Monolayer Behavior of NBD-Labeled Phospholipids at the Air/Water ...

5544 Langmuir, Vol. 18, No. 14, 2002 Tsukanova et al.
one goes from phosphatidylethanolamine to phosphatidylcholine
through the addition of methyl groups: the
former phospholipid was found to bind 7-12 water
molecules, while values ranging from 30 to 35 water
molecules per phospholipid were reported for the latter. 43
Due to the presence of the NBD chromophore, a larger
headgroup size will likely favor a further increase in
hydration. Nevertheless, comparing the hydration potentials
of phosphatidylethanolamine and phosphatidylcholine,
large changes in hydration give rise to a value of
µ H2O/ɛ H2O equivalent to an increase in ∆V of ∼31 mV. Hence,
considering that this simplification leads to some underestimation
of group dipole contributions, we choose to
neglect changes in µ H2O/ɛ H2O due to NBD incorporation in
the phospholipid structure in our calculations as relatively
insignificant. Then, we will consider that for a given
temperature, pH and area per molecule the contributions
of µ CH3 /ɛ CH3 and of µ CdO /ɛ CdO to ∆V are identical to those
pertaining to the DPPE monolayer. 20 Thus, we may ascribe
differences in the surface potentials of DPPE and DPPE-
NBD mainly to the substitution of two phosphatidylethanolamine
headgroup protons by the NBD chromophore.
Then, we may write, after the subtraction of eq 1
from eq 2
µ NBD /ɛ NBD ) ɛ 0 A(∆V DPPE-NBD - ψ 0 - ∆V DPPE ) (3)
where ψ 0 is the double-layer potential of the DPPE-NBD
monolayer. On the basis of the intrinsic pK a of the
phosphatidylethanolamine headgroup, DPPE can be
considered as a zwitterion bearing a permanent positive
charge on the amine group and a negative charge on the
phosphatidyl group over a pH range of 2.3-8. 36 As a result,
the contribution of the ψ 0 potential in ∆V DPPE , in particular,
at pH 5.6 will be negligible.
To calculate µ NBD /ɛ NBD using eq 3, the ψ 0 potential of the
DPPE-NBD monolayer should be determined. As suggested
by Dynarowicz-Latka et al., 44 a portion of ∆V
associated with the double-layer potential can be found
through a comparison of the surface potentials of a
nonionized DPPE-NBD monolayer on an acidic subphase
and an ionized one on pure water. Given an intrinsic pK a
of 0.32-0.7 for the phosphatidyl group 36 and taking into
account the ionization properties of the NBD chromophore,
1,10 it is reasonable to expect that the PE - -NBD
group will be completely neutralized by a proton, probably
in the form of an ion pair, at a subphase pH e 2. Thus,
to obtain the dependence of the ψ 0 potential on the area
per molecule for the DPPE-NBD monolayer on pure water,
the ∆V-A isotherm of the DPPE-NBD monolayer spread
onto a subphase containing HCl at a pH of 1.9 was
measured (curve c in Figure 5) and, then, subtracted from
the ∆V-A isotherm shown in Figure 5A (curve a). The
dependence of the ψ 0 potential on the area per molecule
is presented in Figure 5B. The fact that the resulting ψ 0
potential does not obey the exponential dependence
predicted by the Gouy-Chapman model is worth special
attention and will be discussed below. Since values of the
ψ 0 potential of the DPPE-NBD monolayer at the air/water
interface at different areas per molecule can be obtained
from Figure 5B, the value of µ NBD /ɛ NBD can be readily
assessed using eq 3. In the 0.6-0.4 nm 2 /molecule region,
eq 3 yields µ NBD /ɛ NBD ≈ -0.23 D (to convert the calculated
value of µ NBD /ɛ NBD into Debye units, the conversion factor
1D) 3.335 × 10 -30 C‚m was applied 29 ). We stress that,
although the absolute value of µ NBD /ɛ NBD might be underestimated
due to the simplification of our analysis,
(43) Sen, A.; Hui, S.-W. Chem. Phys. Lipids 1988, 49, 179.
(44) Dynarowicz-Latka, P.; Dhanabalan, A.; Cavalli, A.; Oliveira, O.
N., Jr. J. Phys. Chem. B 2000, 104, 1701.
the contribution of µ NBD /ɛ NBD to ∆V was indeed found to
be negative, which is not unreasonable as explained below.
The NBD heterocyclic chromophore is flat and, because
of the distribution of charges in its structure, bears a
permanent electric dipole moment oriented as drawn
schematically in Figure 6a, making an angle of 43° with
respect to the N-NO 2 axis. 41 With this model, it is thus
possible to discuss the orientation of the dipole moment
MB at the air/water interface on the basis of the ∆V data
analysis. Recent calculations 29,36 showed that a positive
portion of ∆V of the DPPE monolayer originates from two
main dipole moments: µ CH3 that characterizes the aliphatic
chain terminal group and µ CdO corresponding to the
carbonyl CdO group of the phospholipid molecule. Structural
studies on phospholipid monolayers and bilayers 36
indicated that both dipoles in a closely packed state were
oriented essentially parallel to the surface normal with
their strong projected dipole moments µ CH3 and µ CdO
directed into the monolayer (see Figure 6b), thus contributing
positively to the surface potential. By contrast,
the phosphatidylethanolamine dipole with its ethanolamine
group slightly repelled away from the monolayer
toward the water subphase gives the projection µ PE onto
the surface normal antiparallel to µ CH3 and µ CdO as shown
in Figure 6b. As a result, it contributes negatively to the
surface potential. 29,36 Consequently, the negative µ NBD /
ɛ NBD value implies that the orientation of the NBD dipole
moment MB at the air/water interface must be similar to
that of the phosphatidylethanolamine dipole. Therefore,
any orientation of the NBD group as shown in Figure 6c
may be ascribed to the NBD chromophore in the DPPE-
NBD monolayer as long as the projection µ NBD on the
surface normal remains negative. As shown in Figure 6b,
the depiction with the NBD chromophore located underneath
the phosphatidylethanolamine group is consistent
with the conclusion derived from the π-A isotherm: the
NBD group does not require any additional area in the
condensed monolayer. Indeed, given the size of the NBD
group (approximately 0.6 nm × 0.5 nm in the plane and
0.025 nm thick 45 ), such a localization (Figure 6b) will not
alter the area per molecule in the condensed state to any
remarkable extent because the area of ∼0.125 nm 2
required per NBD is sufficiently small to be readily
accommodated underneath the phosphatidylethanolamine
headgroup.
Following a similar approach, the contribution of the
NBD chromophore to the surface potential of the NBD-
(C 12 )-PC monolayer was also estimated. Comparing the
∆V-A isotherm of the NBD(C 12 )-PC monolayer (curve b,
Figure 5A) and that of the parent phospholipid (curve d,
Figure 5A), the attachment of the NBD chromophore
terminally to an aliphatic chain notably causes a decrease
in ∆V of 320 mV. As the headgroup of both monolayerforming
compounds is phosphatidylcholine completely
ionized at pH 5.6 used in the experiments, the presence
of both DPPC and NBD(C 12 )-PC at the interface in
zwitterionic form rules out the possibility for the doublelayer
ψ 0 potential to alter the monolayer ∆V. Moreover,
both monolayers collapse at approximately the same area
per molecule, so that at the end of the isotherm the
terminal CH 3 group, the carbonyl, and the phosphatidylcholine
dipole moments of the DPPC and NBD(C 12 )-
PC molecules are probably all projected alike onto the
surface normal. Consequently, the contributions µ CH3 /ɛ CH3 ,
µ CdO /ɛ CdO and µ PC /ɛ PC as well as the contribution of the
oriented water dipoles, µ H2O/ɛ H2O,to∆V of both monolayers
are almost identical at a given temperature, pH, and area
per molecule. 20 Then, the decrease in ∆V NBD(C12)-PC com-
(45) Flament, C.; Graf, K.; Gallet, F.; Riegler, H. Thin Solid Films
1994, 243, 411.