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Mixed monolayers of valinomycin and dipalmitoylphosphatidic acid
COLLOIDS AND Colloids and Surfaces A: Physicochemicaland EngineeringAspects 94 (1995) 75-83
ELSEVIER
A
SURFACES
Mixed monolayers of valinomycin and dipalmitoylphosphatidic acid Sergei Yu. Zaitsev a,,, Vitaly P. Zubov a, Dietmar MObius b a Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871, Russia b Max-Planck-Institutf~ir Biophysikalische Chemie, Postfach 2841, 37018 Gdttingen, Germany
Received 23 February 1994; accepted 13 May 1994
Abstract
The interactions of mixed monolayers of the hydrophobic peptide valinomycin and the negatively charged phospholipid dipalmitoylphosphatidic acid (DPPA) with alkali metal cations in the aqueous subphase were investigated. For this purpose, the isotherms of surface pressure, surface potential and Brewster reflectivity versus molecular area were measured for the mixed monolayers (valinomycin-DPPA) in the absence and presence of Na + and K + ions, respectively, in the aqueous subphase. Complete miscibility of the components in the monolayers is deduced from Brewster angle microscopy. Two additional states of valinomycin-DPPA monolayers were observed when K + ions were present instead of Na + ions in the aqueous subphase. This phenomenon correlates with the strong complexation between the valinomycin and K + ions in the bulk. The negatively charged phospholipid (DPPA) influences the ability of valinomycin to bind cations at the interface. Keywords: Brewster angle microscopy; Complexation; Dipalmitoylphosphatidic acid; Ionic selectivity: Monolayers; Valinomycin
1. Introduction Mixed lipid monolayers are particularly suited as models of biological membranes, especially because they allow us to observe interactions between surface-active molecules at the interface and those with components in the aqueous subphase. Many investigations have been devoted to the question of miscibility of various lipids in monolayers [ 1-5], but only some of them describe mixed monolayers of phospholipids and biologically active molecules (such as peptides) that are particularly important for the modelling ofbiological membranes,
* Corresponding author,
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Only a few recent studies have dealt with mixed monolayers of lipids and the peptide ionophore valinomycin at the air/water interface [6 91. In most cases [ 6 - 8 ] , the effect of the negatively charged monosialoglycosphingolipids and disialoglycosphingolipids (GM1and GDlagangliosides) on the molecular organization and physicochemical properties of valinomycin in mixed monolayers at the air/water interface was investigated. Deviations from linearity in the plots of the average surface pressure and potential indicate that valinomycin, interacting with the polar head groups of the gangliosides, becomes partly embedded within the lipid interface [8]. In contrast, no deviation from the additivity rule occurred for the mixture of valinomycin with zwitterionic lipids:bovine brain lecithin ['7] and dimyristoyl-
76
S. Y. Zaitsev et al./Colloids SurJaces A: Physicochem. Eng. Aspects 94 (1995) 75 83
phosphatidylcholine (DMPC) E9]. The authors [9] conclude that valinomycin does not perturb the properties of the phospholipid monolayer, whatever the value of the ionophore-to-lipid ratio in the membranes above or below the phase transition temperature T~ of DMPC. However, they did not specify which of the two possibilities (ideal miscibility or complete immiscibility) occurs in this case. Mixtures of valinomycin with stearic acid [ 10-12], arachidic acid [ 13,14], eicosanol [-8] and cholesterol [12] were also investigated. Mixed monolayers with fatty acids were studied for the preparation of Langmuir-Blodgett films on various solid supports with the aim of applying such systems as biosensors [-10,13]. Most of the lipid valinomycin monolayers mentioned above were prepared on a pure water subphase. Thus the influence of various cations on the valinomycinlipid interactions was not investigated in detail. On the other hand, much more work has been carried out previously with pure valinomycin monolayers [-15 21] on various salt-containing subphases. Two additional states of valinomycin monolayers were observed when K + and Rb + ions were present instead of the other alkali metal cations [-15,19 21]. This phenomenon correlates with the strong complexation between the valinomycin and these cations in bulk [-22]. An influence of the size of the anion on the stability of valinomycin-cation complexes at the interface was found [19 21]. Valinomycin is one of the most important and widely investigated ionophores which can selectively increase the cation permeability through biological and synthetic membranes [22]. Taking into consideration the importance of interfacial layers for membrane studies, it is necessary to investigate in detail the properties of valinomycin in mixed lipid monolayers at the water/air interface. Dipalmitoylphosphatidic acid was chosen for this study as one of the common lipids of biological membranes, having a negative charge that can influencethemonolayercharacteristicsofthevalinomycin and its interaction with various cations from the aqueous subphase. The methods of surface pressure and surface potential measurements versus molecular area [ 1,5], as well as the recently developed technique of Brewster angle reflectivity
and microscopy of monolayers at the water/air interface [23-25] were chosen for this investigation. Reflectivity measurements under the Brewster angle of incident light with respect to the pure water surface have already provided extremely important information about the molecular organization of lipids [-23-25] and pure valinomycin molecules in monolayers [19]. Therefore it was very interesting to apply such a new technique to the investigation of mixed monolayers of biologically active molecules at the interface. This study is mainly devoted to the observation of the interactions between valinomycin in mixed monolayers with DPPA and various alkali metal cations present in the aqueous subphase.
2. Experimental Dipalmitoylphosphatidic acid (DPPA) and valinomycin (Sigma, USA) were used as 1.0mM solutions in chloroform (HPLC grade; Fluka, FRG). All inorganic salts and organic solvents were of analytical grade. Monolayers of DPPA and valinomycin were prepared and investigated on a commercial film balance, using the Langmuir principle (horizontal detection [ 1,5]) for the surface pressure measurements (Lauda, FRG) and on a self-made rectangular Teflon trough (area, 355 cm2; depth, 1 cm) using the Wilhelmy principle (vertical detection [1,5]) for the surface pressure measurements and a vibrating plate condenser for the surface potential measurements respectively. Portions 10-20 p,1) of the valinomycin-DPPA solutions in various ratios were spread with a microsyringe onto distilled water or aqueous salt subphases. As a standard procedure, monolayers were rested 5 min before compression to allow sufficient solvent evaporation and to reach equilibrium with respect to the initial surface pressure and surface potential. The surface pressure (~)-molecular area (A) and surface potential (AV) molecular area (A) isotherms were recorded simultaneously during continuous cornpression of the monolayer by a movable barrier at a constant rate of 20cm 2 min 1. The temperature was 18.0 _+0.5°C. The change in surface potential of the air/water
S. Y. Zaitsev et al./Colloids Surfaces A: Physicochem. Eng. Aspects 94 (1995) 75-83
interface after monolayer formation expressed by the Helmholz equation:
(AV)
is
77
7060-
ii
A V = n#(1/eo) where n is the number of molecules in the monolayer, p is the mean change of the effective dipole moment per amphiphile molecule normal to the air/water interface, eo is the permittivity of the vacuum. Using the identity n = l / A , where A is the mean area per molecule, this equation can be transformed to the form
~s41dz ~ ; ! ]
30~ 1~
, .. , _, .. ,
~ ~o lo
# = e o AVA
o
,, ,
.....
,
0
1
2
3
Area/MoleculeInm21
in order to calculate the change of the effective dipole moment per molecule, which reflects changes in molecular orientation at the interface [ 1,4-8]. The reflectivity R = I/I o (where I is the reflected intensity and Io is the incident intensity) of the water surface at the Brewster angle under p-polarization w a s recorded simultaneously with
(a)
the
~ 400 .~. Si i:,¢ : ~'""",.,,ii, "'X'-, 4,."; .......-..6"-"" ......
surface pressure ( ~ ) a s a function of the area per molecule (A). The reflected light from a laser diode (power, below 1 mW; wavelength, 670 nm; light spot diameter, about 1 mm) in combination with a polarizer was detected with a photomultiplier (Hamamatsu R1635). The angles of incidence and observation were fixed to the Brewster angle for the pure water subphase (53°). The whole
coo ~ 600 _~ ~
===========__ ============= ...===......... ======.~.. .
~ 2~ ~ 200-
~.~ii~(...
- ~......
.......................
0 1 (b)
2 Area/Molecule[nm2]
3
system was fixed onto a tripod. By simply tilting
Fig. 1. Surface pressure area (a) and surface potential area
the tripod, small angle adjustments were possible, T h e optimal position was found by minimizing the background signal of the pure water. The system was mounted above a rectangular Teflon film balance. All data were recorded and processed by an IBM-compatible personal computer (type 386). A more detailed description of the system and technique has been published previously [25].
isotherms of the mixed monolayers of D P P A valinomycin at ratios 1 : 0 (curve 1), 10:1 (curve 2) 5 : 1 (curve 31, 3 : 1 (curve 4), 1:1 (curve 5), 1:3 (curve 6), and 0:1 (curve 7) on water
3. Results and discussion It was shown that valinomycin forms stable monolayers on water and various aqueous salt subphases [19]. Surface pressure and potential isotherms for pure valinomycin, phosphatidic acid and their various mixtures on water are shown in Fig. 1(a) The isotherms for pure DPPA and valino-
at 18°c. mycin are completely different. In the case of DPPA, the surface pressure increases very steeply at small monolayer areas (liquid-condensed state), whereas for valinomycin such an increase is relatively slow and is observed already at large areas (approximately ten times larger than for DPPA). These large differences are very important for the investigation of mixed monolayers of DPPA and valinomycin, because they allow us to determine the contribution of each component to the mixed monolayer characteristics. The area per molecule (extrapolated to zero surface pressure) for pure DPPA is around 0.4 nm 2 (Fig. 1 (a), curve 1 }; and
S. Y. Zaitsev et al./Colloids Surfaces A." Physicochem. Eng. Aspects 94 (1995) 75 83
78
for valinomycin, 3.2 nm 2 (Fig. l(a), curve 7); this is in good agreement with the literature data [1,4,5,15,21]. The surface pressure isotherms of mixed D P P A - v a l i n o m y c i n monolayers lie in the range between these isotherms (Fig. l(a), curves 2 6). The same differences are observed for the potential isotherms of pure valinomycin, phosphatidic acid and their various mixtures on water (Fig. l(b)). The surface potential increases steeply (total increase is about 250 mV) at low areas in the case of pure D P P A monolayers (Fig. l(b), curve 1) and very smoothly at large areas for valinomycin (Fig. l(b), curve 7). The potential isotherms of mixed D P P A - v a l i n o m y c i n m o n o layers on water lie in between these limits (Fig. 1 (b), curves 2-6)• The partial contribution of the valinomycin and D P P A in the total values is clearly seen from these mixed isotherms. It is remarkable that the surface potential decreases after passing a m a x i m u m upon compression for molar ratios of D P P A valinomycin from 10 : 1 to 1"1 (curves 2 5). This behaviour of deviations from the additivity of dipole m o m e n t s indicates a particular interaction between the two components. was shown earlier [13,17-19] that surface pressure and potential isotherms of pure valinomycin monolayers depend on the kind of aqueous salt subphases. The cations can be divided into
70-
,
6o -£ s0 v
40
~ ~: 30 ~ ~ 20 ,o
~i .~i ............. '
)
~
' ' , "'.
",,, 0 ......... (a)
, ......... , ......... Area/Mo~culel'~m2l
1
2
, ..........
3
800
E ~ ~ 400
:~:;~,.,,,, •....... •............... "~" ", ..... "' ,, -,.. ,-, ~ ,.
a,
~
..... ._
~ 200
0-
It
tWO groups. Firstly, surface pressure and potential isotherms for valinomycin on LiC1, NaC1, CsC1 and NH4CI (see Fig. 2 for the case of NaC1) are very similar to each other and are close to the same isotherms on distilled water (Fig. 1). Only the liquid-expanded state I is observed. Secondly, surface pressure and potential isotherms for valinomycin on KC1 and RbC1 subphases (Fig. 3 for the case of KC1) have some similarity to those in Figs. 1 and 2 only at low surface pressures and high areas (state I), but are completely different upon further compression. On 1.0 M solutions of these salts, both ~-A and A V-A isotherms have a special region in the surface pressure range 2 0 - 3 0 m M m -~ with corresponding areas 3.0-1•7 nm 2 per molecule (state II) as well as at 30 40 m N m '-1 and 1.7 0.6 nm 2 per molecule (state Ill)(see Fig. 3). It is i m p o r t a n t to stress that all three break points
0 (b)
. . . .
J
. . . .
~ i
. . . .
2 A~e~/~ol~'In~:~ i
. . . .
i
,
~ ,
i . . . .
3 i
. . . .
J
. . . .
Fig. 2. Surface pressure-area (a} and surface potential area isotherms of the mixed monolayers of DPPA-valinomycin at ratios 1:0 (curve 1), 10:1 (curve 21, 5:1 (curve 3), 3:1 (curve 4), 1:1 {curve 5}, 1:3 (curve 6), and 0:1 (curve 7) on 1.0 M solutions of NaCl at 18~C. in the g A isotherms, which can be assigned to the transitions between states I and II, II and III, and III and collapse of the monolayer, respectively, occur at the same areas as in the A V-A isotherms (Fig. 3). The break point between states I and II can be explained at the onset of the transition from an "expanded" conformation of the polypeptide ring of valinomycin, when the hydrophilic groups are facing the aqueous subphase, to the very special "bangle" conformation, in which all carbonyl and carboxyl groups of the amino acid residues are coordinated to the cation inside the polypeptide ring. The "bangle" conformation is very rigid and
S. Y. Zaitsev et al./Colloids Surfaces A." Physicochem. Eng. Aspects 94 (1995) 75-83
70 \ ~ 60 }'~:i~,i,, -g so.. r:~',, ~'~ 4030 20 L ,0 o,
1
2
/a)
3
Area/MoleculeInm~'l
1200lo0o
/"
_
800 •~ 600
-'"
..... ; -", 4 ,k ..", ~ ,' ...#.,' , ~...... '-, "'-..
" " ..
.........
d(~ iii ......."ii iiiiiiiii!!:::> ....iii i i
400 200 ' ' ........ (b)
.............. "..............
, . . . . . . . . . 2, . . . . . . . . . . . . .3 . . . . . . Area/Molecule[,,m:l
1
Fig. 3. Surface pressure-area (a) and surface potential-area isotherms of the mixed monolayers of DPPA- valinomycin 1:0 (curve 1), 10:1 (curve 2), 5:1 (curve 3), 3:1 (curve 4), 1:1 (curve 5), 1:3 (curve 6), and 0:1 (curve 7) on 1.0 M solutions of KC1 at 18C.
can be formed spontaneously even in solution in the presence of K + or Rb + ions, as was shown first by Ovchinnikov et al. [22]. It is correlated with the liquid-condensed character of the state II of valinomycin monolayers, in particular with its low compressibility. The surface pressure of the I - I I transition depends strongly on the concentration of K + ions in the aqueous subphase, while the pressure of the I I - l l I transition is always the same in the range 32.0-34.0 m N m - ~ at various concentrations of KC1 in the aqueous subphase. The transition I I - I I I may be attributed to a reori-
79
entation of the valinomycin molecules with the "bangle" conformation in the monolayer with a reorientation of the ring plane from parallel to perpendicular with respect to the interface. The monolayer in state III has a collapse in the range 38.0-40.0 m N m -1. This unusual behaviour of valinomycin monolayers on KCI subphases can be explained as complex formation of ionophore at the interface with a cation from the subphase. With this behaviour of valinomycin in mind, it was particularly interesting to investigate mixed monolayers of D P P A and valinomycin on various aqueous salt subphases. NaC1 and KC1 subphases were chosen as being typical for the two groups of cations with different influences on valinomycin monolayers, as was discussed above. As shown in Fig. 2, surface pressure and potential isotherms of DPPA-valinomycin on NaC1 aqueous subphase are almost the same as on the distilled water. Some quantitative differences are the following: the area per molecule and potential values for pure D P P A monolayers on the 1.0 M NaC1 subphase are larger (0.5 nm 2 and 350 mV respectively) compared with those on distilled water (0.4nm 2 and 2 5 0 m V respectively). The more positive A V may be due to a reduced negative contribution of the head group region via reduction of the thickness of the dielectric double layer. The slopes of these isotherms for NaC1 are not so steep as for distilled water, indicating a higher compressibility of the monolayer on this subphase. The individual contributions of DPPA and valinomycin are clearly seen as two parts of the isotherms (Fig. 2(a)): first, the liquidexpanded state of valinomycin at low surface pressures and high monolayer areas; second, the liquidcondensed state of D P P A at high surface pressures and small monolayer areas. Surface pressure and potential isotherms for DPPA-valinomycin monolayers on KC1 subphases (Fig. 3) show all three states: liquid-expanded I, liquid-condensed II and Ill, which are typical for pure valinomycin. These states become more pronounced (especially the transitions I - I I and I I - I I l ) with an increase of the valinomycin molar fraction in the mixed monolayers. The parameters of state Ill are changed drastically with the changes of the D P P A : valinomycin ratio, probably because of the strong interaction between the positively charged
S. Y. Zaitsev et aZ/'Colloids"Surfaces A: Physicochem. Eng. Aspects 94 (1995) 75 83
80
valinomycin-K + ion complex and the negatively charged DPPA. In order to characterize miscibility and molecular interactions in the DPPA-valinomycin monolayers, the diagrams of the mean molecular areas, potentials and dipole moments at 20 m N m -1 versus the molar fraction of valinomycin in these mixed films on water, 1.0 M NaC1 and 1.0 M KC1 were plotted in Fig. 4. A surface pressure of 20 mN m - 1 was chosen because of the linear character of all isotherms in this region (from 10 to around 25 m N m - l ) . As shown in Fig. 4(a), the mean molecu3. E = _~ _~ o E ~, " ~' ~, o~ .~
2.
f
[] ~
[]
[]
D o zx
9~
D
[] ~ ~ ~
0
NaCI
[]
KCi water
~
>
. . . . . . . . . . 0.2 0.4 0.6 0.8 1.0 tool. fraction of v a l i n o m y c i n
0
o. (a) 5 .-~, E oE
4
[] o
¢-
"a "
3
1
A
A
O
~ 0
[] O
[]
2
[] 0 zx
A
~ ~ ~
O NaCI KCI A water []
< -1
0.
(b)
•
l
0.2 moh
,
J
,
0.4 fraction
r
0.6 of
,
i
0 iS
f. 0
valinomycin
Fig. 4. Average molecular area (a) and average dipole moment (b) of the mixed monolayers of DPPA-valinomycin at 20 mN lm vs. the molar fraction of valinomycin,
lar areas are higher than expected for additivity. A deviation from linearity in the plot of mean areas indicates an interaction between the two components. These deviations are very small in the case of mixed monolayers on the NaCI subphase, more pronounced on distilled water and highest on the KC1 subphase. This effect can be explained as additional electrostatic interactions between the positively charged valinomycin-K + ion complex and the negatively charged DPPA in the monolayer compared with the interaction of the neutral valinomycin ring and negatively charged phosphate groups of DPPA. The less pronounced interactions in the case of mixed monolayers on the NaC1 subphase can be explained by a competition of the interactions between the valinomycin DPPA in the monolayer and DPPA with Na + ions from the aqueous subphase. The largest deviations from additivity are observed for low molar fractions of valinomycin (0.10-0.25), when the total area of monolayer occupied by valinomycin is close to the total monolayer area occupied by lipid molecules. In this case one can imagine that the valinomycin molecule is completely surrounded by lipids, so the heteromolecular interactions can be higher than lipid lipid, or valinomycin-valinomycin, interactions. The same explanation is valid for the deviations from linearity of the mean surface potential and dipole moments (Fig. 4(b)). All these DPPA-valinomycin mixtures were also investigated with the newly developed methods of Brewster angle microscopy and reflectivity measurement. The microscopic images of DPPA valinomycin monolayers at a ratio of 10' 1 and higher show the same small-domain structures (liquidcondensed state) as pure DPPA (Fig. 5). The total areas occupied by lipid molecules in these mixed monolayers are higher than those for valinomycin molecules. Therefore it is not surprising that the "overall" morphology of these mixed monolayers is determined by the lipid molecules. In contrast, the images of DPPA-valinomycin monolayers at a ratio of 5 : 1 and lower show only very fine points (no well-defined structures) which are typical for the liquid-expanded films of pure valinomycin (Fig. 6). At these amounts of valinomycin in the mixed monolayer, the fractional area of the lipid is almost the same or smaller than that of the
81
S. Y. Zaitsev et al./Colloids Surfaces A." Physicochem. Eng. Aspects 94 (1995) 75 83
0.i m m
=l.0mN/m
~
=
1.0
mN/m
o11 m m
= = 20.0 mN/m Fig. 5. Brewsterangle microscopicimages of mixed monolayers of DPPA-valinomycin (10: 1) on water at pressures 1.0 and 20.0 mN m -1.
valinomycin. Under such conditions, DPPA-valinomycin monolayers start to be miscible and acquire the liquid-expanded morphology that is typical for the pure valinomycin monolayers. Thus, the ratios of DPPA-valinomycin between 10:1 and 5 : 1, where the total monolayer areas occupied by the lipid and valinomycin molecules are close to each other, are the starting range of miscibility of these mixed monolayers, The mixed DPPA-valinomycin monolayers were
20.0
mN/m
Fig. 6. Brewsterangle microscopicimages of mixed monolayers of DPPA valinomycin(5 : 1) on water at pressures 1.0 and 20.0 mN m -1.
also investigated by Brewster angle reflectivity measurements. As shown in Fig. 7 and 8, the Brewster reflectivity for the monolayers of D P P A valinomycin(10: 1) and (5: 1) is increasing very slowly and in the same manner for both mixtures on water. That is similar to the Brewster reflectivity isotherms on water for DPPA. Probably, the longchain lipids hide the flat valinomycin molecules, so the contribution of the peptide to the reflection signal is much less that in the mixed monolayer. The only difference with the Brewster reflectivity
S. Y. Zaitsev et al./Colloids' Surfaces A: Physicochem. Eng. Aspects 94 (1995) 75-83
82
I
o.3.
ii()i 3 . ~', ~ \. ~ ~ "
0.2. :_~ "~ 0.~.
.
\ ~
o.o
........................
0.0
....
I . . . .
0.5
I
. . . .
I
1.0
. . . .
15
Area/Mo~ec~l~lnE]
20
Fig. 7. Brewsterreflectivity-areaisotherms of DPPA valinomycin(10:1) monolayerson distilledwater (curve 1) and 1.0 M solutions of KC1 (curve 2) and NaCI (curve 3) at 18°C.
04-
\ i "~ i \ \ -)
_
° 03 ~"
'~ '~ ' ~
0.2
9
"'. ~',
iiiii
= o.,-
00
. . . .
0.0
I
0.~
. . . .
I.
10
'---'
'
'
I
1.5
. . . .
20
Area/M°lecule[nmZ) Fig. 8. Brewsterreflectivityarea isotherms of DPPA valinomycin(5:1) monolayerson distilledwater (curve 1) and 1.0 M solution of KC1 (curve 2) and NaC1 (curve 3) at 18°C.
for pure DPPA is the occurrence of much higher fluctuations in the reflection signal. This can be explained by the fluctuations of the reflectivity for the different domains in the pure DPPA monolayers. The Brewster reflectivity isotherms on the salt subphases for the DPPA-valinomycin (10:1) mixtures (Fig. 7) are similar to the isotherms for the pure DPPA [ 2 3 - 2 5 ] , whereas for the DPPAvalinomycin (5:1) mixtures (Fig. 8) are close to
the isotherms for pure valinomycin on the same subphases [ 19]. This fact provides additional evidence for a critical ratio for valinomycin-DPPA miscibility in the monolayers. It was very difficult to obtain more quantitative results for these systems from the Brewster reflectivity isotherms, because of the large differences in the structure and morphology of the lipid and peptide in the monolayers. A general description of these isotherms can be as follows: (1) for the DPPAvalinomycin (10:1) mixtures and higher lipid fractions, the lipid molecules with relatively thick hydrocarbon chains dominate the behaviour (the Brewster reflectivity isotherms are close to that for pure DPPA); (2) for the DPPA-valinomycin (5' 1) mixtures and lower lipid fractions, the peptide determines the Brewster reflectivity changes (especially pronounced on the concentrated salt subphases), because valinomycin molecules can be reoriented in the complexed form ("bangle" conformation) from the parallel to the perpendicular position with respect to the interface. This gives rise to a remarkable (2-3 times) increase in the Brewster reflectivity compared with that of the pure DPPA monolayers. Taking into consideration the above discussed results of the non-linearity of the average area and potential from the mixed monolayer composition, one can call the range between 10:1 and 5:1 the critical ratio for the DPPA-valinomycin interactions at the interfaces. We hope that further experiments can clarify the conformational changes of valinomycin in monolayers in the presence of various cations. An understanding of this phenomenon will be useful for future application of such thin films in ionoselective membrane electrodes.
4.
Conclusions
The behaviour of mixed DPPA-valinomycin monolayers depends strongly on the nature of the cations in the aqueous subphases. Two additional monolayer states of valinomycin exist when K + ions are present rather than Na + ions, owing to strong complexation ofvalinomycin at the interface with K + ions from the aqueous subphase.
S. Y. Zaitsev et al./Colloids Surfaces A: Physicochem. Eng. Aspects 94 (1995) 75 83
Acknowledgements The authors are grateful to J. Maack, G. Overbeck and W. Zeiss (MPI for Biophysikalische Chemie, G6ttingen) and N, Kalabina (Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow) for fruitful discussions and technical assistance. This work was supported by a grant from the "Volkswagen-Stiftung" (FRG).
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83
[9] T. Mita and Y. Sairyo, Biosci. Biotech. Biochem., 56 11992) 1971. [10] S. Pathirana, W. Neely, L. Myers and V. Vodyanoy, Langmuir, 8 (1992) 1984. [11] G. Gabrielli, M. Puggelli and G. Prelazzi Prog Colloid Polym. Sci., 84(1991)232. [12] H.E. Ries and H. Swift, J. Colloid Interface Sci., 64 (1978) 111. [13] V.A. Howarth, M.C. Petty, G.H. Davies and J. Jarwood, Langmuir, 5 (1989)330. [14] V.A. Howarth, M.C. Petty and Yu. Lvov, Makromol. Chem., Macromol. Syrup., 51 (1991) 175. [15] G. Kemp and C.E. Wenner, Biochim. Biophys. Acta, 282 (1972) 1. [16] G. Kemp, V. Jacobson and C.E. Wenner, Biochim. Biophys. Acta, 255 (1972) 493. [17] B.M. Abraham and J.B. Ketterson, Langmuir, 1 (1985) 461. [18] J.B. Peng, B.M. Abraham, P. Dutta, J.B. Ketterson and H.F. Gibbard, Langmuir, 3 (1987) 104. [19] S.Yu. Zaitsev, V.P. Zubov and D. MObius, Biochim. Biophys. Acta, 1148 (1993) 191. [20] S.Yu. Zaitsev and V.P. Zubov, Biol. Membr., 6 (1989) 883 (in Russian). [21] T. Mita, Bull. Chem. Soc. Jpn., 66 (1993) 1490. [22] Yu.A. Ovchinnikov, V.T. Ivanov and A.M. Shkrob, Membrane-Active Complexones, Elsevier, Amsterdam, 1974. [23] D. HOnig and D. MObius J. Phys. Chem., 95 (1991) 4590. [24] D. HOnig and D. MObius, Thin Solid Films. 210/211 (1992) 64. [25] D. HOnig, G. Overbeck and D. MObius, Adv. Mater., 4 (1992) 419.
ELSEVIER
A
SURFACES
Mixed monolayers of valinomycin and dipalmitoylphosphatidic acid Sergei Yu. Zaitsev a,,, Vitaly P. Zubov a, Dietmar MObius b a Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871, Russia b Max-Planck-Institutf~ir Biophysikalische Chemie, Postfach 2841, 37018 Gdttingen, Germany
Received 23 February 1994; accepted 13 May 1994
Abstract
The interactions of mixed monolayers of the hydrophobic peptide valinomycin and the negatively charged phospholipid dipalmitoylphosphatidic acid (DPPA) with alkali metal cations in the aqueous subphase were investigated. For this purpose, the isotherms of surface pressure, surface potential and Brewster reflectivity versus molecular area were measured for the mixed monolayers (valinomycin-DPPA) in the absence and presence of Na + and K + ions, respectively, in the aqueous subphase. Complete miscibility of the components in the monolayers is deduced from Brewster angle microscopy. Two additional states of valinomycin-DPPA monolayers were observed when K + ions were present instead of Na + ions in the aqueous subphase. This phenomenon correlates with the strong complexation between the valinomycin and K + ions in the bulk. The negatively charged phospholipid (DPPA) influences the ability of valinomycin to bind cations at the interface. Keywords: Brewster angle microscopy; Complexation; Dipalmitoylphosphatidic acid; Ionic selectivity: Monolayers; Valinomycin
1. Introduction Mixed lipid monolayers are particularly suited as models of biological membranes, especially because they allow us to observe interactions between surface-active molecules at the interface and those with components in the aqueous subphase. Many investigations have been devoted to the question of miscibility of various lipids in monolayers [ 1-5], but only some of them describe mixed monolayers of phospholipids and biologically active molecules (such as peptides) that are particularly important for the modelling ofbiological membranes,
* Corresponding author,
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Only a few recent studies have dealt with mixed monolayers of lipids and the peptide ionophore valinomycin at the air/water interface [6 91. In most cases [ 6 - 8 ] , the effect of the negatively charged monosialoglycosphingolipids and disialoglycosphingolipids (GM1and GDlagangliosides) on the molecular organization and physicochemical properties of valinomycin in mixed monolayers at the air/water interface was investigated. Deviations from linearity in the plots of the average surface pressure and potential indicate that valinomycin, interacting with the polar head groups of the gangliosides, becomes partly embedded within the lipid interface [8]. In contrast, no deviation from the additivity rule occurred for the mixture of valinomycin with zwitterionic lipids:bovine brain lecithin ['7] and dimyristoyl-
76
S. Y. Zaitsev et al./Colloids SurJaces A: Physicochem. Eng. Aspects 94 (1995) 75 83
phosphatidylcholine (DMPC) E9]. The authors [9] conclude that valinomycin does not perturb the properties of the phospholipid monolayer, whatever the value of the ionophore-to-lipid ratio in the membranes above or below the phase transition temperature T~ of DMPC. However, they did not specify which of the two possibilities (ideal miscibility or complete immiscibility) occurs in this case. Mixtures of valinomycin with stearic acid [ 10-12], arachidic acid [ 13,14], eicosanol [-8] and cholesterol [12] were also investigated. Mixed monolayers with fatty acids were studied for the preparation of Langmuir-Blodgett films on various solid supports with the aim of applying such systems as biosensors [-10,13]. Most of the lipid valinomycin monolayers mentioned above were prepared on a pure water subphase. Thus the influence of various cations on the valinomycinlipid interactions was not investigated in detail. On the other hand, much more work has been carried out previously with pure valinomycin monolayers [-15 21] on various salt-containing subphases. Two additional states of valinomycin monolayers were observed when K + and Rb + ions were present instead of the other alkali metal cations [-15,19 21]. This phenomenon correlates with the strong complexation between the valinomycin and these cations in bulk [-22]. An influence of the size of the anion on the stability of valinomycin-cation complexes at the interface was found [19 21]. Valinomycin is one of the most important and widely investigated ionophores which can selectively increase the cation permeability through biological and synthetic membranes [22]. Taking into consideration the importance of interfacial layers for membrane studies, it is necessary to investigate in detail the properties of valinomycin in mixed lipid monolayers at the water/air interface. Dipalmitoylphosphatidic acid was chosen for this study as one of the common lipids of biological membranes, having a negative charge that can influencethemonolayercharacteristicsofthevalinomycin and its interaction with various cations from the aqueous subphase. The methods of surface pressure and surface potential measurements versus molecular area [ 1,5], as well as the recently developed technique of Brewster angle reflectivity
and microscopy of monolayers at the water/air interface [23-25] were chosen for this investigation. Reflectivity measurements under the Brewster angle of incident light with respect to the pure water surface have already provided extremely important information about the molecular organization of lipids [-23-25] and pure valinomycin molecules in monolayers [19]. Therefore it was very interesting to apply such a new technique to the investigation of mixed monolayers of biologically active molecules at the interface. This study is mainly devoted to the observation of the interactions between valinomycin in mixed monolayers with DPPA and various alkali metal cations present in the aqueous subphase.
2. Experimental Dipalmitoylphosphatidic acid (DPPA) and valinomycin (Sigma, USA) were used as 1.0mM solutions in chloroform (HPLC grade; Fluka, FRG). All inorganic salts and organic solvents were of analytical grade. Monolayers of DPPA and valinomycin were prepared and investigated on a commercial film balance, using the Langmuir principle (horizontal detection [ 1,5]) for the surface pressure measurements (Lauda, FRG) and on a self-made rectangular Teflon trough (area, 355 cm2; depth, 1 cm) using the Wilhelmy principle (vertical detection [1,5]) for the surface pressure measurements and a vibrating plate condenser for the surface potential measurements respectively. Portions 10-20 p,1) of the valinomycin-DPPA solutions in various ratios were spread with a microsyringe onto distilled water or aqueous salt subphases. As a standard procedure, monolayers were rested 5 min before compression to allow sufficient solvent evaporation and to reach equilibrium with respect to the initial surface pressure and surface potential. The surface pressure (~)-molecular area (A) and surface potential (AV) molecular area (A) isotherms were recorded simultaneously during continuous cornpression of the monolayer by a movable barrier at a constant rate of 20cm 2 min 1. The temperature was 18.0 _+0.5°C. The change in surface potential of the air/water
S. Y. Zaitsev et al./Colloids Surfaces A: Physicochem. Eng. Aspects 94 (1995) 75-83
interface after monolayer formation expressed by the Helmholz equation:
(AV)
is
77
7060-
ii
A V = n#(1/eo) where n is the number of molecules in the monolayer, p is the mean change of the effective dipole moment per amphiphile molecule normal to the air/water interface, eo is the permittivity of the vacuum. Using the identity n = l / A , where A is the mean area per molecule, this equation can be transformed to the form
~s41dz ~ ; ! ]
30~ 1~
, .. , _, .. ,
~ ~o lo
# = e o AVA
o
,, ,
.....
,
0
1
2
3
Area/MoleculeInm21
in order to calculate the change of the effective dipole moment per molecule, which reflects changes in molecular orientation at the interface [ 1,4-8]. The reflectivity R = I/I o (where I is the reflected intensity and Io is the incident intensity) of the water surface at the Brewster angle under p-polarization w a s recorded simultaneously with
(a)
the
~ 400 .~. Si i:,¢ : ~'""",.,,ii, "'X'-, 4,."; .......-..6"-"" ......
surface pressure ( ~ ) a s a function of the area per molecule (A). The reflected light from a laser diode (power, below 1 mW; wavelength, 670 nm; light spot diameter, about 1 mm) in combination with a polarizer was detected with a photomultiplier (Hamamatsu R1635). The angles of incidence and observation were fixed to the Brewster angle for the pure water subphase (53°). The whole
coo ~ 600 _~ ~
===========__ ============= ...===......... ======.~.. .
~ 2~ ~ 200-
~.~ii~(...
- ~......
.......................
0 1 (b)
2 Area/Molecule[nm2]
3
system was fixed onto a tripod. By simply tilting
Fig. 1. Surface pressure area (a) and surface potential area
the tripod, small angle adjustments were possible, T h e optimal position was found by minimizing the background signal of the pure water. The system was mounted above a rectangular Teflon film balance. All data were recorded and processed by an IBM-compatible personal computer (type 386). A more detailed description of the system and technique has been published previously [25].
isotherms of the mixed monolayers of D P P A valinomycin at ratios 1 : 0 (curve 1), 10:1 (curve 2) 5 : 1 (curve 31, 3 : 1 (curve 4), 1:1 (curve 5), 1:3 (curve 6), and 0:1 (curve 7) on water
3. Results and discussion It was shown that valinomycin forms stable monolayers on water and various aqueous salt subphases [19]. Surface pressure and potential isotherms for pure valinomycin, phosphatidic acid and their various mixtures on water are shown in Fig. 1(a) The isotherms for pure DPPA and valino-
at 18°c. mycin are completely different. In the case of DPPA, the surface pressure increases very steeply at small monolayer areas (liquid-condensed state), whereas for valinomycin such an increase is relatively slow and is observed already at large areas (approximately ten times larger than for DPPA). These large differences are very important for the investigation of mixed monolayers of DPPA and valinomycin, because they allow us to determine the contribution of each component to the mixed monolayer characteristics. The area per molecule (extrapolated to zero surface pressure) for pure DPPA is around 0.4 nm 2 (Fig. 1 (a), curve 1 }; and
S. Y. Zaitsev et al./Colloids Surfaces A." Physicochem. Eng. Aspects 94 (1995) 75 83
78
for valinomycin, 3.2 nm 2 (Fig. l(a), curve 7); this is in good agreement with the literature data [1,4,5,15,21]. The surface pressure isotherms of mixed D P P A - v a l i n o m y c i n monolayers lie in the range between these isotherms (Fig. l(a), curves 2 6). The same differences are observed for the potential isotherms of pure valinomycin, phosphatidic acid and their various mixtures on water (Fig. l(b)). The surface potential increases steeply (total increase is about 250 mV) at low areas in the case of pure D P P A monolayers (Fig. l(b), curve 1) and very smoothly at large areas for valinomycin (Fig. l(b), curve 7). The potential isotherms of mixed D P P A - v a l i n o m y c i n m o n o layers on water lie in between these limits (Fig. 1 (b), curves 2-6)• The partial contribution of the valinomycin and D P P A in the total values is clearly seen from these mixed isotherms. It is remarkable that the surface potential decreases after passing a m a x i m u m upon compression for molar ratios of D P P A valinomycin from 10 : 1 to 1"1 (curves 2 5). This behaviour of deviations from the additivity of dipole m o m e n t s indicates a particular interaction between the two components. was shown earlier [13,17-19] that surface pressure and potential isotherms of pure valinomycin monolayers depend on the kind of aqueous salt subphases. The cations can be divided into
70-
,
6o -£ s0 v
40
~ ~: 30 ~ ~ 20 ,o
~i .~i ............. '
)
~
' ' , "'.
",,, 0 ......... (a)
, ......... , ......... Area/Mo~culel'~m2l
1
2
, ..........
3
800
E ~ ~ 400
:~:;~,.,,,, •....... •............... "~" ", ..... "' ,, -,.. ,-, ~ ,.
a,
~
..... ._
~ 200
0-
It
tWO groups. Firstly, surface pressure and potential isotherms for valinomycin on LiC1, NaC1, CsC1 and NH4CI (see Fig. 2 for the case of NaC1) are very similar to each other and are close to the same isotherms on distilled water (Fig. 1). Only the liquid-expanded state I is observed. Secondly, surface pressure and potential isotherms for valinomycin on KC1 and RbC1 subphases (Fig. 3 for the case of KC1) have some similarity to those in Figs. 1 and 2 only at low surface pressures and high areas (state I), but are completely different upon further compression. On 1.0 M solutions of these salts, both ~-A and A V-A isotherms have a special region in the surface pressure range 2 0 - 3 0 m M m -~ with corresponding areas 3.0-1•7 nm 2 per molecule (state II) as well as at 30 40 m N m '-1 and 1.7 0.6 nm 2 per molecule (state Ill)(see Fig. 3). It is i m p o r t a n t to stress that all three break points
0 (b)
. . . .
J
. . . .
~ i
. . . .
2 A~e~/~ol~'In~:~ i
. . . .
i
,
~ ,
i . . . .
3 i
. . . .
J
. . . .
Fig. 2. Surface pressure-area (a} and surface potential area isotherms of the mixed monolayers of DPPA-valinomycin at ratios 1:0 (curve 1), 10:1 (curve 21, 5:1 (curve 3), 3:1 (curve 4), 1:1 {curve 5}, 1:3 (curve 6), and 0:1 (curve 7) on 1.0 M solutions of NaCl at 18~C. in the g A isotherms, which can be assigned to the transitions between states I and II, II and III, and III and collapse of the monolayer, respectively, occur at the same areas as in the A V-A isotherms (Fig. 3). The break point between states I and II can be explained at the onset of the transition from an "expanded" conformation of the polypeptide ring of valinomycin, when the hydrophilic groups are facing the aqueous subphase, to the very special "bangle" conformation, in which all carbonyl and carboxyl groups of the amino acid residues are coordinated to the cation inside the polypeptide ring. The "bangle" conformation is very rigid and
S. Y. Zaitsev et al./Colloids Surfaces A." Physicochem. Eng. Aspects 94 (1995) 75-83
70 \ ~ 60 }'~:i~,i,, -g so.. r:~',, ~'~ 4030 20 L ,0 o,
1
2
/a)
3
Area/MoleculeInm~'l
1200lo0o
/"
_
800 •~ 600
-'"
..... ; -", 4 ,k ..", ~ ,' ...#.,' , ~...... '-, "'-..
" " ..
.........
d(~ iii ......."ii iiiiiiiii!!:::> ....iii i i
400 200 ' ' ........ (b)
.............. "..............
, . . . . . . . . . 2, . . . . . . . . . . . . .3 . . . . . . Area/Molecule[,,m:l
1
Fig. 3. Surface pressure-area (a) and surface potential-area isotherms of the mixed monolayers of DPPA- valinomycin 1:0 (curve 1), 10:1 (curve 2), 5:1 (curve 3), 3:1 (curve 4), 1:1 (curve 5), 1:3 (curve 6), and 0:1 (curve 7) on 1.0 M solutions of KC1 at 18C.
can be formed spontaneously even in solution in the presence of K + or Rb + ions, as was shown first by Ovchinnikov et al. [22]. It is correlated with the liquid-condensed character of the state II of valinomycin monolayers, in particular with its low compressibility. The surface pressure of the I - I I transition depends strongly on the concentration of K + ions in the aqueous subphase, while the pressure of the I I - l l I transition is always the same in the range 32.0-34.0 m N m - ~ at various concentrations of KC1 in the aqueous subphase. The transition I I - I I I may be attributed to a reori-
79
entation of the valinomycin molecules with the "bangle" conformation in the monolayer with a reorientation of the ring plane from parallel to perpendicular with respect to the interface. The monolayer in state III has a collapse in the range 38.0-40.0 m N m -1. This unusual behaviour of valinomycin monolayers on KCI subphases can be explained as complex formation of ionophore at the interface with a cation from the subphase. With this behaviour of valinomycin in mind, it was particularly interesting to investigate mixed monolayers of D P P A and valinomycin on various aqueous salt subphases. NaC1 and KC1 subphases were chosen as being typical for the two groups of cations with different influences on valinomycin monolayers, as was discussed above. As shown in Fig. 2, surface pressure and potential isotherms of DPPA-valinomycin on NaC1 aqueous subphase are almost the same as on the distilled water. Some quantitative differences are the following: the area per molecule and potential values for pure D P P A monolayers on the 1.0 M NaC1 subphase are larger (0.5 nm 2 and 350 mV respectively) compared with those on distilled water (0.4nm 2 and 2 5 0 m V respectively). The more positive A V may be due to a reduced negative contribution of the head group region via reduction of the thickness of the dielectric double layer. The slopes of these isotherms for NaC1 are not so steep as for distilled water, indicating a higher compressibility of the monolayer on this subphase. The individual contributions of DPPA and valinomycin are clearly seen as two parts of the isotherms (Fig. 2(a)): first, the liquidexpanded state of valinomycin at low surface pressures and high monolayer areas; second, the liquidcondensed state of D P P A at high surface pressures and small monolayer areas. Surface pressure and potential isotherms for DPPA-valinomycin monolayers on KC1 subphases (Fig. 3) show all three states: liquid-expanded I, liquid-condensed II and Ill, which are typical for pure valinomycin. These states become more pronounced (especially the transitions I - I I and I I - I I l ) with an increase of the valinomycin molar fraction in the mixed monolayers. The parameters of state Ill are changed drastically with the changes of the D P P A : valinomycin ratio, probably because of the strong interaction between the positively charged
S. Y. Zaitsev et aZ/'Colloids"Surfaces A: Physicochem. Eng. Aspects 94 (1995) 75 83
80
valinomycin-K + ion complex and the negatively charged DPPA. In order to characterize miscibility and molecular interactions in the DPPA-valinomycin monolayers, the diagrams of the mean molecular areas, potentials and dipole moments at 20 m N m -1 versus the molar fraction of valinomycin in these mixed films on water, 1.0 M NaC1 and 1.0 M KC1 were plotted in Fig. 4. A surface pressure of 20 mN m - 1 was chosen because of the linear character of all isotherms in this region (from 10 to around 25 m N m - l ) . As shown in Fig. 4(a), the mean molecu3. E = _~ _~ o E ~, " ~' ~, o~ .~
2.
f
[] ~
[]
[]
D o zx
9~
D
[] ~ ~ ~
0
NaCI
[]
KCi water
~
>
. . . . . . . . . . 0.2 0.4 0.6 0.8 1.0 tool. fraction of v a l i n o m y c i n
0
o. (a) 5 .-~, E oE
4
[] o
¢-
"a "
3
1
A
A
O
~ 0
[] O
[]
2
[] 0 zx
A
~ ~ ~
O NaCI KCI A water []
< -1
0.
(b)
•
l
0.2 moh
,
J
,
0.4 fraction
r
0.6 of
,
i
0 iS
f. 0
valinomycin
Fig. 4. Average molecular area (a) and average dipole moment (b) of the mixed monolayers of DPPA-valinomycin at 20 mN lm vs. the molar fraction of valinomycin,
lar areas are higher than expected for additivity. A deviation from linearity in the plot of mean areas indicates an interaction between the two components. These deviations are very small in the case of mixed monolayers on the NaCI subphase, more pronounced on distilled water and highest on the KC1 subphase. This effect can be explained as additional electrostatic interactions between the positively charged valinomycin-K + ion complex and the negatively charged DPPA in the monolayer compared with the interaction of the neutral valinomycin ring and negatively charged phosphate groups of DPPA. The less pronounced interactions in the case of mixed monolayers on the NaC1 subphase can be explained by a competition of the interactions between the valinomycin DPPA in the monolayer and DPPA with Na + ions from the aqueous subphase. The largest deviations from additivity are observed for low molar fractions of valinomycin (0.10-0.25), when the total area of monolayer occupied by valinomycin is close to the total monolayer area occupied by lipid molecules. In this case one can imagine that the valinomycin molecule is completely surrounded by lipids, so the heteromolecular interactions can be higher than lipid lipid, or valinomycin-valinomycin, interactions. The same explanation is valid for the deviations from linearity of the mean surface potential and dipole moments (Fig. 4(b)). All these DPPA-valinomycin mixtures were also investigated with the newly developed methods of Brewster angle microscopy and reflectivity measurement. The microscopic images of DPPA valinomycin monolayers at a ratio of 10' 1 and higher show the same small-domain structures (liquidcondensed state) as pure DPPA (Fig. 5). The total areas occupied by lipid molecules in these mixed monolayers are higher than those for valinomycin molecules. Therefore it is not surprising that the "overall" morphology of these mixed monolayers is determined by the lipid molecules. In contrast, the images of DPPA-valinomycin monolayers at a ratio of 5 : 1 and lower show only very fine points (no well-defined structures) which are typical for the liquid-expanded films of pure valinomycin (Fig. 6). At these amounts of valinomycin in the mixed monolayer, the fractional area of the lipid is almost the same or smaller than that of the
81
S. Y. Zaitsev et al./Colloids Surfaces A." Physicochem. Eng. Aspects 94 (1995) 75 83
0.i m m
=l.0mN/m
~
=
1.0
mN/m
o11 m m
= = 20.0 mN/m Fig. 5. Brewsterangle microscopicimages of mixed monolayers of DPPA-valinomycin (10: 1) on water at pressures 1.0 and 20.0 mN m -1.
valinomycin. Under such conditions, DPPA-valinomycin monolayers start to be miscible and acquire the liquid-expanded morphology that is typical for the pure valinomycin monolayers. Thus, the ratios of DPPA-valinomycin between 10:1 and 5 : 1, where the total monolayer areas occupied by the lipid and valinomycin molecules are close to each other, are the starting range of miscibility of these mixed monolayers, The mixed DPPA-valinomycin monolayers were
20.0
mN/m
Fig. 6. Brewsterangle microscopicimages of mixed monolayers of DPPA valinomycin(5 : 1) on water at pressures 1.0 and 20.0 mN m -1.
also investigated by Brewster angle reflectivity measurements. As shown in Fig. 7 and 8, the Brewster reflectivity for the monolayers of D P P A valinomycin(10: 1) and (5: 1) is increasing very slowly and in the same manner for both mixtures on water. That is similar to the Brewster reflectivity isotherms on water for DPPA. Probably, the longchain lipids hide the flat valinomycin molecules, so the contribution of the peptide to the reflection signal is much less that in the mixed monolayer. The only difference with the Brewster reflectivity
S. Y. Zaitsev et al./Colloids' Surfaces A: Physicochem. Eng. Aspects 94 (1995) 75-83
82
I
o.3.
ii()i 3 . ~', ~ \. ~ ~ "
0.2. :_~ "~ 0.~.
.
\ ~
o.o
........................
0.0
....
I . . . .
0.5
I
. . . .
I
1.0
. . . .
15
Area/Mo~ec~l~lnE]
20
Fig. 7. Brewsterreflectivity-areaisotherms of DPPA valinomycin(10:1) monolayerson distilledwater (curve 1) and 1.0 M solutions of KC1 (curve 2) and NaCI (curve 3) at 18°C.
04-
\ i "~ i \ \ -)
_
° 03 ~"
'~ '~ ' ~
0.2
9
"'. ~',
iiiii
= o.,-
00
. . . .
0.0
I
0.~
. . . .
I.
10
'---'
'
'
I
1.5
. . . .
20
Area/M°lecule[nmZ) Fig. 8. Brewsterreflectivityarea isotherms of DPPA valinomycin(5:1) monolayerson distilledwater (curve 1) and 1.0 M solution of KC1 (curve 2) and NaC1 (curve 3) at 18°C.
for pure DPPA is the occurrence of much higher fluctuations in the reflection signal. This can be explained by the fluctuations of the reflectivity for the different domains in the pure DPPA monolayers. The Brewster reflectivity isotherms on the salt subphases for the DPPA-valinomycin (10:1) mixtures (Fig. 7) are similar to the isotherms for the pure DPPA [ 2 3 - 2 5 ] , whereas for the DPPAvalinomycin (5:1) mixtures (Fig. 8) are close to
the isotherms for pure valinomycin on the same subphases [ 19]. This fact provides additional evidence for a critical ratio for valinomycin-DPPA miscibility in the monolayers. It was very difficult to obtain more quantitative results for these systems from the Brewster reflectivity isotherms, because of the large differences in the structure and morphology of the lipid and peptide in the monolayers. A general description of these isotherms can be as follows: (1) for the DPPAvalinomycin (10:1) mixtures and higher lipid fractions, the lipid molecules with relatively thick hydrocarbon chains dominate the behaviour (the Brewster reflectivity isotherms are close to that for pure DPPA); (2) for the DPPA-valinomycin (5' 1) mixtures and lower lipid fractions, the peptide determines the Brewster reflectivity changes (especially pronounced on the concentrated salt subphases), because valinomycin molecules can be reoriented in the complexed form ("bangle" conformation) from the parallel to the perpendicular position with respect to the interface. This gives rise to a remarkable (2-3 times) increase in the Brewster reflectivity compared with that of the pure DPPA monolayers. Taking into consideration the above discussed results of the non-linearity of the average area and potential from the mixed monolayer composition, one can call the range between 10:1 and 5:1 the critical ratio for the DPPA-valinomycin interactions at the interfaces. We hope that further experiments can clarify the conformational changes of valinomycin in monolayers in the presence of various cations. An understanding of this phenomenon will be useful for future application of such thin films in ionoselective membrane electrodes.
4.
Conclusions
The behaviour of mixed DPPA-valinomycin monolayers depends strongly on the nature of the cations in the aqueous subphases. Two additional monolayer states of valinomycin exist when K + ions are present rather than Na + ions, owing to strong complexation ofvalinomycin at the interface with K + ions from the aqueous subphase.
S. Y. Zaitsev et al./Colloids Surfaces A: Physicochem. Eng. Aspects 94 (1995) 75 83
Acknowledgements The authors are grateful to J. Maack, G. Overbeck and W. Zeiss (MPI for Biophysikalische Chemie, G6ttingen) and N, Kalabina (Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow) for fruitful discussions and technical assistance. This work was supported by a grant from the "Volkswagen-Stiftung" (FRG).
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83
[9] T. Mita and Y. Sairyo, Biosci. Biotech. Biochem., 56 11992) 1971. [10] S. Pathirana, W. Neely, L. Myers and V. Vodyanoy, Langmuir, 8 (1992) 1984. [11] G. Gabrielli, M. Puggelli and G. Prelazzi Prog Colloid Polym. Sci., 84(1991)232. [12] H.E. Ries and H. Swift, J. Colloid Interface Sci., 64 (1978) 111. [13] V.A. Howarth, M.C. Petty, G.H. Davies and J. Jarwood, Langmuir, 5 (1989)330. [14] V.A. Howarth, M.C. Petty and Yu. Lvov, Makromol. Chem., Macromol. Syrup., 51 (1991) 175. [15] G. Kemp and C.E. Wenner, Biochim. Biophys. Acta, 282 (1972) 1. [16] G. Kemp, V. Jacobson and C.E. Wenner, Biochim. Biophys. Acta, 255 (1972) 493. [17] B.M. Abraham and J.B. Ketterson, Langmuir, 1 (1985) 461. [18] J.B. Peng, B.M. Abraham, P. Dutta, J.B. Ketterson and H.F. Gibbard, Langmuir, 3 (1987) 104. [19] S.Yu. Zaitsev, V.P. Zubov and D. MObius, Biochim. Biophys. Acta, 1148 (1993) 191. [20] S.Yu. Zaitsev and V.P. Zubov, Biol. Membr., 6 (1989) 883 (in Russian). [21] T. Mita, Bull. Chem. Soc. Jpn., 66 (1993) 1490. [22] Yu.A. Ovchinnikov, V.T. Ivanov and A.M. Shkrob, Membrane-Active Complexones, Elsevier, Amsterdam, 1974. [23] D. HOnig and D. MObius J. Phys. Chem., 95 (1991) 4590. [24] D. HOnig and D. MObius, Thin Solid Films. 210/211 (1992) 64. [25] D. HOnig, G. Overbeck and D. MObius, Adv. Mater., 4 (1992) 419.