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Protamine-induced adsorption of sodium pyrene-3-sulphonate (NaPyS) on the lipid monolayers
COLLOIDS ELSEVIER
Colloidsand Surfaces A: Physicochemicaland EngineeringAspects 135 (1998) 165-174
SURFACES
A
Protamine-induced adsorption of sodium pyrene-3-sulphonate (NaPyS) on the lipid monolayers Z. Kozarac a,,, D. M6bius b a Ruder Bogkovik Institute, Center for Marine Research Zagreb, P.O.Box 1016, HR-IO001 Zagreb, Croatia b Max-Planck-Institutfiir biophysikalische Chemie, Postfach 2841, D-37018 G6ttingen, Germany
Received 3 March 1997; accepted 7 August 1997
Abstract
The interaction of sodium pyrene-3-sulphonate(NaPyS) with different lipid monolayers at the air-solution interface was studied by monolayer techniques (surface pressure and surface potential measurements) and by spectroscopic techniques (fluorescence and reflection measurements). The uptake of negatively charged NaPyS by non-charged and negatively charged phospholipid monolayers was induced by the presence of positively charged protein protamine in bulk solution. © 1998 Elsevier Science B.V. Keywords: Air-water interface; Lipid monolayers; Pyrene salt; Monolayer techniques; Spectroscopic techniques;
Synergetic effects
1. Introduction
Natural aquatic systems contain a large number of organic substances with different functional groups and different hydrophobic properties. Adsorption at natural interfaces (air-water, watermineral, oil-water) is determined by hydrophobic and/or electrostatic interactions of adsorbate and interface. In the case of attractive interactions between two or more components, either in the solution or at the interface, a synergetic effect can be expected. It is known that in the organic matter adsorbed at model and natural phase boundaries, hydrophobic fractions usually dominate [1]. Electrostatic interactions of hydrophilic ions and/or molecules from the bulk solution with functional groups of adsorbed hydrophobic sub* Correspondingauthor. Tel:00385 14561105; fax: 00385 1420437;e-mail:[email protected] 0927-7757/98/$19.00 © 1998ElsevierScienceB.V.All rightsreserved. PII S0927-7757 (97) 00242-2
stances may be responsible for the surface excess of these solute species at the natural interfaces. In our previous adsorption studies of compounds whose mutual interactions can cause enhanced adsorption and uptake to the interfacial layers at natural phase boundaries we used a para-nitrophenol (PNP)-lipid system [2]. It was found that PNP does not accumulate from bulk solution to the free air-solution interface and does not accumulate in non-charged phospholipid monolayers spread at the air-solution interface either. The attachment of PNP to the neutral phospholipid monolayers was enhanced and accelerated at the air-water interface by the presence of quaternary ammonium salts in the solution. Considerable attention has recently been given to the studies of pollutant-surfactant interactions in water and studies of pollutant solubilization by micelles [3-6]. Recently, we have started systematic physicochemical studies of adsorption of pyrene and
166
Z. Kozarac, D. M6bius / Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
pyrene derivatives at different model phase boundaries [7-9]. It is known that pyrene and its derivatives belong to the highly dangerous pollutants in the environment [10]. Our results showed that sodium pyrene sulphonate (NaPyS) does not adsorb at the free water surface and does not interact with dipalmitoylphosphatidylcholine (DPPC) monolayers, but interacts strongly with positively charged dioctadecyldimethylammonium bromide (DOMA) monolayers [8]. Accordingly, we expected surfactant-mediated adsorption when positively charged surface-active molecules are present in the subphase. In natural aquatic systems, among the positively charged lipids the artificial cationic detergents (quaternary ammonium salts) are predominant, whereas among naturally occurring substances, especially in marine waters, protamines can be found. They represent a group of simple proteins which occur in combination with nucleic acids in the sperm of fish. Here, we report on adsorption studies of NaPyS at the air-water interface and its interaction with charged and neutral lipids in the presence of protamine in the subphase.
2. Experimental 2.1. Procedure Surface pressure-molecular area (vr-A) and surface potential-molecular area (AV-A) isotherms have been measured in a rectangular Teflon trough with the inner dimensions of 56 × 18 cm 2 and a depth of 1 cm which was enclosed in a tight box and thermostatically controlled. The monolayers were compressed by a movable Teflon barrier with compression velocity between 0.08 and 0.12nmZmin-lmolecule -1 at which the best reproducibility was achieved, although other rates of compression could also be used. A Wilhelmy balance (20 mm wide filter paper) was used to measure the surface pressure, and the surface potential was measured using a vibrating plate condenser. Fluorescence measurements have been performed using a Perkin-Elmer Luminescence LS-5 spectrometer with xenon lamp modified for in situ
measurements at the air-water interface in combination with a Teflon trough [11]. The monolayer was excited at 350 rim, and the emission spectrum was measured in the 375-575nm range. Fluorescence spectra have been normalized for a constant lipid surface density. Reflection spectroscopic measurements have been performed using a reflection spectrometer for measurements under normal incidence of light [12]. The reflection was measured and expressed as the difference AR in the reflectivity from the surface covered with a monolayer and from the monolayer-free-solution surface. 2.2. Chemicals NaPyS was obtained from Molecular Probes. DOMA, DPPC and dimyristoylphosphatidic acid (DMPA) were purchased from Sigma Chemical Co. and used as-received. The main reason for choosing these three lipids was their charge and not the length of hydrophobic chains. DPPC and DMPA or DPPA are usually used as neutral and negatively charged monolayers, and no significant difference in the behaviour of DMPA and DPPA monolayers was noticed. DOMA was chosen because it forms a very stable water insoluble monolayer due to the presence of two hydrophobic C18 chains. Deionized water from a Milli-Q system (Millipore Corp.) was used for preparing the subphase. Chloroform (HPLC) p.a. grade was used as spreading solvent and was obtained from Baker Chemicals, Holland. Protamine sulphate, isolated from Salmine, was obtained from Serva, Heidelberg.
3. Results and discussion 3.1. Surface pressure and surface potential measurements 3.1.1. n-A isotherms We have already reported that no appreciable accumulation of NaPyS at the monolayer free air-water interface and no interaction with neutral zwitterionic phospholipid monolayers of DPPC
Z. Kozarac, D. MObius/ Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
has been observed [8]. If adding protamine, which is very hydrophilic and carries positive charge to NaPyS solution, a weak adsorption to the water s u r f a c e is detected during 2 h accumulation time, as evidenced by a slight increase in surface pressure n in Fig. 1, subphase 2 (full line). For comparison, the time dependence of n for a pure protamine solution is also shown in Fig. 1, subphase 1 (dotted line). However, the attachment of NaPyS to the DPPC monolayers is considerably enhanced and accelerated in the presence of protamine in solution (Fig. 1). A similar effect was observed in the presence of a DMPA monolayer (data not shown here). An increase of the surface pressure due to the adsorption in the presence of a DPPC monolayer at initial surface pressures of n = 0 and n = 5.5 mN m - 1 (surface pressure change of An = 3.8 and A n = 6 . 7 m N m -~) was observed after 50 min accumulation time, as shown in Fig. 2. The An values in the presence of DMPA monolayer on a subphase containing protamine and NaPyS at the initial surface pressures of 0 and 5.37 mN m-1 and after 50 min accumulation time were 4.6 mN m - 1 and 6.7 mN m - ~ respectively. From the surface pressure-area (n-A) isotherm of the material accumulated at the subphase 2-air interface, shown in Fig. 3, it can be seen that even a self-assembled mixed monolayer, although rather unstable, has been formed.
20E Z
DPPC on subphasecontaining protamineand NaPyS
15-
E •" l 8 100. O
~ co
5-
DPPC
on
subphase 2
0
108-
DPPC on subphase 1
642 -
subphase 1 and 2
o.~.....~
o
50
Fig. 2. Time dependence of surface pressure of DPPC monolayers spread on solution of 10-SM protamine and 10-SM NaPyS. Initial surface pressure n = 0 m N m -1 and n = 5.5 mN m - 1 respectively.
s0~ 407 -,
400
O~
Protamlne + NaPyS without monolayer
300
30-
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loo 3 '" ................
<
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i
i
i
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i
100
150
200
250
300
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Fig. 3. Surface-pressure- and surface-potential-area isotherms for pure solution containing 10-SM protamine and 10 -5 M NaPyS.
~E' 12Z
I:1..
4'0
Time [min]
Area [era2]
14-
(/)
3'0
1'0
50
E ,=
167
2'0
...............
do
8'o
i
100
120
Time [min] Fig. 1. Time dependence of surface pressure of the subphase-air interface and of DPPC monolayers spread on subphases 1 and 2. Subphase 1:10 -5 M protamine. Subphase 2:10 -5 M protamine and 10 -5 M NaPyS.
The surface pressure-molecular area (n-A) isotherms for DPPC, DOMA, and DMPA monolayers spread on water (curve 1), on a subphase containing 10-SM protamine (curve 2), and a subphase containing both 10-SM protamine and 10 -5 M NaPyS (curve 3) are shown in Figs. 4-6. The thermodynamic parameters compressional modulus C~- 1 and compressibility 1/C~ ~ have been calculated and are given in Table 1. DPPC spread on water exhibits a rather condensed film with a phase transition around 5 mN m -~ (Fig. 4, curve 1). The isotherm for the
Z. Kozarac, D. MObius/Colloids Surfaces A." Physicochem. Eng. Aspects 135 (1998) 165-174
168
50-
50~
E
"~ Z
40-
4030-
u) I~- 2 0 -
~,
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8 ~= 10-
¢:
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1
lO--
i"~"~"~3
......... ~.~
", 2
........ \
0-
0-
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&
,io
" 1.5 .
.
.
.
2.0
Fig. 4. Surface pressure-area (n-A) isotherms for DPPC monolayers spread on water (1) and on subphases containing 10-SM protamine (2) and 10 -5 M protamine and 10-SM NaPyS (3). 50-
~ Z
\
40-
",~
PyS-
3o¢t) •~
.,.
20-
-% %.. 2
"%'-.. 3
09 0 0.0
110
00
0.5
1.0
1.5
2.0
Area/DMPA Molecule [nm 2 ]
Area/DPPC Molecule [nm 2 ]
2.0
Area/DOMA Molecule [nm 2 ] Fig. 5. Surface pressure-area (n-A) isotherms for DOMA monolayers spread on water (1) and on subphases containing 10 -5 M protamine (2), and 10 -5 M NaPyS and 10 5 M protamine (3).
DPPC monolayer shows only small differences if protamine (curve 2) is present in the subphase. The same was previously observed with a DPPC monolayer spread on the subphase containing NaPyS [8]. When a DPPC monolayer was spread on the subphase containing protamine and NaPyS, both 10 -5 M, an extreme expansion of the area per DPPC molecule was observed (Fig. 4, curve 3). The physical state of DPPC is altered from the
Fig. 6. Surface pressure area (n A) isotherms for DMPA monolayers spread on water (1) and on subphases containing 10-SM protamine (2) and 10 -5 M protamine and 10-SM NaPyS (3).
condensed to the liquid-expanded film simultaneously with disappearance of phase transition. This can also be seen from the change in compressibility values given in Table 1. The area per DPPC spread on the subphase containing protamine and NaPyS is much larger until a surface pressure of 25 mN m -1, and is increased by approximately 0.07 nm 2 even at collapse pressure. The n-A isotherm of a DOMA monolayer spread on water is of the liquid-expanded type, with a phase transition around 14 mN m-1 (Fig. 5, curve 1). If protamine is present in the solution, the DOMA monolayer isotherm does not change much. It still shows a liquid-expanded phase at low surface pressure with a phase transition at 18 m N m -a (Fig. 5, curve 2). The isotherms in a more densely packed phase (n>25 mN m -1) are identical. The isotherm for a DOMA monolayer spread on the subphase containing 10-SM NaPyS shows a contraction with respect to DOMA on water at larger areas, A > 1.0 nm 2 (5 mNm-1). Further compression until A =0.85 nm 2 leads to a strong increase in surface pressure, and the phase transition disappears. The area/DOMA molecule for the monolayer in the dense packed state (40 mN m -a) is larger by approximately 0.32 nm 2 compared with the DOMA monolayer spread on
Z. Kozarac, D. M6bius / Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
169
Table 1 Thermodynamicparameters of DPPC, DOMA and DMPA monolayers on different subphases Subphase
Limiting specificarea A0 (nmz molecule-1)
Compressiionalmodulus C~-1 (mN m- 1)
CompressibilityC~-1 (m N - l)
0.5 0.52 0.65
148.1 146.1 13.0
0.0067 0.0068 0.077
0.62 0.97 1.25
32.2 171.3 12.5
0.031 0.0058 0.08
0.40 0.47 0.51
422.3 48.57 35.0
0.0024 0.021 0.029
DPPC
water 10-5 M protamine 10-s M protamine and 10-5 M NaPyS DOMA water 10-5 M NaPyS 10-5 M protamine and 10-5 M NaPyS DMPA
water 10-5 M protamine t0 -5 M protamine and 10-5 M NaPyS
water. This is in agreement with previously observed behaviour of D O M A monolayers spread on NaPyS solution (2 x 1 0 - 6 M ) and measured after 2 h accumulation time [8]. The condensation of the D O M A monolayer isotherm at small surface pressures was attributed to the decrease of repulsive forces between the head groups of D O M A due to the electrostatic interaction with P y S - from solution. The condensation effect is also indicated by a decreasing of compressibility from 0.031 for D O M A on water to 0.0058 for D O M A on NaPyS. However, the a r e a / D O M A molecule at high surface pressures is much larger than the area/DOMA molecule on water subphase, which clearly shows the lipid-solute interaction. This can be interpreted in terms of penetration and/or incorporation of solute molecules in the monolayer [13-15] but also in the terms of binding the solute molecules underneath the lipid monolayer [7,12,16]. Very interesting behaviour was observed when a D O M A monolayer was spread on the subphase containing both solutes, protamine and NaPyS. An extreme expansion of the area per D O M A molecule at all surface pressures was observed with respect to the D O M A monolayers spread on water, and in the tightly packed state (40 m N m - i ) the area increase is 0.2 nm z. However, if we compare the isotherm for D O M A spread on this subphase to the isotherm for D O M A spread on subphase containing only NaPyS, the expansion of area per D O M A molecule is very large at surface area until
A =0.92 nm 2. By further compressing the D O M A monolayer, a smaller area per matrix molecule for D O M A spread on subphase containing both solutes with respect to D O M A spread on NaPyS solution is obtained. At high surface pressures (40 m N m - ~) the difference in the area is approximately 0.12 nm 2. Compressibility is larger than for D O M A on water, which was expected because of the expansion of the isotherm. The surface pressure-area (n-A) isotherms for DMPA monolayers spread on water and on NaPyS solution are of the condensed type (Fig. 6, curve 1). In the presence of protamine in the subphase, a more expanded isotherm with a phase transition around 7.0 m N m -~ is obtained (Fig. 6, curve 2). Even at very high surface densities of monolayer ( ~ 40 m N m - 1) area/DMPA molecule is larger by approximately 0.05 nm 2. By addition of NaPyS to the protamine subphase, the isotherm for DMPA monolayer expanded to much higher area values until a pressure around 22 m N m -1. By further compression, the area reached the same value as for the DMPA monolayer spread on the protamine subphase (Fig. 6, curve 3). Compressibility values for DMPA monolayers spread on both subphases increases by ten times compared with the value for the monolayer on water. 3.1.2. AV-A isotherms
The surface potential of an amphiphilic lipid monolayer at the air-water interface may be
Z. Kozarac, D. M6bius / Colloids Surfaces A." Physicochem. Eng. Aspects 135 (1998) 165 174
170
expressed [17] using the following relation:
1000-
AV=AVo + AVp+ yo The A Vo term is the value of surface potential for the clean air-water interface, the A Vp term represents the contribution of the dipole moment of the lipid molecule and may be divided into the head group and the tail region dipole moments. Yo is the so-called double layer potential due to the charges in the lipid. The value of Yo is dependent on the degree of dissociation, on the density of head groups, and the nature and concentration of counterions in the subphase. It can be estimated from the Gouy-Chapman theory [18]. The value of Yo is expected to change in the case of specific adsorption of any counterion at the monolayer-subphase interface. Surface potential-area isotherms for DPPC, DOMA and DMPA spread on the subphase containing protamine and NaPyS measured simultaneously with the rc-A isotherms, which are presented in Figs. 4-6, are shown in Figs. 7-9. DPPC monolayer (Fig. 7) on water (curve 1) shows positive values of the surface potential in the whole range of the isotherm. The isotherm also shows the phase transition between A =0.95 and A--0.6 nm 2, i.e. for values of the surface pressure around 3 0 m N m -a. The isotherm for DPPC on the protamine subphase (curve 2) is
> ~ BOO-
1
~5 600o_ oo ~ 400o~ "t
2o00.o
015
110
115
2.0
Area/DOMA Molecule [nm 2 ] Fig. 8. Surface potential area (AV-A) isotherms for DOMA monolayers spread on water (1) and on subphases containing 10 5M NaPyS (2) and 10-SM protamine and 10-SM NaPyS (3).
600 -
~ 500400-
-~ E 300-
',2
o. 200o ~: 100-,
09
0-
800 -100 0.0
0.5
1.0
1.5
2.0
AreaJDMPA Molecule [nm 2 ]
600. m .i..,
\
~) 400-
"
Q.
Fig. 9. Surface potential-area (AV-A) isotherms for DMPA monolayers spread on water (1) and on subphases containing 10-SM protamine (2) and 10-SM protamine and 10-SM NaPyS (3).
2 ';
3
o 200-
0-
00
015
1'0
l's
2.0
Area/DPPC Molecule [nm2 ] Fig. 7. Surface potential area (AV-A) isotherms for DPPC monolayers spread on water (1) and on subphases containing 10 -5 M protamine (2), 10 -5 M NaPyS (3) and 10 -5 M protamine and 10 -5 M NaPyS (4).
almost identical with that on water, while the isotherm for DPPC on NaPyS solution (curve 3) is shifted towards more positive values until A = l . 0 n m z and by further compression also becomes almost identical with the isotherm on water. Since DPPC, as a non-charged lipid, probably does not interact with protamine or NaPyS from solution via coulombic interactions, we did not expect any appreciable change in surface
Z. Kozarac, D. Mrbius / Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
potential either. However, the surface potential values for DPPC monolayers spread on the subphase containing both solutes, i.e. NaPyS and protamine (curve 4), are dramatically less positive than the values for DPPC monolayers on water and on subphases containing only a single component, i.e. either NaPyS or protamine. The difference in A V is approximately 500 mV in the dense packed monolayer. It can be assumed that the binding of NaPyS to DPPC monolayers is enabled via complex hydrophobic and electrostatic interactions of DPPC and NaPyS with protamine. Surface potential-area isotherms for DOMA on water, on NaPyS solution and on subphase containing NaPyS and protamine are shown in Fig. 8. The surface potential for DOMA spread on water (curve 1) is positive in the whole area range, indicating the dominance of positive head group charge in the A V values. Since NaPyS interacts with DOMA monolayers via coulombic interactions [7], the decrease in AV values for DOMA monolayers spread on NaPyS subphase with respect to DOMA on water is expected and has also been observed (Fig. 8, curve 2). The surface potential of DOMA on water at A =0.56 nm 2 was 900 mV and was 670 mV on NaPyS. The decrease in positive surface potential due to the charge compensation of DOMA head group with PySgroup is 230 mV, which is in good correlation with the recently reported value of 200 mV assigned to the dipole moment of the DOMA head group [19]. DOMA spread on the subphase containing both solutes showed more positive values of surface potential at larger areas until A ~ 1.0 nm 2. By further compression, the surface potential is shifted towards less positive values in comparison with DOMA on water and on NaPyS. The surface potential-area isotherm for DMPA on water exhibits negative values for A V until the molecular area 0.65 nm 2, which shows the predominant role of the negative head group charge in the A V values (Fig. 9, curve 1). Further compression of the film is followed by the increase in A V. It can be expected that the adsorption of positively charged protamine to the negatively charged DMPA influences the A V-A isotherms. As a result of charge compensation, the surface potential A V should be shifted towards more posi-
171
tive values. The A V-A isotherms corresponding to the 7r-A isotherms (Fig. 6) are shown in Fig. 9. It can be seen that A V values for the DMPA spread on protamine (curve 2) are more positive compared with the reference DMPA. The maximum increase in A V as a result of DMPA-protamine interaction is ca. 300 mV, which is in good agreement with previously reported surface potential data for DMPA interacting with positively charged bisbipyridinium tetracations [ 16]. At areas larger than A =0.65 nm 2, surface potential values for DMPA on subphase containing protamine and NaPyS are equal to the A V values for DMPA on water. By further compression until A = 0.4 nm 2, the potential values of DMPA monolayer on subphase containing both solutes are shifted towards negative values in comparison with the values of DMPA on water and on protamine solution. 3.2. Fluorescence and reflection spectroscopy From the surface pressure and surface potential measurements it is obvious that the presence of protamine in solution influences strongly the binding of PyS- to the non-charged and negatively charged phospholipids. The complex adsorption of two components from the solution to the phospholipid monolayers at the interface takes place due to the synergetic effects. The interaction of the water-soluble components, NaPyS and protamine, can take place in bulk solution and/or at the interface. In order to achieve a better understanding of the mechanisms involved in these interactions, spectroscopic measurements have been made. Fluorescence measurements are very often used in the studies of adsorption of unsubstituted and substituted pyrene. The pyrene chromophore has a relatively long excited state lifetime, high quantum yield of fluorescence and a pronounced tendency to form excimers, e.g. the molecular complexes in the excited state [20-22]. Furthermore, the structured monomer emission spectrum and the quantum yield of fluorescence are very sensitive to the environment polarity and pyrene-pyrene interactions. The proximity of pyrene molecules is easily detected by the presence of excimer emission. This pyrene-pyrene inter-
Z. Kozarac, D. M6bius / Colloids Surfaces A." Physicochem. Eng. Aspects 135 (1998) 165-174
172
action causes a reduction of the monomer emission in the 360-430 nm range and the appearance of a red-shifted broad structureless emission band with a maximum at ca. 480 nm. The location of pyrene molecules at the lipid monolayer-subphase interface and the intermolecular association processes are expected to be influenced by the nature (dipole moment, charge) of lipid head groups and by the subphase composition. Fig. 10 shows the emission spectra (range 400-600nm) of the pure subphase containing 3 × 10 - 6 M NaPyS and 3 × 10 - 6 M protamine (full line), and the spectra of DPPC and DOMA monolayers spread on this subphase and compressed to 20 mN m-1 (dashed and dotted lines). The monomer fluorescence is predominant in all emission spectra, but an additional excimer band at 475 nm for DOMA monolayers can be seen. However, this excimer emission is much weaker than the excimer emission of DOMA/PyS- co-spread monolayers [7]. The same weak excimer emission was observed when DOMA monolayers were spread on subphase containing only NaPyS [8]. The excimer formation requires direct contact between PySchromophores. When NaPyS is dissolved in the solution, the possibility of interaction of an unexcited pyrene with an excited pyrene during the excited state lifetime is decreased, and thus no, or only very weak, excimer emission can be observed.
However, monomer emission can be expected, since both pyrene chromophores present in the solution and at the interface contribute to the measured fluorescence. Reflection spectroscopy is based on the enhanced reflection due to the presence of a chromophore in a monolayer at an air-water interface without any contribution of the chromophores from the bulk solution [12]. Therefore, reflection measurements provide better insight into processes at the interface and have been performed to prove the presence of PyS- at the interface. The reflection spectra AR measured at normal inqidence o f ~ the solution containing 3 x 10-6M NaPyS and 3 × 10-6M protamine in the absence and in the presence of DPPC, DMPA and DOMA monolayers, at an initial surface pressure n ~ = 5 m N m -~ are presented in Fig. 11. Reflection spectra with the maximum at 346 nm are obtained from all three monolayers, confirming the presence of pyrene chromophores at the interface. Since no enhanced reflection signal was recorded from the DPPC and DMPA monolayers spread on the pure NaPyS subphase [8], these results provide evidence for increased accumulation of NaPyS at the lipid monolayer-solution interface from the solution containing protamine. The AR-A (at 346 nm) isotherms of DMPA,
0.20 120DOMA~DOMA
¢- 100-
0.15 -
f ~ A /
/
o~
0 o
80-
o t-
60-
o
o.10-
<1
0.05 -
o,~
S u b p h a s e
offl 40-
0 20-
0J
400
0.00
I
I
I
I
450
500
550
600
650
Wavelength [nm] Fig. 10. Fluorescence spectra for D O M A and DPPC monolayers spread on the subphase containing 3 x 10 6 M protamine and 3 x 10 6 M NaPyS. Fluorescence spectrum from pure subphase is also given. Surface pressure ~t = 20 mN m 1.
-
300
s~,~
- v
v-~_,.,_.~,.
I
I
I
I
I
~
I
320
340
360
380
400
420
440
Wavelenght [nm] Fig. l 1. Reflection spectra from solution of 3 x 10 -6 M protamine and 3 x 10 -6 M NaPyS, and from DPPC, D O M A and DMPA monolayers spread on the same subphase. Initial surface pressure of monolayers g = 5 m N m - 1.
Z. Kozarac, D. M6bius / Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
changes during the penetration and/or squeeze-out processes.
0.5-
E t-
0.4-
0.3 -
173
\
\~ .9o~
4. Conclusion
t-
.g O
0.2 -
" ...........
DMPA
m
n-
o.,_ i?; 0.00.5
" i" 1.0
1.5
2.0
2.5
3.0
3.5
Area/Matrix Molecule [ nm 2] Fig. 12. Surface reflection AR (at 346 nm) area isotherms for DPPC, DOMA and DMPA monolayers spread on the subphase of 3 × 10 -6 M protamine and 3 x 10 -6 M NaPyS.
DOMA and DPPC monolayers on the subphase containing 3 x 10 - 6 M NaPyS and 3 x 10 - 6 M protamine are shown in Fig. 12. The results show that AR values obtained by reflection from DPPC and DOMA monolayers increase sharply until ADppc=3.0nm 2 and ADOMA=2.5nm 2, and afterwards remain constant until ADPPC= 1.7 nm 2 and ADOMA= 1.0 nm 2. AR values for DMPA are lower and constant until A = 1.3 n m 2. During further compression, AR values for DPPC and DMPA decrease and those for DOMA increase. Reflection spectroscopy is a very sensitive method with regard to surface densities, and the dependence of AR on area A can be very informative. If no orientational change or loss of NaPyS during the compression process takes place, it can be expected that the AR values should remain constant. If AR increases at higher surface densities, this can be attributed to the adsorption of the adsorbate molecules to the solid phase of the lipid monolayer [23-25]. AR for DOMA increases at areas smaller than 1.0 nm 2, indicating a formation of adsorbed submonolayer under the positively charged head groups of the densely packed DOMA monolayer in accordance with the model of molecular organization of PyS- in the co-spread P y S - / D O M A monolayers [7]. AR values for DPPC and DMPA monolayers decrease by further compression, indicating that adsorbate molecules undergo orientational
The interaction of the hydrophilic, negatively charged pyrene derivative NaPyS with noncharged and hydrophobic interfaces such as lipid membranes and/or air-water interface is enhanced strongly by the presence of the positively charged protein protamine in the solution. Studies of synergetic effects caused by the complex adsorption of two components from the subphase to the lipid monolayers at the interface are of considerable interest because of the co-occurrence of such components in the natural environment.
Acknowledgment
We express our thanks to Werner Zeiss for technical assistance. Financial support to Zlatica Kozarac from the Osteuropa-Verbindungsbtiro des BMBF bei der DLR, Bonn, within the bilateral agreement between the Federal Republic of Germany and the Republic of Croatia is gratefully acknowledged. The work was also funded by the Ministry of Science, Republic of Croatia.
References [1] B. (%sovi6, V. Vojvodi6, Mar. Chem. 28 (1989) 183. [2] Z. Kozarac, B. Cosovi6, B. Ga~parovi6, A. Dhathathreyan, D. M6bius, Langmuir 7 (1991) 1076. [3] D.E. Kile, C.T. Chiou, R.S. Helburn, Environ. Sci. Technol. 24 (1990) 205. [4] C.T. Jafvert, Environ. Sci. Technol. 25 (1991) 1039. [5] D.A.L. Edwards, R.G. Luthy, Z. Liu, Environ. Sci. Technol. 25 (1991) 127. [6] C.T. Jafvert, P.L. van Hoof, J.K. Heath, Water Res. 28 (1994) 1009. [7]Z. Kozarac, R.C. Ahuja, D. M6bius, Langmuir 11 (1995) 568. [8] Z. Kozarac, B. Cosovi6, R.C. Ahuja, D. M6bius, W. Budach, Langmuir 12 (1996) 5387. [9] Z. Kozarac, B. ¢~osovi6, W. Budach, D. M6bius, Croat. Chem. Acta 70 (1997) 125.
174
Z. Kozarae, D. MObius/Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
[10] H.L. Keith, W.A. Telliard, Environ. Sci. Technol. 13 (1979) 416. [11] W. Budach, Elektrostatische, dynamische und optische Aufbaus yon Sensor-Schichtsystemen, Ph.D. Thesis, GOttingen, 199l. [12] H. Gr6niger, D. MObius, H. Meyer, J. Chem. Phys. 79 (1983) 3701. [13] R. Verger, F. Pattus, Chem. Phys. Lipids 30 (1982) 189. [14] L. Ter-Minassian Saraga, Langmuir 1 (1985) 391. [15] M.G. Ivanova, R. Verger, A.G. Bois, I. Panaiotov, Colloids Surf. 54 (1991) 279. [16] R.C. Ahuja, P.L. Caruso, D. MObius, G. Wildburg, H. Ringsdorf, D. Philp, J.A. Preece, J.F. Stoddart, Langmuir 9 (1993) 1534. [17] D.M. Taylor, O.N. Oliveira Jr.,, H. Morgan, J. Colloid Interface Sci. 139 (1990) 508.
[18] J.T. Davies, E.K. Rideal, lnterfacial Phenomena, Academic Press, New York, 1963. [19] R.C. Ahuja, P.L. Caruso, D. MObius, Thin Solid Films 242 (1994) 195. [20] G.P.L. Heureux, M. Fragata, J. Photochem. Photobiol. B: 3 (1989) 53. [21] B. Stevens, E. Hutton, Nature 186 (1960) 1045. [22] Th. FOrster, Angew. Chem. 81 (1969) 364. [23] Z. Kozarac, A. Dhathathreyan, D. Mfbius, FEBS Lett. 229 (1988) 372. [24] Z. Kozarac, A. Dhathathreyan, D. MObius, Eur. Biophys. J. 15 (1987) 193. [25] Z. Kozarac, D. MObius, Colloids Surf. A: 83 (1994) 99.
Colloidsand Surfaces A: Physicochemicaland EngineeringAspects 135 (1998) 165-174
SURFACES
A
Protamine-induced adsorption of sodium pyrene-3-sulphonate (NaPyS) on the lipid monolayers Z. Kozarac a,,, D. M6bius b a Ruder Bogkovik Institute, Center for Marine Research Zagreb, P.O.Box 1016, HR-IO001 Zagreb, Croatia b Max-Planck-Institutfiir biophysikalische Chemie, Postfach 2841, D-37018 G6ttingen, Germany
Received 3 March 1997; accepted 7 August 1997
Abstract
The interaction of sodium pyrene-3-sulphonate(NaPyS) with different lipid monolayers at the air-solution interface was studied by monolayer techniques (surface pressure and surface potential measurements) and by spectroscopic techniques (fluorescence and reflection measurements). The uptake of negatively charged NaPyS by non-charged and negatively charged phospholipid monolayers was induced by the presence of positively charged protein protamine in bulk solution. © 1998 Elsevier Science B.V. Keywords: Air-water interface; Lipid monolayers; Pyrene salt; Monolayer techniques; Spectroscopic techniques;
Synergetic effects
1. Introduction
Natural aquatic systems contain a large number of organic substances with different functional groups and different hydrophobic properties. Adsorption at natural interfaces (air-water, watermineral, oil-water) is determined by hydrophobic and/or electrostatic interactions of adsorbate and interface. In the case of attractive interactions between two or more components, either in the solution or at the interface, a synergetic effect can be expected. It is known that in the organic matter adsorbed at model and natural phase boundaries, hydrophobic fractions usually dominate [1]. Electrostatic interactions of hydrophilic ions and/or molecules from the bulk solution with functional groups of adsorbed hydrophobic sub* Correspondingauthor. Tel:00385 14561105; fax: 00385 1420437;e-mail:[email protected] 0927-7757/98/$19.00 © 1998ElsevierScienceB.V.All rightsreserved. PII S0927-7757 (97) 00242-2
stances may be responsible for the surface excess of these solute species at the natural interfaces. In our previous adsorption studies of compounds whose mutual interactions can cause enhanced adsorption and uptake to the interfacial layers at natural phase boundaries we used a para-nitrophenol (PNP)-lipid system [2]. It was found that PNP does not accumulate from bulk solution to the free air-solution interface and does not accumulate in non-charged phospholipid monolayers spread at the air-solution interface either. The attachment of PNP to the neutral phospholipid monolayers was enhanced and accelerated at the air-water interface by the presence of quaternary ammonium salts in the solution. Considerable attention has recently been given to the studies of pollutant-surfactant interactions in water and studies of pollutant solubilization by micelles [3-6]. Recently, we have started systematic physicochemical studies of adsorption of pyrene and
166
Z. Kozarac, D. M6bius / Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
pyrene derivatives at different model phase boundaries [7-9]. It is known that pyrene and its derivatives belong to the highly dangerous pollutants in the environment [10]. Our results showed that sodium pyrene sulphonate (NaPyS) does not adsorb at the free water surface and does not interact with dipalmitoylphosphatidylcholine (DPPC) monolayers, but interacts strongly with positively charged dioctadecyldimethylammonium bromide (DOMA) monolayers [8]. Accordingly, we expected surfactant-mediated adsorption when positively charged surface-active molecules are present in the subphase. In natural aquatic systems, among the positively charged lipids the artificial cationic detergents (quaternary ammonium salts) are predominant, whereas among naturally occurring substances, especially in marine waters, protamines can be found. They represent a group of simple proteins which occur in combination with nucleic acids in the sperm of fish. Here, we report on adsorption studies of NaPyS at the air-water interface and its interaction with charged and neutral lipids in the presence of protamine in the subphase.
2. Experimental 2.1. Procedure Surface pressure-molecular area (vr-A) and surface potential-molecular area (AV-A) isotherms have been measured in a rectangular Teflon trough with the inner dimensions of 56 × 18 cm 2 and a depth of 1 cm which was enclosed in a tight box and thermostatically controlled. The monolayers were compressed by a movable Teflon barrier with compression velocity between 0.08 and 0.12nmZmin-lmolecule -1 at which the best reproducibility was achieved, although other rates of compression could also be used. A Wilhelmy balance (20 mm wide filter paper) was used to measure the surface pressure, and the surface potential was measured using a vibrating plate condenser. Fluorescence measurements have been performed using a Perkin-Elmer Luminescence LS-5 spectrometer with xenon lamp modified for in situ
measurements at the air-water interface in combination with a Teflon trough [11]. The monolayer was excited at 350 rim, and the emission spectrum was measured in the 375-575nm range. Fluorescence spectra have been normalized for a constant lipid surface density. Reflection spectroscopic measurements have been performed using a reflection spectrometer for measurements under normal incidence of light [12]. The reflection was measured and expressed as the difference AR in the reflectivity from the surface covered with a monolayer and from the monolayer-free-solution surface. 2.2. Chemicals NaPyS was obtained from Molecular Probes. DOMA, DPPC and dimyristoylphosphatidic acid (DMPA) were purchased from Sigma Chemical Co. and used as-received. The main reason for choosing these three lipids was their charge and not the length of hydrophobic chains. DPPC and DMPA or DPPA are usually used as neutral and negatively charged monolayers, and no significant difference in the behaviour of DMPA and DPPA monolayers was noticed. DOMA was chosen because it forms a very stable water insoluble monolayer due to the presence of two hydrophobic C18 chains. Deionized water from a Milli-Q system (Millipore Corp.) was used for preparing the subphase. Chloroform (HPLC) p.a. grade was used as spreading solvent and was obtained from Baker Chemicals, Holland. Protamine sulphate, isolated from Salmine, was obtained from Serva, Heidelberg.
3. Results and discussion 3.1. Surface pressure and surface potential measurements 3.1.1. n-A isotherms We have already reported that no appreciable accumulation of NaPyS at the monolayer free air-water interface and no interaction with neutral zwitterionic phospholipid monolayers of DPPC
Z. Kozarac, D. MObius/ Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
has been observed [8]. If adding protamine, which is very hydrophilic and carries positive charge to NaPyS solution, a weak adsorption to the water s u r f a c e is detected during 2 h accumulation time, as evidenced by a slight increase in surface pressure n in Fig. 1, subphase 2 (full line). For comparison, the time dependence of n for a pure protamine solution is also shown in Fig. 1, subphase 1 (dotted line). However, the attachment of NaPyS to the DPPC monolayers is considerably enhanced and accelerated in the presence of protamine in solution (Fig. 1). A similar effect was observed in the presence of a DMPA monolayer (data not shown here). An increase of the surface pressure due to the adsorption in the presence of a DPPC monolayer at initial surface pressures of n = 0 and n = 5.5 mN m - 1 (surface pressure change of An = 3.8 and A n = 6 . 7 m N m -~) was observed after 50 min accumulation time, as shown in Fig. 2. The An values in the presence of DMPA monolayer on a subphase containing protamine and NaPyS at the initial surface pressures of 0 and 5.37 mN m-1 and after 50 min accumulation time were 4.6 mN m - 1 and 6.7 mN m - ~ respectively. From the surface pressure-area (n-A) isotherm of the material accumulated at the subphase 2-air interface, shown in Fig. 3, it can be seen that even a self-assembled mixed monolayer, although rather unstable, has been formed.
20E Z
DPPC on subphasecontaining protamineand NaPyS
15-
E •" l 8 100. O
~ co
5-
DPPC
on
subphase 2
0
108-
DPPC on subphase 1
642 -
subphase 1 and 2
o.~.....~
o
50
Fig. 2. Time dependence of surface pressure of DPPC monolayers spread on solution of 10-SM protamine and 10-SM NaPyS. Initial surface pressure n = 0 m N m -1 and n = 5.5 mN m - 1 respectively.
s0~ 407 -,
400
O~
Protamlne + NaPyS without monolayer
300
30-
...g 20O O
t0
AV
0. 20~ i |l ~:-~ 09
t-
O
=.. "--,....
loo 3 '" ................
<
11[
i
i
i
i
i
100
150
200
250
300
0 350
Fig. 3. Surface-pressure- and surface-potential-area isotherms for pure solution containing 10-SM protamine and 10 -5 M NaPyS.
~E' 12Z
I:1..
4'0
Time [min]
Area [era2]
14-
(/)
3'0
1'0
50
E ,=
167
2'0
...............
do
8'o
i
100
120
Time [min] Fig. 1. Time dependence of surface pressure of the subphase-air interface and of DPPC monolayers spread on subphases 1 and 2. Subphase 1:10 -5 M protamine. Subphase 2:10 -5 M protamine and 10 -5 M NaPyS.
The surface pressure-molecular area (n-A) isotherms for DPPC, DOMA, and DMPA monolayers spread on water (curve 1), on a subphase containing 10-SM protamine (curve 2), and a subphase containing both 10-SM protamine and 10 -5 M NaPyS (curve 3) are shown in Figs. 4-6. The thermodynamic parameters compressional modulus C~- 1 and compressibility 1/C~ ~ have been calculated and are given in Table 1. DPPC spread on water exhibits a rather condensed film with a phase transition around 5 mN m -~ (Fig. 4, curve 1). The isotherm for the
Z. Kozarac, D. MObius/Colloids Surfaces A." Physicochem. Eng. Aspects 135 (1998) 165-174
168
50-
50~
E
"~ Z
40-
4030-
u) I~- 2 0 -
~,
\\
~.~
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8 ~= 10-
¢:
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1
lO--
i"~"~"~3
......... ~.~
", 2
........ \
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.
.
.
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Fig. 4. Surface pressure-area (n-A) isotherms for DPPC monolayers spread on water (1) and on subphases containing 10-SM protamine (2) and 10 -5 M protamine and 10-SM NaPyS (3). 50-
~ Z
\
40-
",~
PyS-
3o¢t) •~
.,.
20-
-% %.. 2
"%'-.. 3
09 0 0.0
110
00
0.5
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1.5
2.0
Area/DMPA Molecule [nm 2 ]
Area/DPPC Molecule [nm 2 ]
2.0
Area/DOMA Molecule [nm 2 ] Fig. 5. Surface pressure-area (n-A) isotherms for DOMA monolayers spread on water (1) and on subphases containing 10 -5 M protamine (2), and 10 -5 M NaPyS and 10 5 M protamine (3).
DPPC monolayer shows only small differences if protamine (curve 2) is present in the subphase. The same was previously observed with a DPPC monolayer spread on the subphase containing NaPyS [8]. When a DPPC monolayer was spread on the subphase containing protamine and NaPyS, both 10 -5 M, an extreme expansion of the area per DPPC molecule was observed (Fig. 4, curve 3). The physical state of DPPC is altered from the
Fig. 6. Surface pressure area (n A) isotherms for DMPA monolayers spread on water (1) and on subphases containing 10-SM protamine (2) and 10 -5 M protamine and 10-SM NaPyS (3).
condensed to the liquid-expanded film simultaneously with disappearance of phase transition. This can also be seen from the change in compressibility values given in Table 1. The area per DPPC spread on the subphase containing protamine and NaPyS is much larger until a surface pressure of 25 mN m -1, and is increased by approximately 0.07 nm 2 even at collapse pressure. The n-A isotherm of a DOMA monolayer spread on water is of the liquid-expanded type, with a phase transition around 14 mN m-1 (Fig. 5, curve 1). If protamine is present in the solution, the DOMA monolayer isotherm does not change much. It still shows a liquid-expanded phase at low surface pressure with a phase transition at 18 m N m -a (Fig. 5, curve 2). The isotherms in a more densely packed phase (n>25 mN m -1) are identical. The isotherm for a DOMA monolayer spread on the subphase containing 10-SM NaPyS shows a contraction with respect to DOMA on water at larger areas, A > 1.0 nm 2 (5 mNm-1). Further compression until A =0.85 nm 2 leads to a strong increase in surface pressure, and the phase transition disappears. The area/DOMA molecule for the monolayer in the dense packed state (40 mN m -a) is larger by approximately 0.32 nm 2 compared with the DOMA monolayer spread on
Z. Kozarac, D. M6bius / Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
169
Table 1 Thermodynamicparameters of DPPC, DOMA and DMPA monolayers on different subphases Subphase
Limiting specificarea A0 (nmz molecule-1)
Compressiionalmodulus C~-1 (mN m- 1)
CompressibilityC~-1 (m N - l)
0.5 0.52 0.65
148.1 146.1 13.0
0.0067 0.0068 0.077
0.62 0.97 1.25
32.2 171.3 12.5
0.031 0.0058 0.08
0.40 0.47 0.51
422.3 48.57 35.0
0.0024 0.021 0.029
DPPC
water 10-5 M protamine 10-s M protamine and 10-5 M NaPyS DOMA water 10-5 M NaPyS 10-5 M protamine and 10-5 M NaPyS DMPA
water 10-5 M protamine t0 -5 M protamine and 10-5 M NaPyS
water. This is in agreement with previously observed behaviour of D O M A monolayers spread on NaPyS solution (2 x 1 0 - 6 M ) and measured after 2 h accumulation time [8]. The condensation of the D O M A monolayer isotherm at small surface pressures was attributed to the decrease of repulsive forces between the head groups of D O M A due to the electrostatic interaction with P y S - from solution. The condensation effect is also indicated by a decreasing of compressibility from 0.031 for D O M A on water to 0.0058 for D O M A on NaPyS. However, the a r e a / D O M A molecule at high surface pressures is much larger than the area/DOMA molecule on water subphase, which clearly shows the lipid-solute interaction. This can be interpreted in terms of penetration and/or incorporation of solute molecules in the monolayer [13-15] but also in the terms of binding the solute molecules underneath the lipid monolayer [7,12,16]. Very interesting behaviour was observed when a D O M A monolayer was spread on the subphase containing both solutes, protamine and NaPyS. An extreme expansion of the area per D O M A molecule at all surface pressures was observed with respect to the D O M A monolayers spread on water, and in the tightly packed state (40 m N m - i ) the area increase is 0.2 nm z. However, if we compare the isotherm for D O M A spread on this subphase to the isotherm for D O M A spread on subphase containing only NaPyS, the expansion of area per D O M A molecule is very large at surface area until
A =0.92 nm 2. By further compressing the D O M A monolayer, a smaller area per matrix molecule for D O M A spread on subphase containing both solutes with respect to D O M A spread on NaPyS solution is obtained. At high surface pressures (40 m N m - ~) the difference in the area is approximately 0.12 nm 2. Compressibility is larger than for D O M A on water, which was expected because of the expansion of the isotherm. The surface pressure-area (n-A) isotherms for DMPA monolayers spread on water and on NaPyS solution are of the condensed type (Fig. 6, curve 1). In the presence of protamine in the subphase, a more expanded isotherm with a phase transition around 7.0 m N m -~ is obtained (Fig. 6, curve 2). Even at very high surface densities of monolayer ( ~ 40 m N m - 1) area/DMPA molecule is larger by approximately 0.05 nm 2. By addition of NaPyS to the protamine subphase, the isotherm for DMPA monolayer expanded to much higher area values until a pressure around 22 m N m -1. By further compression, the area reached the same value as for the DMPA monolayer spread on the protamine subphase (Fig. 6, curve 3). Compressibility values for DMPA monolayers spread on both subphases increases by ten times compared with the value for the monolayer on water. 3.1.2. AV-A isotherms
The surface potential of an amphiphilic lipid monolayer at the air-water interface may be
Z. Kozarac, D. M6bius / Colloids Surfaces A." Physicochem. Eng. Aspects 135 (1998) 165 174
170
expressed [17] using the following relation:
1000-
AV=AVo + AVp+ yo The A Vo term is the value of surface potential for the clean air-water interface, the A Vp term represents the contribution of the dipole moment of the lipid molecule and may be divided into the head group and the tail region dipole moments. Yo is the so-called double layer potential due to the charges in the lipid. The value of Yo is dependent on the degree of dissociation, on the density of head groups, and the nature and concentration of counterions in the subphase. It can be estimated from the Gouy-Chapman theory [18]. The value of Yo is expected to change in the case of specific adsorption of any counterion at the monolayer-subphase interface. Surface potential-area isotherms for DPPC, DOMA and DMPA spread on the subphase containing protamine and NaPyS measured simultaneously with the rc-A isotherms, which are presented in Figs. 4-6, are shown in Figs. 7-9. DPPC monolayer (Fig. 7) on water (curve 1) shows positive values of the surface potential in the whole range of the isotherm. The isotherm also shows the phase transition between A =0.95 and A--0.6 nm 2, i.e. for values of the surface pressure around 3 0 m N m -a. The isotherm for DPPC on the protamine subphase (curve 2) is
> ~ BOO-
1
~5 600o_ oo ~ 400o~ "t
2o00.o
015
110
115
2.0
Area/DOMA Molecule [nm 2 ] Fig. 8. Surface potential area (AV-A) isotherms for DOMA monolayers spread on water (1) and on subphases containing 10 5M NaPyS (2) and 10-SM protamine and 10-SM NaPyS (3).
600 -
~ 500400-
-~ E 300-
',2
o. 200o ~: 100-,
09
0-
800 -100 0.0
0.5
1.0
1.5
2.0
AreaJDMPA Molecule [nm 2 ]
600. m .i..,
\
~) 400-
"
Q.
Fig. 9. Surface potential-area (AV-A) isotherms for DMPA monolayers spread on water (1) and on subphases containing 10-SM protamine (2) and 10-SM protamine and 10-SM NaPyS (3).
2 ';
3
o 200-
0-
00
015
1'0
l's
2.0
Area/DPPC Molecule [nm2 ] Fig. 7. Surface potential area (AV-A) isotherms for DPPC monolayers spread on water (1) and on subphases containing 10 -5 M protamine (2), 10 -5 M NaPyS (3) and 10 -5 M protamine and 10 -5 M NaPyS (4).
almost identical with that on water, while the isotherm for DPPC on NaPyS solution (curve 3) is shifted towards more positive values until A = l . 0 n m z and by further compression also becomes almost identical with the isotherm on water. Since DPPC, as a non-charged lipid, probably does not interact with protamine or NaPyS from solution via coulombic interactions, we did not expect any appreciable change in surface
Z. Kozarac, D. Mrbius / Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
potential either. However, the surface potential values for DPPC monolayers spread on the subphase containing both solutes, i.e. NaPyS and protamine (curve 4), are dramatically less positive than the values for DPPC monolayers on water and on subphases containing only a single component, i.e. either NaPyS or protamine. The difference in A V is approximately 500 mV in the dense packed monolayer. It can be assumed that the binding of NaPyS to DPPC monolayers is enabled via complex hydrophobic and electrostatic interactions of DPPC and NaPyS with protamine. Surface potential-area isotherms for DOMA on water, on NaPyS solution and on subphase containing NaPyS and protamine are shown in Fig. 8. The surface potential for DOMA spread on water (curve 1) is positive in the whole area range, indicating the dominance of positive head group charge in the A V values. Since NaPyS interacts with DOMA monolayers via coulombic interactions [7], the decrease in AV values for DOMA monolayers spread on NaPyS subphase with respect to DOMA on water is expected and has also been observed (Fig. 8, curve 2). The surface potential of DOMA on water at A =0.56 nm 2 was 900 mV and was 670 mV on NaPyS. The decrease in positive surface potential due to the charge compensation of DOMA head group with PySgroup is 230 mV, which is in good correlation with the recently reported value of 200 mV assigned to the dipole moment of the DOMA head group [19]. DOMA spread on the subphase containing both solutes showed more positive values of surface potential at larger areas until A ~ 1.0 nm 2. By further compression, the surface potential is shifted towards less positive values in comparison with DOMA on water and on NaPyS. The surface potential-area isotherm for DMPA on water exhibits negative values for A V until the molecular area 0.65 nm 2, which shows the predominant role of the negative head group charge in the A V values (Fig. 9, curve 1). Further compression of the film is followed by the increase in A V. It can be expected that the adsorption of positively charged protamine to the negatively charged DMPA influences the A V-A isotherms. As a result of charge compensation, the surface potential A V should be shifted towards more posi-
171
tive values. The A V-A isotherms corresponding to the 7r-A isotherms (Fig. 6) are shown in Fig. 9. It can be seen that A V values for the DMPA spread on protamine (curve 2) are more positive compared with the reference DMPA. The maximum increase in A V as a result of DMPA-protamine interaction is ca. 300 mV, which is in good agreement with previously reported surface potential data for DMPA interacting with positively charged bisbipyridinium tetracations [ 16]. At areas larger than A =0.65 nm 2, surface potential values for DMPA on subphase containing protamine and NaPyS are equal to the A V values for DMPA on water. By further compression until A = 0.4 nm 2, the potential values of DMPA monolayer on subphase containing both solutes are shifted towards negative values in comparison with the values of DMPA on water and on protamine solution. 3.2. Fluorescence and reflection spectroscopy From the surface pressure and surface potential measurements it is obvious that the presence of protamine in solution influences strongly the binding of PyS- to the non-charged and negatively charged phospholipids. The complex adsorption of two components from the solution to the phospholipid monolayers at the interface takes place due to the synergetic effects. The interaction of the water-soluble components, NaPyS and protamine, can take place in bulk solution and/or at the interface. In order to achieve a better understanding of the mechanisms involved in these interactions, spectroscopic measurements have been made. Fluorescence measurements are very often used in the studies of adsorption of unsubstituted and substituted pyrene. The pyrene chromophore has a relatively long excited state lifetime, high quantum yield of fluorescence and a pronounced tendency to form excimers, e.g. the molecular complexes in the excited state [20-22]. Furthermore, the structured monomer emission spectrum and the quantum yield of fluorescence are very sensitive to the environment polarity and pyrene-pyrene interactions. The proximity of pyrene molecules is easily detected by the presence of excimer emission. This pyrene-pyrene inter-
Z. Kozarac, D. M6bius / Colloids Surfaces A." Physicochem. Eng. Aspects 135 (1998) 165-174
172
action causes a reduction of the monomer emission in the 360-430 nm range and the appearance of a red-shifted broad structureless emission band with a maximum at ca. 480 nm. The location of pyrene molecules at the lipid monolayer-subphase interface and the intermolecular association processes are expected to be influenced by the nature (dipole moment, charge) of lipid head groups and by the subphase composition. Fig. 10 shows the emission spectra (range 400-600nm) of the pure subphase containing 3 × 10 - 6 M NaPyS and 3 × 10 - 6 M protamine (full line), and the spectra of DPPC and DOMA monolayers spread on this subphase and compressed to 20 mN m-1 (dashed and dotted lines). The monomer fluorescence is predominant in all emission spectra, but an additional excimer band at 475 nm for DOMA monolayers can be seen. However, this excimer emission is much weaker than the excimer emission of DOMA/PyS- co-spread monolayers [7]. The same weak excimer emission was observed when DOMA monolayers were spread on subphase containing only NaPyS [8]. The excimer formation requires direct contact between PySchromophores. When NaPyS is dissolved in the solution, the possibility of interaction of an unexcited pyrene with an excited pyrene during the excited state lifetime is decreased, and thus no, or only very weak, excimer emission can be observed.
However, monomer emission can be expected, since both pyrene chromophores present in the solution and at the interface contribute to the measured fluorescence. Reflection spectroscopy is based on the enhanced reflection due to the presence of a chromophore in a monolayer at an air-water interface without any contribution of the chromophores from the bulk solution [12]. Therefore, reflection measurements provide better insight into processes at the interface and have been performed to prove the presence of PyS- at the interface. The reflection spectra AR measured at normal inqidence o f ~ the solution containing 3 x 10-6M NaPyS and 3 × 10-6M protamine in the absence and in the presence of DPPC, DMPA and DOMA monolayers, at an initial surface pressure n ~ = 5 m N m -~ are presented in Fig. 11. Reflection spectra with the maximum at 346 nm are obtained from all three monolayers, confirming the presence of pyrene chromophores at the interface. Since no enhanced reflection signal was recorded from the DPPC and DMPA monolayers spread on the pure NaPyS subphase [8], these results provide evidence for increased accumulation of NaPyS at the lipid monolayer-solution interface from the solution containing protamine. The AR-A (at 346 nm) isotherms of DMPA,
0.20 120DOMA~DOMA
¢- 100-
0.15 -
f ~ A /
/
o~
0 o
80-
o t-
60-
o
o.10-
<1
0.05 -
o,~
S u b p h a s e
offl 40-
0 20-
0J
400
0.00
I
I
I
I
450
500
550
600
650
Wavelength [nm] Fig. 10. Fluorescence spectra for D O M A and DPPC monolayers spread on the subphase containing 3 x 10 6 M protamine and 3 x 10 6 M NaPyS. Fluorescence spectrum from pure subphase is also given. Surface pressure ~t = 20 mN m 1.
-
300
s~,~
- v
v-~_,.,_.~,.
I
I
I
I
I
~
I
320
340
360
380
400
420
440
Wavelenght [nm] Fig. l 1. Reflection spectra from solution of 3 x 10 -6 M protamine and 3 x 10 -6 M NaPyS, and from DPPC, D O M A and DMPA monolayers spread on the same subphase. Initial surface pressure of monolayers g = 5 m N m - 1.
Z. Kozarac, D. M6bius / Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
changes during the penetration and/or squeeze-out processes.
0.5-
E t-
0.4-
0.3 -
173
\
\~ .9o~
4. Conclusion
t-
.g O
0.2 -
" ...........
DMPA
m
n-
o.,_ i?; 0.00.5
" i" 1.0
1.5
2.0
2.5
3.0
3.5
Area/Matrix Molecule [ nm 2] Fig. 12. Surface reflection AR (at 346 nm) area isotherms for DPPC, DOMA and DMPA monolayers spread on the subphase of 3 × 10 -6 M protamine and 3 x 10 -6 M NaPyS.
DOMA and DPPC monolayers on the subphase containing 3 x 10 - 6 M NaPyS and 3 x 10 - 6 M protamine are shown in Fig. 12. The results show that AR values obtained by reflection from DPPC and DOMA monolayers increase sharply until ADppc=3.0nm 2 and ADOMA=2.5nm 2, and afterwards remain constant until ADPPC= 1.7 nm 2 and ADOMA= 1.0 nm 2. AR values for DMPA are lower and constant until A = 1.3 n m 2. During further compression, AR values for DPPC and DMPA decrease and those for DOMA increase. Reflection spectroscopy is a very sensitive method with regard to surface densities, and the dependence of AR on area A can be very informative. If no orientational change or loss of NaPyS during the compression process takes place, it can be expected that the AR values should remain constant. If AR increases at higher surface densities, this can be attributed to the adsorption of the adsorbate molecules to the solid phase of the lipid monolayer [23-25]. AR for DOMA increases at areas smaller than 1.0 nm 2, indicating a formation of adsorbed submonolayer under the positively charged head groups of the densely packed DOMA monolayer in accordance with the model of molecular organization of PyS- in the co-spread P y S - / D O M A monolayers [7]. AR values for DPPC and DMPA monolayers decrease by further compression, indicating that adsorbate molecules undergo orientational
The interaction of the hydrophilic, negatively charged pyrene derivative NaPyS with noncharged and hydrophobic interfaces such as lipid membranes and/or air-water interface is enhanced strongly by the presence of the positively charged protein protamine in the solution. Studies of synergetic effects caused by the complex adsorption of two components from the subphase to the lipid monolayers at the interface are of considerable interest because of the co-occurrence of such components in the natural environment.
Acknowledgment
We express our thanks to Werner Zeiss for technical assistance. Financial support to Zlatica Kozarac from the Osteuropa-Verbindungsbtiro des BMBF bei der DLR, Bonn, within the bilateral agreement between the Federal Republic of Germany and the Republic of Croatia is gratefully acknowledged. The work was also funded by the Ministry of Science, Republic of Croatia.
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174
Z. Kozarae, D. MObius/Colloids Surfaces A: Physicochem. Eng. Aspects 135 (1998) 165-174
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