equations can be analyzed
statistically by multiple regression method to obtain the coefficient
(a0, a1, and
a2) in Equation 1. We obtain Equation 2
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(Eq. 2)
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with a correlation coefficient of 0.9988 and a root mean square
error of 0.7009. This high value of correlation coefficient and low
value of root mean square error indicate that Equation 2 is indeed an
excellent Tm structural parameter expression for
dienoic PE. Based on Equation 2, the calculated Tm values for the 10 lipid species can be obtained, and they are also
presented in Table II as Tmcal. The
largest difference between Tmcal and
Tmobs is 1.4 °C for
C(18):C(20:2
11,14)PE, which amounts to a relatively
small change of about 0.5% in kelvin. The agreement between
Tmcal and
Tmobs for these 10 dienoic PE is clearly
excellent. Such an excellent agreement is expected, since the
Tm values of these 10 lipid species belong to the
set from which Equation 2 was derived. Hence, comparisons between
Tmcal and
Tmobs have to be sought from other
dienoic PEs. Table II shows the Tmobs
values for six other lipid species ranging from
C(22):C(18:2
9,12)PE to
C(24):C(22:2
13,16)PE (4). The agreement between
Tmcal and
Tmobs values is again excellent for
these six lipid species, with the largest relative difference between
them being about 0.5% in kelvin. Equation 2 thus appears to be
effective in predicting the Tm value for mixed chain
sn-1 saturated/sn-2 dienoic PE. It should be
emphasized that Equation 2 was derived on the basis of experimental Tm values obtained with
C(X):C(Y:2
n,n+3)PE with
n
0.5 Y; hence, it should not be used to
calculate Tm values for mixed chain dienoic PE with
n < 0.5 Y.

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Fig. 5.
Molecular graphics representations of the
energy-minimized structures of monomeric
C(20):C(20:2 11,14)PE (A) and transbilayer
dimer of C(20):C(20:2 11,14)PE (B). The
three structural parameters, SS, C, and N, described in
the text are indicated. All three structural parameters have the same
unit of C-C bond length. The sn-2 acyl chain is kinked. In
this view, the zigzag planes of the long and short segments are seen to
be nearly parallel, but not coplanar, with the lower plane extending in
front of the zigzag plane of the long segment. A different view of the
same kinked sn-2 acyl chain is presented in Fig.
2A.
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Table II
The structure parameters (SS, C, and N) and Tm values
for various dienoic PE
All structure parameters have a common unit of C-C bond length.
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The Tm values for
C(X):C(20:2
10,13)PE with X = 18, 20, and 22 can be estimated from Equation 2. These values, given in Table II, can now be combined with other Tm values
shown in Table I to obtain more precisely defined Tm
profiles for the three series of
C(X):C(20:2
n,n+3)PE with
X = 18, 20, 22 and n = 5, 8, 10, 11, and 14. The resulting profiles are illustrated in Fig.
6. Clearly, the dip of each of the
V-shaped profiles is observed to be at
10,13, a position
near the middle of the sn-2 acyl chain as shown in Fig.
2H. For comparison, the published Tm
profiles for monoenoic PE with various positions of the single double
bond,
n, are also shown in the inset in Fig.
6.

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Fig. 6.
The plot of the gel-to-liquid crystalline
phase transition temperature (Tm)
versus the position of the two cis double bonds
in the sn-2 acyl chain of
C(X):C(20:2 n,n+3)PE, where
X = 18, 20, and 22, and n = 5, 8, 10, 11, and 14. With the exception of
C(X):C(20:2 10,13)PE, all
Tm values are calorimetrically determined values
derived from Fig. 4. The Tm values for
C(X):C(20:2 10,13)PE are calculated values
based on Equation 2 and are listed in Table II. In the
inset, the experimental Tm of monoenoic
C(X):C(18:1 n)PE is plotted against the position
of the single cis double bond, n, in the
sn-2 acyl chain, where X = 18 and 20 and
n = 6, 7, 9, 11, 12, 13, and 15. With the exception of
C(X):C(18:1 15)PE, all Tm
values are calorimetrically determined values taken from the literature
(1, 2). The Tm values of
C(18):C(18:1 15)PE and C(20):C(18:1 15)PE
are 54.3 and 54.8 °C, respectively, determined recently in this
laboratory by high resolution DSC.
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DISCUSSION |
Based on the molecular structure of monounsaturated PE obtained
with molecular mechanics (MM) simulations, a molecular model has been
invoked to interpret qualitatively the Tm-lowering effect of sn-2 acyl chain unsaturation for monoenoic PE (1). This MM-based molecular model has been subsequently extended to explain
qualitatively the similar Tm-lowering effect observed for trienoic PE (3). Before we present this model and apply it
to interpret the Tm-lowering effect for dienoic PE
observed in the present study, it is appropriate to mention that upon
heating the lipid bilayer, the L
L
phase transition occurring abruptly at Tm involves
fundamentally the trans
gauche
isomerizations, which proceed with rotations of carbon atoms about the
C-C single bonds in the acyl chains of the lipid (14). Consequently,
the thermally induced phase transition can be discussed in terms of
trans
gauche isomerizations at
Tm.
The MM-based molecular model that we have devised originally to
describe the Tm-lowering effect of acyl chain
monounsaturation makes the following three assumptions. 1) The
monoenoic sn-2 acyl chain in the gel-state bilayer is
assumed to adopt a kinked motif. Specifically, the monoenoic chain is
considered to consist of a long linear segment and a short disordered
segment at T < Tm. The two segments
are linked by the cis double bond containing kink. 2) The
short segment does not contribute significantly to the conformational
disordering process of trans
gauche
isomerizations of C-C bonds at Tm, since it is
already partially disordered at T < Tm. The large Tm-lowering effect
of acyl chain monounsaturation can be accounted for quite simply by the presence of a partially disordered short segment of methylene groups in
the sn-2 acyl chain at T < Tm. 3) The long linear segment of the
sn-2 acyl chain is assumed to undergo a favorable van der
Waals contact interaction with the neighboring all-trans
sn-1 acyl chain in the gel-state bilayer; hence, unlike the short
disordered segment, its contribution to the chain melting process at
Tm is significant. The strength of the contact interaction, however, depends on the length of the long linear all-trans segment. When the single cis double
bond is located near the middle of the sn-2 acyl chain, the
long linear all-trans segment has a minimal length. As a
result, the van der Waals contact interaction with the sn-1
acyl chain is also minimal. As the cis double bond moves
away from the chain center toward either the carboxyl or the methyl
end, the length of the long linear all-trans segment
increases progressively, leading to a proportionally increased van der
Waals contact interaction and hence a gradual increase in
Tm. The observed V-shaped Tm
profile can thus be largely explained by the third assumption of the
MM-based molecular model.
On the basis of our earlier MM calculations (4), an energy-minimized
monomeric structure of C(20):C(20:2
11,14)PE is presented
in Fig. 5A. The sn-2 acyl chain of this
energy-minimized structure is observed to be kinked with a
s
s+s+
s
sequence in the kinked region of
C(10)-C(11)=C(12)-C(13)-C(14)=C(15)-C(16), where
s± refer to skew (±) conformations
with torsion angles of about ± 110° and
denotes
cis double bond with torsion angle of about 0° (see also
Fig. 2A). Consequently, the first assumption of the MM-based
molecular model can be applied to the dienoic PE. Equation 2 derived
earlier implicitly implies that the structural parameter SS acts
antagonistically against N, the thickness of the bilayer's hydrocarbon
core. As a result, when N and
C are constant, the larger the value
of SS, the smaller Tmcal becomes (Table
II). Hence, the short segment of the kinked sn-2 acyl chain
is basically a perturbation term. The mathematic expression of Equation 2 is thus in complete accord with the second assumption of the MM-based
molecular model, implying that the short segment is largely disorder at
T < Tm. An important question that
needs an answer at this point is, "Are the experimentally observed
Tm values shown in Figs. 3 and 4 consistent with the
phase transition process involving primarily the sn-1 acyl
chain and the long linear segment of the sn-2 acyl chain in
the dienoic PE bilayer"? If the answer is yes, then the third assumption of the MM-based molecular model is also applicable to
dienoic PE, and hence the model as a whole can be considered as a more
general one which can explain the Tm-lowering effect
of acyl chain mono- and di-unsaturation.
The answer to the question just raised does not appear to be a simple
one. This is due in part to the possible complication that for dienoic
PE the chain melting process of trans
gauche isomerizations at Tm may involve a fraction of the
short segment as its length approaches that of the long segment.
Nevertheless, we can test the third assumption by comparing the
Tm of
C(X):C(Y:2
n,n+3)PE with
that of a selected species of saturated
C(X):C(Y')PE. This selected lipid species must be
characterized by a (Y'
3) chain length that is equivalent
to the length of the long linear all-trans segment of the
kinked C(Y:2
n,n+3) chain. If the
third assumption is valid, we can expect that the lipid bilayer
composed of
C(X):C(Y:2
n,n+3)PE will
exhibit calorimetrically the L
L
phase transition with a Tm value that is equivalent
to the Tm associated with the L
L
phase transition of the
C(X):C(Y')PE bilayer.
When the two methylene-interrupted cis double bonds are
located mostly in the lower half of the sn-2 acyl chain, the
long linear all-trans segment of
C(Y:2
n,n+3) chain can be calculated
to extend from C(3) to C(n
1) with a segment length
of (n
4) carbon-carbon bond lengths (Fig.
2A). This calculation has taken the following two structural
features into consideration. 1) The sn-2 acyl chain bends
sharply at C(2). 2) The C(n
1)
C(n)
single bond constitutes the initial part of the kink sequence of
s
s+s+
s
.
Let us consider C(18):C(18:2
9,12)PE as an example. The
long linear all-trans segment of its sn-2 acyl
chain contains 6 carbons, extending from C(3) to C(n
1) or C(8) atom. The Tm value of this unsaturated
lipid species can, therefore, be compared directly with that of
saturated C(18):C(8)PE. If, on the other hand, the two
methylene-interrupted cis double bonds are located mostly in
the upper half of the sn-2 acyl chain, the long linear
all-trans segment begins with the C(n + 5) atom
and ends at the methyl carbon with (Y
n
5) carbon-carbon bond lengths.
C(18):C(18:2
6,9)PE can serve as an example. Its long
linear all-trans segment in the dienoic sn-2 acyl
chain can be shown to extend from C(11) to C(18). The corresponding
C(18):C(Y')PE is thus C(18):C(10)PE with its saturated
sn-2 acyl chain containing an all-trans segment of 7 C-C bond lengths. It should be mentioned that the lipid bilayer composed of highly asymmetric C(18):C(8)PE or C(18):C(10)PE undergoes the mixed-interdigitated (L
M) to the
liquid-crystalline (L
) phase transition upon heating (15). Nevertheless, the Tm value associated with the fictive L
L
phase transition for the
bilayer composed of highly asymmetric PE can be calculated (12).
For a homologous series of C(18):C(18:2
n,n+3)PE
with n varying stepwise from 5 to 8, the Y'
values of the corresponding C(18):C(Y')PE can be shown to
decrease stepwise from 9 to 6. The Tm values
associated with the fictive L
L
phase
transitions for C(18):C(Y')PE with Y' = 9, 8, 7, and 6 are 29.1, 18.3, 6.0, and
8.0 °C, respectively (12). In
contrast, as n in the dienoic C(18:2
n,n+3) chain further increases
progressively from 9 to 12, the number of carbon atoms in the
sn-2 acyl chain of C(18):C(Y')PE increases successively from 6 to 9. The Tm value of the
C(18):C(Y')PE also increases correspondingly with increasing
n. These Tm values of
C(18):C(Y')PE are plotted in Fig.
7A as a function of n of the corresponding
C(18):C(18:2
n,n+3)PE. Clearly, the connected
Tm curve exhibits the expected V-shaped profile as
the two methylene-interrupted cis double bonds move along
the chain as a unit from the
5,8 position to the
3 or
12,15 position. Similarly, a calculated
Tm profile can also be derived for
C(20):C(Y')PE from the long linear all-trans
segment of the sn-2 acyl chain in
C(20):C(18:
n,n+3)PE. The resulting
Tm curve is nearly superimposable over the one shown
in Fig. 7A; hence, it is not illustrated. It should be
emphasized that the calculated V-shaped Tm profile
shown in Fig. 7A is the hypothetical Tm
curve for C(18):C(18:2
n,n+3)PE based on the
supposition that only the long all-trans segment of the
sn-2 acyl chain and the all-trans sn-1 acyl chain
contribute to the chain melting process of trans
gauche isomerizations at Tm.

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Fig. 7.
The comparison between the experimental and
calculated Tm values in the plot of
Tm versus the position of the double
bonds. A, the dienoic PEs are
C(18):C(18:2 n,n+3)PE and
C(20):C(18:2 n,n+3)PE with n = 6, 9, and 12. The experimental Tm values are
represented by two sets of symbols, and the solid V-shaped
curve is calculated on the assumption that only all-trans
sn-1 acyl chain and the long all-trans segment of the
kinked sn-2 acyl chain undergo the trans gauche isomerizations at Tm.
B, three series of
C(X):C(20:2 n,n+3)PE are compared,
where X = 18, 20, and 22 and n = 5, 8, 11, and 14. The calculated Tm values are connected
by a V-shaped solid curve, and the experimental
Tm values are individually represented by three sets
of symbols. C, the Tm values of two
series of monoenoic PE, C(18):C(18:1 n)PE, and
C(20):C(18:1 n)PE, are compared. Experimental values are
individually represented by two sets of symbols, and the calculated
Tm values are connected by a solid V-shaped
curve.
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The experimental Tm values listed in Table I for
C(X):C(18:2
n,n+3)PE with
X = 18 and 20 and n = 6, 9, and 12 are
presented in Fig. 7A. It is evident that the experimental
Tm values agree well with the corresponding points
on the calculated Tm curve for
C(X):C(18:2
6,9)PE and
C(X):C(18:2
12,15)PE, suggesting that the
third assumption of the MM-based molecular model is indeed valid for
these two sets of dienoic PE species. However, the
Tm values of C(18):C(18:2
9,12)PE and
C(20):C(18:2
9,12)PE shown in Fig. 7A deviate
significantly from the predicted Tm values. These
deviations, nonetheless, should not be taken as a surprising result.
The long segment of the C(18:2
9,12) chain has 6 consecutive C-C single bonds (Fig. 2D); however, its
all-trans segment from C(3) to C(8) is only 5 C-C bond
lengths long. The short segment from C(13) to C(18) also has 5 single C-C bonds (Fig. 2D). The difference in the C-C single bond
number is, therefore, zero between the long linear all-trans
segment and the short segment. In this case, it is most likely that the entire length of the short segment is not completely disordered at
T < Tm; hence, it contributes
somewhat to the chain melting process at Tm, leading
to a higher Tm than expected.
The calculated Tm curve and the experimental
Tm values for
C(X):C(20:2
n,n+3)PE with
X = 18, 20, and 22 and n = 5, 8, 11, and 14 are shown in Fig. 7B. The
3 or
14,17 lipids with highly dynamic short segments are seen
to exhibit Tm values that match nearly perfectly
with the calculated Tm curve. As the
methylene-interrupted two cis double bonds move upward along
the chain to the
11,14 position, the experimental and
calculated Tm values begin to deviate; however, the
deviation is not too severe. This moderate deviation can be explained
based on the difference in the number of the C-C single bonds between
the long all-trans segment and the short segment which is
merely 2. As a result, the short segment may provide an additional
contribution to the chain melting process of the trans
gauche isomerizations, leading to a higher
Tm. In the case of
C(X):C(20:2
8,11)PE, the difference in the
single C-C bond number between the long linear all-trans
segment and the short segment is also 2. However, the short segment is
located between C(3) and C(8) without a free end; the methylene units
are thus rotationally less dynamic at T < Tm. Consequently, this short segment can be expected to have a higher additive contribution to the chain melting process of
trans
gauche isomerizations at
Tm. The experimental values of
C(X):C(20:2
8,11)PE are indeed observed to
deviate more from the calculated Tm in comparison
with C(X):C(20:2
11,14)PE as shown in Fig.
7B. In the same figure, the experimental and calculated
Tm values for
C(X):C(20:2
5,8)PE are also different.
Interestingly, all the experimental values are observed to be less than
the calculated ones. It should be mentioned that in
C(X):C(20:2
5,8)PE the two
methylene-interrupted cis double bonds are very near to the
H2O/hydrocarbon interface. Water molecules can, therefore, penetrate into the upper kinked region, which can cause a decrease in
the lateral chain-chain interaction. The lower values of experimental Tm values for
C(X):C(20:2
5,8)PE shown in Fig. 7B
may be explained simply by an increased interchain hydration.
In the foregoing discussion of the deviation of experimental
Tm values from the calculated one, we have
introduced a simple concept which can be restated as follows. The short
segment of the kinked sn-2 acyl chain in dienoic PE may
contribute somewhat to the chain melting process of trans
gauche isomerizations at Tm when its
length approaches that of the long segment. In principle, this same
concept should be equally applicable to describe the
Tm-lowering phenomenon exhibited by any series of
positional isomers of sn-1 saturated/sn-2
monoenoic PE. With this in mind, we now focus on the calorimetrically
determined Tm values for the two series of
C(18):C(18:1
n)PE and C(20):C(18:1
n)PE seen in the
inset of Fig. 6. These Tm values are replotted in Fig. 7C where a predicted V-shaped
Tm profile is also illustrated. When the concept of
the contribution of the short segment to Tm is
considered, one can expect two characteristic features to be observed
for monoenoic PE as follows. 1) The experimental Tm
value must deviate increasingly from the predicted one as the single
cis double bond moves progressively along the
sn-2 acyl chain from either end at
6 or
15 toward the chain center at
10. This is
due to the fact that the chain length of the short segment is largest
when the single double bond is at
10 for sn-2
cis-octadecaenoyl acyl chain. 2) When the short segments of
two positional isomers have the same length, the magnitude of the
Tm deviation depends on the location of the short segment. Specifically, the lipid species with the short segment located
in the lower half of the sn-2 acyl chain is expected to exhibit a calorimetric Tm value that deviates less
from the calculated one. For instance, C(18):C(18:1
9)PE
and C(18):C(18:1
11)PE have a common length of the short
segment. When the experimental Tm values of
C(18):C(18:1
11)PE and C(18):C(18:1
9)PE
are compared, the former is expected to deviate less from the
calculated Tm. This expectation is based on the fact that the short segment of the sn-2 acyl chain of
C(18):C(18:1
11)PE is in the lower half of the lipid
molecule with a free methyl end; hence, this short segment is more
disordered at T < Tm. Consequently,
it contributes less to the chain melting process at
Tm in comparison with the short segment of its
positional isomer of C(18):C(18:1
9)PE. A close
inspection of Fig. 7C reveals that the two expected features
discussed above can indeed be identified, indicating that the
fundamentally simple concept of the contribution of the short chain to
Tm is supported by our calorimetric results. Based
on data shown in Fig. 7, A-C, we can conclude
that the simple concept of the contribution of the short chain to
Tm does account for the deviation of experimental
Tm from the expected one for both mono- and
di-unsaturated PE systems.
To sum up, we have determined by DSC the Tm values
of aqueous dispersions prepared individually from two subclasses of
sn-1 saturated/sn-2 dienoic PE with a total of 18 lipid species. The experimental Tm values are
plotted in Fig. 7, A and B, as a function of the
positions of the cis double bonds. The calculated
Tm curves are also included in Fig. 7, A
and B, which are generated based on the third assumption of
our previously proposed molecular model (1, 3). Clearly, the
experimental and calculated Tm values do not all
agree, indicating that our earlier proposed molecular model is not an
optimal one and hence it needs to be refined. Here, we offer the
following two refinements. 1) Although the all-trans sn-1
acyl chain and the long all-trans segment of the kinked
sn-2 acyl chain are assumed to contribute mostly to the
chain melting process of trans
gauche isomerizations at Tm, the short segment may also
contribute somewhat to the chain melting process, especially if the
length of the short segment approaches that of the long segment. 2) If the positions of the two cis double bonds such as
5,8 are located near to the H2O/hydrocarbon
interface, the interchain hydration is postulated to increase, leading
to a decreased Tm. With these refinements, the
observed Tm values in Fig. 7, A and
B, can be reasonably explained in comparison with the calculated Tm values. Support for the assumption
that the short segment may contribute somewhat to the chain melting process at Tm comes from the experimental and
calculated Tm values of monoenoic PE as shown in
Fig. 7C. Finally, it should be emphasized that the revised
molecular model can serve best as a qualitative means to explain the
Tm-lowering effect of acyl chain unsaturation at the
present time. It remains a challenge to develop a simple and unified
mathematical expression in terms of structural parameters in describing
precisely the Tm profile observed in the plot of
Tm versus
n,n+3 or
n. Nevertheless, the refined MM-based molecular model
proposed in this investigation may be further improved in the future to meet the challenge, when the data base is expanded continuously.