(Received for publication, September 21, 1995; and in revised form, November 14, 1995)
From the
In lipid bilayers, pyrene and pyrene-labeled lipids form
excimers in a concentration-dependent manner. The aromatic amine N,N-diethylaniline (DEA), which has a high
membrane-to-medium partition coefficient, quenches the monomers only,
and therefore it is expected that under conditions in which the
monomers are in equilibrium with the excimers due to the mass law, the
Stern-Volmer coefficient (K) for monomers (M),
defined as K
, should be identical to that of the
excimer (E), defined as K
, and K
/K
= 1.0. This is
indeed the case for pyrene and pyrene valerate in egg
phosphatidylcholine small unilamellar vesicles. However, for pyrene
decanoate and pyrene dodecanoate in these vesicles, and for N-[12-(1-pyrenyl)dodecanoyl]sphingosylphosphocholine
in a matrix of either N-stearoyl sphingosylphosphocholine or
1-palmitoyl-2-oleoyl phosphatidylcholine, K
< K
. This can be explained either by the existence
of (a) two subpopulations of excimers, one in fast equilibrium
with the monomers and the other, related to ground-state
protoaggregates of pyrene lipids; (b) two monomer
subpopulations where part of M cannot be quenched by DEA; or (c) two monomer subpopulations, both quenched by DEA, but only
one of which produces excimers. The good agreement between the
photophysical processes determined by steady state and time-resolved
measurements supports the third explanation for the bilayers containing
pyrene phospholipids. It also suggests that the main factors
determining the immiscibility of pyrene lipids in phospholipid bilayers
are the temperature, the difference in the gel-to-liquid-crystalline
phase transition temperature (
T
)
between the matrix and the pyrene lipid, and the structural differences
between the matrix lipid and the pyrene-labeled lipid. These results
indicate that the K
/K
ratio
can serve as a very sensitive tool to quantify isothermal microscopic
immiscibility in membranes. This novel approach has the following
advantages: applicability to fluid phase immiscibility, requirement of
a relatively low mol fraction of pyrene lipids, and conceivably,
applicability to biological membranes.
Biological membranes are complex multicomponent assemblies. It
is well established that the matrix of these membranes, the lipid
bilayer, has two faces that are compositionally distinct (for review,
see (1, 2, 3) ). At the level of resolution
of the wavelength of visible light (0.5 µm), studies based on
determination of the immobile fraction in a fluorescence recovery after
photobleaching experiment indicate that biological membranes are
laterally heterogeneous and have in-plane domains distributed in a
homogeneous lipid continuum(4, 5, 6) . More
precise fluorescence recovery after photobleaching studies were
recently performed in lipid bilayers of defined composition in which
solid and fluid phases coexist, and the phase diagrams are well
characterized ( (7) and (8) and references therein).
However, not much information is available on lateral organization at
the submicron range. The functional significance of lateral
heterogeneity must be related to creation of microenvironments that
enable better control of functional assemblies in the membranes such as
channels and signal transduction systems. It is expected that the
kinetics of interaction between membrane components should be very
different when the interaction occurs in a restricted domain or a
continuum(9) . Since most of these assemblies are relatively
small (<100 nm), it is important to study how they are affected by
both the micron and submicron scales of lateral heterogeneity. Our goal
is to better understand the lateral organization of biological
membranes. Since they are complex multicomponent systems, it is
necessary first to study well-defined one-, two-, and three-component
lipid bilayers in the liquid, liquid-ordered, and solid-ordered phases (10) as well as systems in which various combinations of these
phases coexist. The model systems to be used to characterize lateral
organization at the submicron range should be applicable to both lipid
bilayers and biological membranes, which is the case for the
fluorescence recovery after photobleaching approach used for the micron
range organization (5, 7, 8, 11) .
Most of the approaches available for quantification of lateral
heterogeneity at the submicron
range(1, 12, 13, 14) , although
informative, suffer from either theoretical or practical drawbacks such
as low sensitivity, inability to measure fluid-phase immiscibility, and
inability to be used in parallel in both model systems and biological
membranes(13, 15) .
Here we evaluate a novel
approach based on the ability to detect and quantify the lateral
organization of pyrene-labeled lipids present in lipid bilayers of
defined compositions. This method can be applied to biological
membranes that can be labeled by introducing a pyrene fluorophore
either metabolically (for review, see (16) ), or enzymatically
by phospholipid exchange proteins (17) using liposomes as
donors. A third approach of spontaneous diffusion from a labeled donor
is rather limited due to the very low desorption rate (k), which is the rate-limiting step in the
exchange process (for review, see (3) ).
Pyrene-labeled
lipids were extensively used in membrane research to determine the
dynamics and organization of lipid membranes and biological membranes ( (18) and references therein). The most common use of pyrene and
pyrene lipid is based on the ability of one excited pyrene moiety,
together with a nonexcited pyrene moiety, to form an excited-state
dimer (excimer). This reaction was studied extensively in organic
solvents and proved to be a diffusion-controlled bimolecular
reaction(19, 20) . This assumption was used to study
the dynamics of lipid bilayers(21) . The approach of simple
controlled diffusion was slightly modified by Galla et
al.(22) . However, the importance of the diffusion became
more of a controversial issue as recent data suggested that pyrene
excimer formation in membranes may not be diffusion controlled. The
explanation for the rate-limiting step in this process is not clear and
may be dependent on the exact pyrene lipid and method used (compare
Blatt et al.(23) and Lemmetyinen et
al.(24) ). It was found that for binary systems in which
the temperature-dependent determination of the ratio between the level
of excimers and monomers (E/M ratio) ()was studied, most of
the methods and systems have only limited use to determine the lateral
organization of the pyrene lipid in bilayers due to their inability to
be applied to cases in which the matrix lipid has a lower transition
temperature than the pyrene-labeled lipid(15) . An important
contribution is the finding of Martins and Melo(25) , which
supports previous suggestions that the Birks three-dimensional
formalism is not applicable for systems of reduced dimensionality such
as lipid bilayers. Of special importance in the two-dimensional system
is the time dependence of the fluorescence decay rate coefficient,
which eliminates the need to assume the long term association of
ground-state (``static'') pyrene lipids in the bilayer plane.
The product of this association is referred to as the pyrene
ground-state protoaggregate. Recently Chong and
co-workers(26, 27) , using pyrene lipids, showed that
the fluorescent lipids were organized into hexagonal super-lattices in
the bilayer plane, information that could not be obtained by other
methods.
In most studies done so far, the ability to distinguish between two monomer populations in bilayers in which solid and fluid domains coexist is based on the discrepancy between the decay of the excited monomer and excimer populations. This was used to show that a fraction of the pyrene probes is isolated and does not participate in the domain formation(28) . In this study, we present another approach, which is based on using aromatic amines as pyrene (or pyrene moiety) quenchers. The aromatic amines selectively quench only the pyrene monomers, thereby enabling one to determine lateral immiscibility of pyrene and pyrene-labeled lipids in lipid bilayers.
Figure 2:
The dependence of excimer to monomer
fluorescence intensity ratio on the pyrene or pyrene-fatty acid mole
fraction in egg PC SUV at 25 °C. The pyrene fatty acids are 5-(1
pyrenyl valeric acid (), 10-(1-pyrenyl decaneoic acid (
),
and 16-(1-pyrenyl hexadecanoic acid (
). Pyrene (
) was 99.9%
pure. The egg PC and the pyrene or pyrene-labeled lipids were mixed in
chloroform/methanol, 2:1 (v/v) at the desired mol ratio, and SUV were
prepared as described under ``Materials and Methods.'' Egg PC
SUV (1 mM egg PC) were used. The concentration of the pyrene
or pyrene fatty acid was varied between 0.03 mM and 0.1 mM (3-9 mol %). All data were corrected for light scattering,
which was minimal for the SUV used in this study. Oxygen was removed,
and fluorescence measurements were performed as described under
``Materials and Methods.''
The unique property of pyrene (and pyrene-labeled molecules) to form excimers by two mechanisms: (a) as a result of a dynamic collision between a ground-state and an excited-state monomer (M and M*, respectively), producing a ``dynamic'' excimer, and (b) as a result of a static interaction between M and M* in close proximity, producing a ``static'' excimer(20, 23, 24, 26, 27, 33) .
The ability to distinguish between, and separately quantify, quenching of monomers (M*) and excimers (E) from Stern-Volmer plots under conditions that the quencher directly interacts only with M* and not with E.
Therefore, based on the mass law, it is expected that
the Stern-Volmer constant for M* and for E (K and K
, respectively) will be equal. As is demonstrated
below, this is not the rule for all cases studied. Depending on
miscibility of the matrix lipid with the pyrene-labeled lipid and the
temperature of the measurement, it was found that for many systems K
> K
. This can be
explained in three alternative ways. (a) There exists at least
one excimer subpopulation that is not in equilibrium with quenchable
monomers; namely, that this population is of ground-state
protoaggregates referred to as static
excimers(19, 20, 23) . (b) There
exists an equilibrated subpopulation, M* + M &cjs0635; E, where
part of M* is not accessible to the quencher. Or (c) there
exists a monomer subpopulation that does not form excimers. The pattern
of cases in which K
> K
is
not random; it usually occurs under conditions of coexistence of two
phases, such as gel and liquid-crystalline. Use of the ratio K
/K
is proposed to quantify
the two populations, either of excimers or of monomers (see a-c above).
E = E
+ E
and M*
= M*
+ M*
, where M*
are monomers in
equilibrium with excimers, E
are dynamic excimers in
equilibrium with M*, E
are static excimers not in equilibrium with monomers, and M*
are quenchable
monomers that do not form excimers. In all these cases (a-c above), the ratio K
/K
is
defined as X, where X and 1 - X give
the fraction of M*
and M*
, respectively.
These definitions are valid only if all quenchable monomers have the
same k
(bimolecular quenching rate constant) and
(lifetime of the excited state of the monomer in
dilute solution), and only the monomers are quenched by DEA.
First, it was demonstrated that DEA quenches only the monomers. This was then applied to examples of lipid vesicles composed of well characterized binary mixtures of a matrix phospholipid and a pyrene-labeled lipid at defined temperatures. The steady state results were also confirmed by dynamic time-resolved measurements.
Our previous study (18) indicates that the aromatic amine DEA, which quenches
excited pyrene moieties through a charge transfer mechanism, is a more
efficient quencher (k in ethanol at 25 °C is 1
10
M
s
) than heavy atom derivatives such as
bromobenzene and iodide, which quench the pyrene through a spin-orbit
coupling mechanism. One of the advantages of DEA as a quencher is its
high membrane-to-buffer partition coefficient(18) . In order to
study which one of the excited species of pyrene (monomer or excimer)
is quenched by DEA, we characterized the quenching of pyrene in ethanol
under two sets of conditions: (a) pyrene at 3.6
10
M, at which no excimers can be detected
and (b) pyrene at 1.8
10
M, at which most of the fluorescence intensity is
associated with the excimer peak at 480 nm (E/M = 6).
Stern-Volmer plots were obtained using the basic Stern-Volmer
equation(34) .
K is the Stern-Volmer coefficient, and
[DEA] is the quencher concentration, describing F
/F - 1 as a function of DEA
concentration for the two concentrations of pyrene, for monomers and
excimers (the latter for the 1.8
10
M pyrene only). DEA in the range of 4
10
to 1
10
M was used. K
of 4.9
10
M
and 92.6 M
were obtained for 3.6
10
M and 1.8
10
M pyrene,
respectively (Fig. 1). Also, within experimental error, the data
show that K
= K
,
suggesting that no real quenching of excimers takes place and that the K
obtained is related to the dissociation of E to
the monomers. This was further tested as follows. The bimolecular
quenching rate constant (k
) was calculated as
Figure 1:
Quenching of pyrene monomers (M) and
excimers (E) in ethanol by DEA. Pyrene was dissolved in ethanol
(spectral grade) at a concentration of 3.6 10
M (M only,
), and 1.8
10
M (E/M = 6.0) DEA in ethanol at the concentration
range specified in the figure was added.
, monomer;
,
excimer. The fluorescence measurements were performed as described
under ``Materials and Methods.'' The intensity of the
fluorescence emission was determined at the monomer peak (393 nm) and
excimer peak (480 nm) in the absence (F
)
and the presence (F) of the desired DEA concentration. For
more details see text.
(where is the lifetime of the excited state
of the monomer obtained in dilute solution). In the presence of high
concentration of M, the measured lifetime of the excited state of the
monomer (M*) is reduced according to
where k is the first-order rate constant of
the fluorescence, k
is the first-order rate
constant of all the nonradiative processes, k
is
the rate constant for excimerization in ethanol, and [Py] is
the pyrene concentration. Using the values obtained by Birks of 6.0
10
M
s
for k
(20) and
(475 ns) (in the absence of oxygen and at low pyrene
concentration), and for the lifetime of the excimers,
(in the absence of oxygen) = 53 ns permits us to calculate
the sum of k
+ k
. These
are the intrinsic characteristics of pyrene(19) ; from these,
the
for the concentrated pyrene solution was
determined. The bimolecular quenching rate constant, k
, was obtained for the DEA in dilute pyrene
solution using
values, the Stern-Volmer rate
constant, and . k
= 1.04
10
M
s
. For the concentrated pyrene solution,
of 9.1 ns was calculated using (a
shorter lifetime is expected due to the large extent of
excimerization). This
was used in and a k
of 1.02
10
M
s
was obtained
for DEA-pyrene quenching at high pyrene concentration. This value is
almost identical to the k
value of 1.04
10
M
s
obtained for the low pyrene concentration. The identity in k
for the low and high pyrene concentrations
indicates that the excimerization does not interfere with the quenching
of monomers by DEA and that only monomers are quenched by DEA.
Fig. 2shows that the effect of probe mol fraction on its capability to form excimers was in the order pyrene > pyrene hexadecanoate > pyrene decanoate > pyrene valerate. It seems that the attachment of a paraffinic chain to pyrene reduces its ability to form excimers, which may be related to reduced mobility and/or steric restriction, which reduces the ability of the M* and M to interact in a productive way to give excimers. The data presented in Fig. 2are in good agreement with previous data on the ``order parameter'' along phospholipid acyl chains (1, 2) and therefore lend support to a major contribution of steric restriction. Our previous data on a similar system suggest that the steric factor should be more important than the diffusion rate (Table III in (18) ), in agreement with the suggestion that the rotational energy barrier to excimer formation is higher than the translational one(35, 36) .
Fig. 2also shows that the curves for pyrene and pyrene
valerate were linear for all the concentration ranges tested, while for
pyrene decanoate, and, even more so, pyrene hexadecanoate, a strong
positive deviation from linearity was observed. For pyrene
hexadecanoate, the E/M ratio, when extrapolated to zero concentration,
gave a finite value of 0.2, while for the other two pyrene
derivatives and pyrene a value of
0 was obtained. This may be
related to phase separation of pyrene decanoate and more so for pyrene
hexadecanoate in the egg PC bilayer, leading to the formation of static
excimers that exist as dimers (or other aggregates) before the
excitation(20) . Such phase separation is expected from phase
diagrams for phospholipid and fatty acid
mixtures(37, 38) , in agreement with what was
suggested before by Lemmetyinen et al.(24) .
Accordingly, a subpopulation of the excimers is derived from collisions
between M* and M, which are in close proximity at the time of
excitation. Therefore, the high local concentration of monomers exists,
which shifts the local equilibrium to form a high E concentration even
at relatively low total mol fraction of pyrene hexadecanoate. These
excimers are not in equilibrium with the total monomers, and therefore
they are referred to as E
. This favors the existence of
two excimer populations for the pyrene hexadecanoate in egg PC.
Previous studies using pyrene hexadecanoate in PC SUV did not show this behavior(33) . However, these studies were done in bilayers having lower mol fraction of the pyrene hexadecanoate and no data of E/M versus mol fraction of the pyrene hexadecanoate were reported.
Fig. 3describes the DEA Stern-Volmer plots
for M* and E for the same pyrene lipids described in Fig. 2. All
(except the highest) DEA concentrations used were below the DEA/lipid
concentration, which may have a large effect on bilayer properties (18) . For pyrene (a) and pyrene valerate (b)
the slopes of the DEA Stern-Volmer plots for M* and E are identical (K = K
), which is not
the case for pyrene decanoate (c) and pyrene hexadecanoate (d), for which K
> K
. The ratio K
/K
was used to calculate the
mol fraction not in M* &cjs0635; E equilibrium, which was 25 and 39%,
respectively, for these two pyrene-labeled fatty acids. All plots in Fig. 3were fitted using linear regression. However, it is
evident that some of the curves may deviate from linearity and can
better be fitted to a hyperbola. This was not studied in detail,
although such nonlinearity is consistent with the presence of two
populations of fluorophores, one of which is not accessible to the
quencher. This may be an artifact, as the highest concentration of DEA
used (DEA/lipid ratio > 0.4) may have a large effect on membrane
structure(18) . The two different criteria represented by Fig. 2and Fig. 3agree and both describe deviation from
the behavior expected if the systems were governed solely by the
diffusion-dependent collision model. However, the DEA-quenching
criterion seems to be more sensitive since it monitors the deviation at
a lower mol fraction of the pyrene-labeled lipid than the E/M approach.
No DEA-pyrene exciplexes (18) were found in the spectrum at 6
mol % pyrene lipid; that is, no other interaction between DEA and the
pyrene moiety could be detected. Vesicles composed of phospholipids and
fatty acids may represent a special case since there are large
differences in structure and molecular dimensions of the fatty acids
and the matrix PC(38, 39) . Therefore, the study was
extended to other systems in which both the matrix lipid and the
pyrene-labeled lipids are phospholipids of defined acyl chain
composition and gel-to-liquid-crystalline phase transition temperature (T
). (
)The quenching experiments were
conducted using 1 mM total phospholipids. Stern-Volmer plots
were performed over a DEA concentration range of 0-350 µM (below the DEA/lipid ratio that may cause pronounced perturbation
to vesicle structure (18) .
Figure 3:
Stern-Volmer plots for DEA quenching of
pyrene (A), pyrene valerate (B), pyrene decanoate (C), and pyrene hexadecanoate (D). Egg PC SUV
containing the pyrene or pyrene fatty acid were prepared as described
in Fig. 2. DEA in an oxygen-poor ethanol solution was added to
the oxygen-poor egg PC SUV suspension (1 mM egg PC containing
10 mol % pyrene or the specified pyrene fatty acid). The final ethanol
solution did not exceed 0.5%. The DEA is volatile and therefore has to
be added in the final step (18) . The high pH of the medium (pH
8.0, ``Materials and Methods'') ensured that most of the DEA
(pK = 6.5) is in its neutral form
in order to obtain its favorable partition into the lipid bilayer.
Fluorescence measurements were performed and the Stern-Volmer plots for
monomers (
) and excimers (
) were obtained (after
correction for light scattering, Fig. 2) as described in the
legend to Fig. 1and Fig. 2.
At least 10 different DEA
concentrations were used for each curve. All curves were fitted best
for linear plots throughout all the DEA concentration range, with a
correlation coefficient >0.993 (which was not the case in Fig. 3). Table 1clearly demonstrates that in all the
systems there is a deviation from a complete M* &cjs0635; E
equilibrium. However, the degree of deviation varied to a large extent,
from 5% for Py-C-SPM in a matrix of C
-SPM at
50 °C, to complete lack of equilibrium for Py-C
-SPM in
1-palmitoyl-2-oleoyl-sn-PC at 20 °C. The curves describing
the E/M dependence on the mol fraction of pyrene lipids in the matrix
were all linear at the concentration range used in this study
(0-4 mol %), which may suggest that in this example the presence
of two monomer populations explains the deviation from equilibrium. The
ratio between the two can be determined from the K
/K
ratio. This quenching
approach is therefore more informative than the E/M versus temperature (15) since it can be successfully applied also
to bilayers in which the matrix lipid has a lower T
than the pyrene-labeled lipid, such as vesicles composed of
1-palmitoyl-2-oleoyl-sn-PC and Py-C
-SPM. It can
also be used to determine fluid-phase immiscibility, which is difficult
to monitor by most other methods. Also, this method can detect
immiscibility even when the pyrene-labeled lipid is a minor component
composing only 2 mol % of the bilayer, or when the two lipids are only
0.6 °C apart in their T
.
The same photophysical processes were monitored by time-resolved fluorescence emission measurements at both the monomer and excimer emission bands. The results of the time-resolved measurements are summarized in Table 2.
Pyrene monomers have relatively very long fluorescence lifetimes, which makes them ideal probes for time-dependent dynamic intermolecular processes. The results shown in Table 2confirm the conclusions obtained in the steady state measurements.
(a) The lifetime of the monomer was reduced
by increasing the mol % of either pyrene or Py-C-SPM in
vesicles composed of egg PC as the matrix lipid, in agreement with a
dynamic process of excimerization.
(b) DEA was partitioned
into the lipid bilayer and quenched the fluorescence of the monomer
only. The fluorescence lifetime of the monomer in both the free pyrene
and the lipid-bound pyrene (Py-C-SPM) was drastically
reduced by DEA. The lifetime of the fluorescence of the excimer was not
affected for both pyrene and Py-C
-SPM. This confirms the
conclusions obtained from the steady state measurements and indicates
that indeed only monomers are quenched by DEA in homogeneous systems
such as ethanol (Fig. 1) and in lipid bilayers (Table 2).
(c) The dynamic mechanism of the excimer formation was confirmed by the observation of the negative preexponent in the decay process of the excimer fluorescence. Both the free pyrene and the lipid pyrene derivative showed this characteristic of the excited state reaction. The fact that the two preexponents in the decay curve of the excimer emission were not of the same absolute value may be an indication of heterogeneity of the population of monomer molecules. A possible source can be a subpopulation of molecules that does not form excimers (see ``Assumptions and Modeling,'' above).
(d) As was discussed by Martins and Melo(25) , there is no need to assume the presence of ground-state aggregate, explanation a under ``Assumptions and Modeling,'' above. Therefore, our data favor explanation c, namely that there are at least two populations of excited monomers, one of which cannot excimerize.
The good agreement between the photophysical processes of
pyrene and pyrene lipid in lipid bilayers monitored by steady state and
time-resolved measurements described above led us to suggest that the
main factors contributing to immiscibility of the pyrene lipid in the
matrix lipid are (a) the temperatures at which the measurement
was performed, (b) the difference in T (
T
) between the matrix lipid and the
pyrene lipid, and (c) structural factors such as the presence
of pyrene fluorophore on the
position of the acyl chain, which,
due to its bulkiness, increases the volume of the hydrophobic region of
the phospholipid molecule. Therefore c may be the overriding
factor in determining lateral immiscibility. It may be that c is also related to the hexagonal super-lattices found in bilayers
of binary mixtures composed of pyrene PC and dimyristoyl-PC, or in
other PCs ( (26) and (27) and references therein).
Recently (39) it was demonstrated that the immiscibility of
pyrene PC in a PC bilayer is increased by partial dehydration of the
phospholipid headgroup, which further increases the packing
parameter(39, 40) .
The relevancy of bilayers composed of phospholipids and pyrene lipids to understanding the miscibility of lipids in biological membranes is not yet clear. However, biologically relevant structural features that may contribute to fluid (or liquid-ordered) lateral immiscibility do exist, for example, in sphingolipids where the mismatch between the acyl chain and the sphingosine base chain may be expressed as interdigitation or as protrusion of the headgroup above the plane of the headgroup of the symmetric matrix lipid(41, 42) . Indeed, it was demonstrated that partial dehydration increased immiscibility between PC and glucosylceramide in bilayers(45) .