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aqueous solution interface
Colloids and Surfaces B: Biointerfaces 27 (2003) 303 /309 www.elsevier.com/locate/colsurfb
The influence of pH on phosphatidylcholine monolayer at the air/aqueous solution interface Izabela Brzozowska, Zbigniew A. Figaszewski * Laboratory of Interfacial Electrochemistry, Faculty of Chemistry, University of Warsaw, PL / 02-093 Warsaw, Pasteura 1, Poland Institute of Chemistry, University of Bialystok, Al. Pilsudskiego 11/4, PL / 15-443 Bialystok, Poland Received 4 January 2002; received in revised form 10 April 2002; accepted 17 June 2002
Abstract The measurements of the interfacial tension at the air/aqueous subphase interface as the function of pH were performed. The interfacial tension of the air /aqueous subphase interface was divided into contributions of individuals. A simple model of the influence of pH on the phosphatidylcholine monolayer at the air/hydrophobic chains of phosphatidylcholine is presented. The contributions of additive phosphatidylcholine forms (both interfacial tension values and molecular area values) depend on pH. The interfacial tension values and the molecular areas values for LH , LOH forms of phosphatidylcholine were calculated. The assumed model was verified experimentally. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Monolayer; Egg yolk phosphatidylcholine; Interfacial tension; pH
1. Introduction Phospholipids are major fractions of lipids found in biological membranes. Since monolayers*/especially at the air /water interface */ are commonly used as simplified models of biomembrane, many studies have been concentrated on them [1]. The structure of lipid monolayer seems to be apparently simple at the first estimation. However, at closer study, one can distinguish there two different interfaces: the airhydrophobic chains of phospholipid and theirs hydrophilic heads*/aqueous subphase. The prop-
* Corresponding author E-mail address: [email protected] (Z.A. Figaszewski).
erties of such membranes depend strongly on the molecules that built it. Therefore, the knowledge of molecular structure and organisation of phospholipids is necessary. Hence, the detailed researches connected with the influence of pH on lecithin monolayer are required. Phosphatidylcholine (L) is a neutral, zwitterionic phospholipid with an amphiphilic character. The surface pressure /area per molecule (p /A ) curves of dicetyl phosphate, of dipalmitoyl, egg dileoyl lecithin have been reported previously [2]. According to Anderson and Pethica the surface pressure /area isotherms of distearoyl lecithin monolayers are the same in the pH range 1/7 [2]. The pH dependence of amphiphilic substances at the air /water interface was investigated even earlier, at the beginning of the last century [3]. Not
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only the interfacial tension values were recorded but also the ionic properties of a monolayer were studied by means of investigation of its surface potential at a fixed value of area/molecule. According to Schulman et al. the surface potential is not influenced by the pH of the subphase since the DV /pH plot is a straight line with zero slope. This fact can be explained by assuming a mutual interaction between the phosphate and trimethylammonium groups, which neutralised each other. Also the effect of pH on the electrophoretic mobility of lecithin is characteristically similar to the ones obtained by the monolayer technique [4]. In a smaller pH range, 4 /8, dipalmitoyl and egg lecithins also show the properties of uncharged monolayers. There is no net charge on the monolayers between pH 4 /8. This suggests that neutralisation of the phosphate group is related to intermolecular spacing in monolayers. Shah and Schulman propose that the internal neutralisation of ionic charges to be caused by neutralisation of oppositely charged groups of adjacent molecules [5]. However, the related researches concerning the adsorption of Ca2 as a function of pH subphase suggest something quite different. Since below pH 3 only small amount of 45Ca is adsorbed, the films of phospholipid are assumed to be unionised. Ca2 is permanently adsorbed at pH values above 6.5 with an increasing affinity up to pH 11 [6]. While a phosphatidylcholine monolayer is not altered by the pH of subphase in some pH regions, a recent study connected with bilayer has shown that there is a maximum of interfacial tension at a certain pH [7]. A careful study is thus pertinent. Since the changes in interfacial tension values induce the changes of the values in the area per molecules, it is very important, in context of biological membranes, to know the exact molecular packing in various pH solutions [8]. We develop a theory, based on the additivity rule, for the influence of pH on a egg yolk phosphatidylcholine monolayer. Using the derived equations we present a model of ion-monolayer interaction based on the calculations employing p /A curves.
2. Experimental The measuring procedure was described elsewhere [9]. Surface tension measurements were carried out in Langmuir trough equipped with 9000 Nima tensiometer at the water/air interface at 22 8C. Lipids were brought into contact with the interface with a Hamilton syringe. After evaporation of the solvent (10 min) the compression of the monolayer was performed. The rate of compression was 0.1 cm/s. The measurements were carried out using Britton-Robinson buffers in the range of 2/12 [10]. This buffer is often used in the biological studies because of its wide pH range. The pH was altered by the addition of 0.2 M NaOH to the mixture of 0.04 M acetic acid, 0.04 M phosphoric acid and 0.04 M boric acid. The required pH was controlled using Radiometer pH meter. The water used was triply distilled. The egg lecithin (3-sn-phosphatidylcholine of hen eggs yolk) /99% (TLC) from Fluka was used without further purification. The composition of fatty acids in the egg lecithin was 16:0 /33%, 18:0 / 4%, 18:1 /30%, 18:2 /14%, 20:4 /4%. 1-chloropropane was used as a spreading solvent.
3. Theory Since the phosphatidylcholine molecule (L) possess a zwitterionic character, it can participate in equilibrium reactions with both hydrogen ions and hydroxyl anions. LH ULH LOH ULOH LHOH ULHOH
(1) (2) (3)
Consequently, equations of associations Eqs. (1)/(3) can be consider as the description of an adsorption process. As a result of adsorption of H and OH ions on the surface of phosphatidylcholine layer, the phosphatidylcholine molecule can exist in four different forms. We shall consider following forms: LH with H adsorbed, LOH with OH adsorbed and LHOH with both H and OH ions adsorbed on the surface and a free
I. Brzozowska, Z.A. Figaszewski / Colloids and Surfaces B: Biointerfaces 27 (2003) 303 /309
lecithin molecule L i.e. with no ions adsorbed. A phosphatidylcholine monolayer is assumed to consist of these four forms. The relative contributions of above forms are dependent on pH, according to equations Eqs. (1) /(3). One can write three equations for equilibrium Eqs. (1) /(3), containing the equilibrium constants of these equilibria. On the basis of these equations, the activity of following phosphatidylcholine forms can be calculated: aLH KLH aL aH aLOH KLOH aL aOH aLHOH KLHOH aL
(4) (5) (6)
where ai is the surface concentration of ‘i ’ form of phosphatidylcholine (mol/m2); i LH ;/ /LOH ; LHOH; L; aH ; aOH ; the concentrations of ions in the subphase (mol/m3); Ki is the equilibrium constant of adsorption process of H or OH ions on lecithin (m3/mol). The sum of surface concentrations of any phosphatidylcholine forms at the air /water interface has to be equal to total surface concentrations of phosphatidylcholine (S ). This S concentration can be easily measured using the p /A isotherms. Moreover, the sum of the area fractions of these four phosphatidylcholine forms should give unity. These relationships are described by following equations: aL aLH aLHOH aLOH S (7) aL AL aLH ALH aLHOH ALHOH aLOH ALOH 1
(8)
where S , total surface concentration of phosphatidylcholine measured by p/A isotherms (mol/m2); ˚ 2/molec). Ai , area per molecule (A The equations Eqs. (4) /(8) describe quantitatively the model of the influence of pH subphase on a phosphatidylcholine monolayer. The different forms of phosphatidylcholine would give monolayers, built form one component, that have different stability constant. The value of surface concentrations of any phosphatidylcholine forms affects the molecular packing of the head groups, which*/in a consequence */influences the interfacial tension of lipid monolayer. Depending on the pH of subphase the surface concentrations will
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change as the area per molecule changes. Different forms of phosphatidylcholine will have different areas per molecules depending on the contribution of the forms to the total amount of phosphatidylcholine molecules. After elimination of aLH ; aLHOH ; aL and aLOH terms from the Eqs. (4) /(8), one obtains: 1 S
A1 aH ALH KLH aOH ALOH K LOH A2 aH KLH aOH K LOH
(9)
where A1 AL ALHOH KLHOH ; A2 KLHOH 1/ The direct form of the Eq. (9) is not convenient for calculations. After substituting the concentration of OH ions by the quotient of KH2 O and H concentration one can divide the numerator of above polynomial by its denominator. As a result we obtain the series of terms containing the decreasing powers of H ions concentration. The equation obtained by multiplication by aH is in the form where one can treat the negative terms as negligible. In consequence, such equation would have the linear character. For big H concentrations, i.e. when aH 0 ; the Eq. (9) will assume the following form: aH S
ALH aH
A1 A2 ALH KLH
(10)
Eq. (9) can be treated in the analogous way after substitution of H ion concentrations by concentrations of hydroxyl ions. For big OH concentrations i.e. when aOH 0 ; one can obtain: aOHS
ALOH aOH
A1 A2 ALOH KLOH
(11)
Using these latter relationships, one can easily calculate the values of ALH/and ALOH by regression in the region of big H and OH concentration values, respectively. The next Eq. (12) can be used for verification of the calculated values against the experimental ones obtained on the basis of Eq. (10) and Eq. (11). Good agreement between them will mean that the system is well described by the above equations. To verify this agreement the Eq. (9) should be presented in the following form:
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A1 1 S
KLOH
KLH aOH ALOH KLOH K aH LH aOH KLOH
(15) we obtain:
aH ALH A2
KLOH
(12)
KLH value is required for further calculations. KLOH The equation needed to calculate this expression can be obtained from Eqs. (4) and (5). Its value can be calculated using the values of aH/and aOH/ at the isoelectric point. On the basis of the assumed model the interfacial tension can be calculated, provided that the interfacial tension value of phosphatidylcholine layer is the sum of the contributions from all forms i.e. ideal mixing of the different forms of phosphatidylcholine. As was mentioned above, the values of the molecular area of phosphatidylcholine influence the interfacial tension values of the relative phosphatidylcholine forms. The surface concentrations of phosphatidylcholine forms are the same as described by Eqs. (4) / (6). The Eqs. (13) and (14) describe further dependencies in the studied system.
/
Ai
g0i
(14)
ggLH gLHOH gL gLOH
where g0i ; the interfacial tension of the adequate form of phosphatidylcholine (mN/m); g; the measured interfacial tension obtained from the p /A isotherms As the interfacial tension can be treated as the interfacial energy concentrated at the interfaces, we assume */basing on the additivity rule */that the interfacial tension of the lecithin layer is a sum of the interfacial tensions values of the e-PC forms. Then, the relationship between the surface concentration, the total surface concentration S and the interfacial tension values is obtained:
aL S
g1 aH g0LH KLH aOH g0LOH KLOH g2 aH KLH aOH KLOH
aLHOH S
g0LH
aLH S
g0LOH
aLOH S
g0L (15)
After the substitution of Eqs. (4) /(6) into Eq.
(16)
where g1 g0L g0LHOH KLHOH ; g2 KLHOH 1/ In analogy to the above equations describing the areas per molecules, the polynomial Eq. (16) and adequate approximations lead to the following forms depending on the conditions: For big H concentrations, i.e. when aH 0 gaH g0LH aH
KLHOH (g0LHOH g0LH ) (g0L g0LH ) KLH (17)
This approximation enables the calculation of the interfacial tension value of phosphatidylcholine form with adsorbed H ions. Analogously, for basic solutions, when aOH 0 gaOH g0LOH aOH
KLHOH (g0LHOH g0LOH ) (g0L g0LOH ) KLOH
(13)
gS
gg0LHOH
g
(18) The accuracy of the assumed model */the additivity of the phosphatidylcholine forms */can be verified with the help of Eq. (19). g1 g
KLOH
aH g0LH g2
KLOH
KLH KLOH KLH
aH
aLOH g0LOH
KLOH
(19) aOH
4. Results and discussion The measurements of interfacial tension values of lipid monolayer are useful for determination of the surface area per molecule. Dependence of some physical properties on pH is of interest for applications of biological membranes in biology and medical sciences. The dependence of the surface area per phosphatidylcholine molecule vs.
I. Brzozowska, Z.A. Figaszewski / Colloids and Surfaces B: Biointerfaces 27 (2003) 303 /309
pH of the subphase could be obtained with the usage of the p/A isotherms. The plot exhibits interesting properties and will be describe in some details. Fig. 1 presents the measured values of the surface concentration of phosphatidylcholine as a function of pH subphase. The experimental values are denoted as points. The solid curve is calculated using the Eq. (12), as will be discussed later. When subphase is acidic (pH from 2.0 to 3.8), the surface concentration S is nearly constant and approximately equal to 3.58 /105 mol/m2. Values of S increase steeply reaching a maximum close to the isoelectric point of phosphatidylcholine. It is noteworthy that this maximum is not obtained at the isoelectric point (4.15) of egg lecithin, which was established for egg lecithin bilayer [7]. In the case of the monolayers, increasing ionisation caused by the increasing H concentration results in a maximum value for 3.78 /105 mol/m2 at pH 4.36. When pH of subphase increases further, the values of the phosphatidylcholine surface concentration decrease steeply within 4.15 /7 pH range. The pH regions between 7/12, as can be seen from Fig. 1, are characterised by only small variations. Fig. 2 presents the interfacial tension values measured for egg yolk phosphatidylcholine monolayer. As it can be seen, the values in the 2 /6 pH range are only slightly change with increasing pH of subphase. These results are in agreement with the conclusions obtained by Anderson and Pethica [2]. A further decrease in the H concentration results in an abrupt change of the plot. The interfacial tension values start to increase continuously up to pH 6.5 /8.5. For big OH concentra-
Fig. 1. The interfacial tension of the phosphatidylcholine monolayer at the air/hydrophobic chains of lecithin as a function of pH.
307
Fig. 2. The interfacial tension values of the phosphatidylcholine monolayer at the air/aqueous solution vs. pH of this solution.
tions, the interfacial tension values are almost the same. In the Langmuir approach [11], an air-hydrophobic layer interfacial tension value and polar layer */aqueous subphase interfacial tension value make up the monolayer surface tension. The interfacial tension values for the interface of the hydrophobic chains */hydrophobic chains in a phospholipid bilayer is assumed negligible. Moreover, the values of the interfacial tension at the interface of headgroups of phospholipid */subphase are the same in both cases: for a monolayer and for a bilayer. As a result, the difference between monolayer interfacial tension obtained experimentally and the interfacial tension of bilayer equals the interfacial tension of hydrophobic layer /air interface. We proceed in a similar way as Ja¨hning [12] who approximated hydrophobic layer /air by the interfacial tension of n -alkane/ air interface. Since the values of the interfacial tension of the hydrophobic chains /air interface are dominating, these values were applied to the system and used in further calculation [13]. In Fig. 3 points denote the calculated values of interfacial tension of lipid monolayer values for interface of the air /phosphatidylcholine hydro-
Fig. 3. The surface concentration of e-PC as a function of subphase pH.
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phobic chains. As it was written above, these value are calculated as a difference of the experimental values for monolayer and bilayer composed of the same egg lecithin [7]. The calculated values obtained from Eq. (19) are denoted in the same Figure by the solid line. It is worth emphasising that for the hydrophobic chains /air interface this run is dependent on pH. The latter interface is dominating as for as the values of the interfacial tension are concerned. When the pH value approaches the isoelectric point we obtain the minimum of the interfacial tension values. It is equal $/40 mN/m at pH 4.15. With the changes of subphase pH, the interfacial tension values increase until the pH reaches $/9. The interfacial tension value of phosphatidylcholine monolayer on the basic subphase is then approximately 48 mN/m. In similar investigations concerning polylysine a minimum in the interfacial tension values was observed at pH near the pK value for polylysine. The surface tension values change with temperature and pH in poly-lysine aqueous solution. It was concluded that at this pH value, the concentration of hydrophobic side chains at the surface was maximal [14]. The structure and organisation of the lipid layer play an important role in the ionisation processes. It is necessary to examine the layer at a molecular level. Larger molecular areas are obtained at acidic pH than at pH neutral or basic. It is confirmed by means other measurements that the structure of the monolayer is altered by the changes of the pH subphase [15]. Employing on the assumed model, one can calculate the area of LH form, using the Eq. ˚ 2/molec.). (10). Its value is 3.53 /105 m2/mol (59 A However, since the extrapolation from only a few experimental points can produce unreliable results, we have proceeded differently. In order to confirm the obtained result, we calculated the value of LH phosphatidylcholine form by fitting the experimental curve using the algorithm for least square estimation of parameters. Then, it is equal to 3.28 /105 mol/m2. It is worth noting that we can use the extrapolated LOH value of surface concentration for such calculations. From Fig. 1 one can see that in the pH range of 9.5 /12 we can
treat the experimental points as reliable. The respective surface concentration value of LOH ˚ 2/molec.) form is equal to 3.34 /105 m2/mol (54 A We proceed in the same way in order to calculate the interfacial tension values. Then, interfacial tension values of respective forms are: 43.8 and 48.6 mN/m. The calculated values of interfacial tension for pH less than 2 are calculated on the basis of Eq. (17). The results are presented in Fig. 3. Remarkably, the experimental values are in good agreement with the calculated ones presented in Figure as a solid line. The verification of the assumed model is presented in the form of the Eq. (12) for the molecular areas of phosphatidylcholine forms and as Eq. (19) for the interfacial tension values. As can be seen from the Fig. 3 the obtained values (solid line) are very close to the experimental results represented by points. The good agreement between points and the solid lines (representing the calculated values) means that the proposed model is well derived and the obtained values are correct. The results confirm the existence of four phosphatidylcholine forms, no matter whether a phosphatidylcholine exists as a monolayer or a bilayer. A head group of zwitterionic phosphatidylcholine contains two separated oppositely charged moieties. Then, there is a possibility of strong electrostatic attraction between hydrophobic parts and appropriate local charge [16]. The negative charge of the phosphate group is distributed among four oxygen atoms, while the positive charge of ammonium group is concentrated on a single nitrogen atom, which is favourable for electrostatic interaction with anions [16]. The P end of the phosphatidylcholine head group is anchored at the air /water interface, while the hydrocarbon chains are driven towards air, thus the hydrophobic effects drives the methyl and methylene groups around the N charge towards the hydrocarbon. In order to reduce hydrocarbon /water contact, the polar head groups are packed closely. This restricts the freedom of the lipid chains and results in them exerting a lateral pressure on the surroundings. The area of hydrophilic heads of lipids determines the whole value of the area per molecule.
I. Brzozowska, Z.A. Figaszewski / Colloids and Surfaces B: Biointerfaces 27 (2003) 303 /309
The surface charge is dependent on pH and thus loosing protons can easily modify it. As we can see, the influence of pH of subphase results in the molecular packing of the phosphatidylcholine headgroup, and */directly */on the area taken up by chains at the air /chains interface. One can see as comparing Figs. 2 and 3 that the interfacial tension values for air /water interface is no so strongly dependent on pH as it is in the case of air /phospholipid chains. The packing properties can depend significantly on the degree of ionisation of the charged polar headgroups [17]. No changes in surface pressure are observed during changes in the pH range from 2.5 to 11.5. The differences between obtained values presented in Fig. 1 are quite small; however, the agreement between experimental and theoretical values is good. Eibl and Blume studied negatively charged phospholipids, which show changes in the transitions temperature in the region of pH 3/5. It was interpreted as the effect of increasing ionisation on the phase transition temperature of phospholipids [18]. They found that at pH /4, the maximum of transition temperature for negatively charged phospholipids is observed [18]. The differences at the air/hydrophobic parts of molecules are quite small comparing to those observed at the head group level, as the results presented above indicate.
5. Conclusions The assumed model is based on the additivity of the interfacial tension values and molecular area values of phosphatidylcholine forms. The contribution of following phosphatidylcholine forms: LH , L, LHOH, LOH depend on pH of subphase. The value of the molecular area of hydrophilic heads of phosphatidylcholine determines the surface concentration of phosphatidylcholine and, as
309
the result, the interfacial tension values at the air/ hydrophobic chains interface. The difference between monolayer interfacial tension obtained experimentally and the interfacial tension of bilayer equals the interfacial tension of hydrophobic layer /air interface. The assumed model agreed well with the experimental values.
Acknowledgements This work was supported by a grant from Polish Commitee of Scientific Research No 4T09A 153 22.
References [1] M.C. Phillips, D. Chapman, Biochim. Biophys. Acta 163 (1968) 301. [2] P.J. Anderson, B.A. Pethis, in: G. Popjak, E. Le Breton (Eds.), Biochemical Problems of Lipids, Butterworths Scientific Publications, London, 1956. [3] J.H. Schulman, A.H. Hughes, Proc. Roy. Soc. (London) Ser. A138 (1931) 430. [4] D. Papahadjopoulos, Biochim. Biophys. Acta 163 (1968) 240. [5] D.O. Shah, J.H. Schulman, J. Lip. Res. 6 (1965) 341. [6] P.J. Quinn, R.M.C. Dawson, Chem. Phys. Lip. 8 (1972) 1. [7] A.D. Petelska, Z.A. Figaszewski, Biophys. J. 78 (2000) 812. [8] M.M. Standish, B.A. Pethica, Trans. Faraday Soc. 64 (1968) 1113. [9] I. Brzozowska, Z.A. Figaszewski, Colloids & Surfaces B: Biointerfaces 23 (2002) 51. [10] Poradnik Fizykochemiczny, WNT, Warszawa, 1974. [11] I. Langmuir, J. Chem. Phys. 1 (1933) 756. [12] F. Ja¨hning, Biophys. J. 46 (1984) 687. [13] J. Nagle, J. Membrane Biol. 27 (1976) 233. [14] A.W. Neumann, M.A. Moscarello, R.M. Epand, Biopolymers 12 (1973) 1945. [15] L. Dziri, S. Boussaad, N. Tao, R.M. Leblanc, Thin Solid Films 327 /329 (1998) 56. [16] V.L. Shapovalov, Thin Solid Films 327 /329 (1998) 599. [17] P.W.M. Van Dijck, B. de Kruijff, A.J. Verkleij, L.L.M. van Deenen, Biochim. Biophys. Acta 512 (1978) 84. [18] H. Eibl, A. Blume, Biochim. Biophys. Acta 553 (1979) 476.
The influence of pH on phosphatidylcholine monolayer at the air/aqueous solution interface Izabela Brzozowska, Zbigniew A. Figaszewski * Laboratory of Interfacial Electrochemistry, Faculty of Chemistry, University of Warsaw, PL / 02-093 Warsaw, Pasteura 1, Poland Institute of Chemistry, University of Bialystok, Al. Pilsudskiego 11/4, PL / 15-443 Bialystok, Poland Received 4 January 2002; received in revised form 10 April 2002; accepted 17 June 2002
Abstract The measurements of the interfacial tension at the air/aqueous subphase interface as the function of pH were performed. The interfacial tension of the air /aqueous subphase interface was divided into contributions of individuals. A simple model of the influence of pH on the phosphatidylcholine monolayer at the air/hydrophobic chains of phosphatidylcholine is presented. The contributions of additive phosphatidylcholine forms (both interfacial tension values and molecular area values) depend on pH. The interfacial tension values and the molecular areas values for LH , LOH forms of phosphatidylcholine were calculated. The assumed model was verified experimentally. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Monolayer; Egg yolk phosphatidylcholine; Interfacial tension; pH
1. Introduction Phospholipids are major fractions of lipids found in biological membranes. Since monolayers*/especially at the air /water interface */ are commonly used as simplified models of biomembrane, many studies have been concentrated on them [1]. The structure of lipid monolayer seems to be apparently simple at the first estimation. However, at closer study, one can distinguish there two different interfaces: the airhydrophobic chains of phospholipid and theirs hydrophilic heads*/aqueous subphase. The prop-
* Corresponding author E-mail address: [email protected] (Z.A. Figaszewski).
erties of such membranes depend strongly on the molecules that built it. Therefore, the knowledge of molecular structure and organisation of phospholipids is necessary. Hence, the detailed researches connected with the influence of pH on lecithin monolayer are required. Phosphatidylcholine (L) is a neutral, zwitterionic phospholipid with an amphiphilic character. The surface pressure /area per molecule (p /A ) curves of dicetyl phosphate, of dipalmitoyl, egg dileoyl lecithin have been reported previously [2]. According to Anderson and Pethica the surface pressure /area isotherms of distearoyl lecithin monolayers are the same in the pH range 1/7 [2]. The pH dependence of amphiphilic substances at the air /water interface was investigated even earlier, at the beginning of the last century [3]. Not
0927-7765/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 2 ) 0 0 0 9 5 - 4
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only the interfacial tension values were recorded but also the ionic properties of a monolayer were studied by means of investigation of its surface potential at a fixed value of area/molecule. According to Schulman et al. the surface potential is not influenced by the pH of the subphase since the DV /pH plot is a straight line with zero slope. This fact can be explained by assuming a mutual interaction between the phosphate and trimethylammonium groups, which neutralised each other. Also the effect of pH on the electrophoretic mobility of lecithin is characteristically similar to the ones obtained by the monolayer technique [4]. In a smaller pH range, 4 /8, dipalmitoyl and egg lecithins also show the properties of uncharged monolayers. There is no net charge on the monolayers between pH 4 /8. This suggests that neutralisation of the phosphate group is related to intermolecular spacing in monolayers. Shah and Schulman propose that the internal neutralisation of ionic charges to be caused by neutralisation of oppositely charged groups of adjacent molecules [5]. However, the related researches concerning the adsorption of Ca2 as a function of pH subphase suggest something quite different. Since below pH 3 only small amount of 45Ca is adsorbed, the films of phospholipid are assumed to be unionised. Ca2 is permanently adsorbed at pH values above 6.5 with an increasing affinity up to pH 11 [6]. While a phosphatidylcholine monolayer is not altered by the pH of subphase in some pH regions, a recent study connected with bilayer has shown that there is a maximum of interfacial tension at a certain pH [7]. A careful study is thus pertinent. Since the changes in interfacial tension values induce the changes of the values in the area per molecules, it is very important, in context of biological membranes, to know the exact molecular packing in various pH solutions [8]. We develop a theory, based on the additivity rule, for the influence of pH on a egg yolk phosphatidylcholine monolayer. Using the derived equations we present a model of ion-monolayer interaction based on the calculations employing p /A curves.
2. Experimental The measuring procedure was described elsewhere [9]. Surface tension measurements were carried out in Langmuir trough equipped with 9000 Nima tensiometer at the water/air interface at 22 8C. Lipids were brought into contact with the interface with a Hamilton syringe. After evaporation of the solvent (10 min) the compression of the monolayer was performed. The rate of compression was 0.1 cm/s. The measurements were carried out using Britton-Robinson buffers in the range of 2/12 [10]. This buffer is often used in the biological studies because of its wide pH range. The pH was altered by the addition of 0.2 M NaOH to the mixture of 0.04 M acetic acid, 0.04 M phosphoric acid and 0.04 M boric acid. The required pH was controlled using Radiometer pH meter. The water used was triply distilled. The egg lecithin (3-sn-phosphatidylcholine of hen eggs yolk) /99% (TLC) from Fluka was used without further purification. The composition of fatty acids in the egg lecithin was 16:0 /33%, 18:0 / 4%, 18:1 /30%, 18:2 /14%, 20:4 /4%. 1-chloropropane was used as a spreading solvent.
3. Theory Since the phosphatidylcholine molecule (L) possess a zwitterionic character, it can participate in equilibrium reactions with both hydrogen ions and hydroxyl anions. LH ULH LOH ULOH LHOH ULHOH
(1) (2) (3)
Consequently, equations of associations Eqs. (1)/(3) can be consider as the description of an adsorption process. As a result of adsorption of H and OH ions on the surface of phosphatidylcholine layer, the phosphatidylcholine molecule can exist in four different forms. We shall consider following forms: LH with H adsorbed, LOH with OH adsorbed and LHOH with both H and OH ions adsorbed on the surface and a free
I. Brzozowska, Z.A. Figaszewski / Colloids and Surfaces B: Biointerfaces 27 (2003) 303 /309
lecithin molecule L i.e. with no ions adsorbed. A phosphatidylcholine monolayer is assumed to consist of these four forms. The relative contributions of above forms are dependent on pH, according to equations Eqs. (1) /(3). One can write three equations for equilibrium Eqs. (1) /(3), containing the equilibrium constants of these equilibria. On the basis of these equations, the activity of following phosphatidylcholine forms can be calculated: aLH KLH aL aH aLOH KLOH aL aOH aLHOH KLHOH aL
(4) (5) (6)
where ai is the surface concentration of ‘i ’ form of phosphatidylcholine (mol/m2); i LH ;/ /LOH ; LHOH; L; aH ; aOH ; the concentrations of ions in the subphase (mol/m3); Ki is the equilibrium constant of adsorption process of H or OH ions on lecithin (m3/mol). The sum of surface concentrations of any phosphatidylcholine forms at the air /water interface has to be equal to total surface concentrations of phosphatidylcholine (S ). This S concentration can be easily measured using the p /A isotherms. Moreover, the sum of the area fractions of these four phosphatidylcholine forms should give unity. These relationships are described by following equations: aL aLH aLHOH aLOH S (7) aL AL aLH ALH aLHOH ALHOH aLOH ALOH 1
(8)
where S , total surface concentration of phosphatidylcholine measured by p/A isotherms (mol/m2); ˚ 2/molec). Ai , area per molecule (A The equations Eqs. (4) /(8) describe quantitatively the model of the influence of pH subphase on a phosphatidylcholine monolayer. The different forms of phosphatidylcholine would give monolayers, built form one component, that have different stability constant. The value of surface concentrations of any phosphatidylcholine forms affects the molecular packing of the head groups, which*/in a consequence */influences the interfacial tension of lipid monolayer. Depending on the pH of subphase the surface concentrations will
305
change as the area per molecule changes. Different forms of phosphatidylcholine will have different areas per molecules depending on the contribution of the forms to the total amount of phosphatidylcholine molecules. After elimination of aLH ; aLHOH ; aL and aLOH terms from the Eqs. (4) /(8), one obtains: 1 S
A1 aH ALH KLH aOH ALOH K LOH A2 aH KLH aOH K LOH
(9)
where A1 AL ALHOH KLHOH ; A2 KLHOH 1/ The direct form of the Eq. (9) is not convenient for calculations. After substituting the concentration of OH ions by the quotient of KH2 O and H concentration one can divide the numerator of above polynomial by its denominator. As a result we obtain the series of terms containing the decreasing powers of H ions concentration. The equation obtained by multiplication by aH is in the form where one can treat the negative terms as negligible. In consequence, such equation would have the linear character. For big H concentrations, i.e. when aH 0 ; the Eq. (9) will assume the following form: aH S
ALH aH
A1 A2 ALH KLH
(10)
Eq. (9) can be treated in the analogous way after substitution of H ion concentrations by concentrations of hydroxyl ions. For big OH concentrations i.e. when aOH 0 ; one can obtain: aOHS
ALOH aOH
A1 A2 ALOH KLOH
(11)
Using these latter relationships, one can easily calculate the values of ALH/and ALOH by regression in the region of big H and OH concentration values, respectively. The next Eq. (12) can be used for verification of the calculated values against the experimental ones obtained on the basis of Eq. (10) and Eq. (11). Good agreement between them will mean that the system is well described by the above equations. To verify this agreement the Eq. (9) should be presented in the following form:
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A1 1 S
KLOH
KLH aOH ALOH KLOH K aH LH aOH KLOH
(15) we obtain:
aH ALH A2
KLOH
(12)
KLH value is required for further calculations. KLOH The equation needed to calculate this expression can be obtained from Eqs. (4) and (5). Its value can be calculated using the values of aH/and aOH/ at the isoelectric point. On the basis of the assumed model the interfacial tension can be calculated, provided that the interfacial tension value of phosphatidylcholine layer is the sum of the contributions from all forms i.e. ideal mixing of the different forms of phosphatidylcholine. As was mentioned above, the values of the molecular area of phosphatidylcholine influence the interfacial tension values of the relative phosphatidylcholine forms. The surface concentrations of phosphatidylcholine forms are the same as described by Eqs. (4) / (6). The Eqs. (13) and (14) describe further dependencies in the studied system.
/
Ai
g0i
(14)
ggLH gLHOH gL gLOH
where g0i ; the interfacial tension of the adequate form of phosphatidylcholine (mN/m); g; the measured interfacial tension obtained from the p /A isotherms As the interfacial tension can be treated as the interfacial energy concentrated at the interfaces, we assume */basing on the additivity rule */that the interfacial tension of the lecithin layer is a sum of the interfacial tensions values of the e-PC forms. Then, the relationship between the surface concentration, the total surface concentration S and the interfacial tension values is obtained:
aL S
g1 aH g0LH KLH aOH g0LOH KLOH g2 aH KLH aOH KLOH
aLHOH S
g0LH
aLH S
g0LOH
aLOH S
g0L (15)
After the substitution of Eqs. (4) /(6) into Eq.
(16)
where g1 g0L g0LHOH KLHOH ; g2 KLHOH 1/ In analogy to the above equations describing the areas per molecules, the polynomial Eq. (16) and adequate approximations lead to the following forms depending on the conditions: For big H concentrations, i.e. when aH 0 gaH g0LH aH
KLHOH (g0LHOH g0LH ) (g0L g0LH ) KLH (17)
This approximation enables the calculation of the interfacial tension value of phosphatidylcholine form with adsorbed H ions. Analogously, for basic solutions, when aOH 0 gaOH g0LOH aOH
KLHOH (g0LHOH g0LOH ) (g0L g0LOH ) KLOH
(13)
gS
gg0LHOH
g
(18) The accuracy of the assumed model */the additivity of the phosphatidylcholine forms */can be verified with the help of Eq. (19). g1 g
KLOH
aH g0LH g2
KLOH
KLH KLOH KLH
aH
aLOH g0LOH
KLOH
(19) aOH
4. Results and discussion The measurements of interfacial tension values of lipid monolayer are useful for determination of the surface area per molecule. Dependence of some physical properties on pH is of interest for applications of biological membranes in biology and medical sciences. The dependence of the surface area per phosphatidylcholine molecule vs.
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pH of the subphase could be obtained with the usage of the p/A isotherms. The plot exhibits interesting properties and will be describe in some details. Fig. 1 presents the measured values of the surface concentration of phosphatidylcholine as a function of pH subphase. The experimental values are denoted as points. The solid curve is calculated using the Eq. (12), as will be discussed later. When subphase is acidic (pH from 2.0 to 3.8), the surface concentration S is nearly constant and approximately equal to 3.58 /105 mol/m2. Values of S increase steeply reaching a maximum close to the isoelectric point of phosphatidylcholine. It is noteworthy that this maximum is not obtained at the isoelectric point (4.15) of egg lecithin, which was established for egg lecithin bilayer [7]. In the case of the monolayers, increasing ionisation caused by the increasing H concentration results in a maximum value for 3.78 /105 mol/m2 at pH 4.36. When pH of subphase increases further, the values of the phosphatidylcholine surface concentration decrease steeply within 4.15 /7 pH range. The pH regions between 7/12, as can be seen from Fig. 1, are characterised by only small variations. Fig. 2 presents the interfacial tension values measured for egg yolk phosphatidylcholine monolayer. As it can be seen, the values in the 2 /6 pH range are only slightly change with increasing pH of subphase. These results are in agreement with the conclusions obtained by Anderson and Pethica [2]. A further decrease in the H concentration results in an abrupt change of the plot. The interfacial tension values start to increase continuously up to pH 6.5 /8.5. For big OH concentra-
Fig. 1. The interfacial tension of the phosphatidylcholine monolayer at the air/hydrophobic chains of lecithin as a function of pH.
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Fig. 2. The interfacial tension values of the phosphatidylcholine monolayer at the air/aqueous solution vs. pH of this solution.
tions, the interfacial tension values are almost the same. In the Langmuir approach [11], an air-hydrophobic layer interfacial tension value and polar layer */aqueous subphase interfacial tension value make up the monolayer surface tension. The interfacial tension values for the interface of the hydrophobic chains */hydrophobic chains in a phospholipid bilayer is assumed negligible. Moreover, the values of the interfacial tension at the interface of headgroups of phospholipid */subphase are the same in both cases: for a monolayer and for a bilayer. As a result, the difference between monolayer interfacial tension obtained experimentally and the interfacial tension of bilayer equals the interfacial tension of hydrophobic layer /air interface. We proceed in a similar way as Ja¨hning [12] who approximated hydrophobic layer /air by the interfacial tension of n -alkane/ air interface. Since the values of the interfacial tension of the hydrophobic chains /air interface are dominating, these values were applied to the system and used in further calculation [13]. In Fig. 3 points denote the calculated values of interfacial tension of lipid monolayer values for interface of the air /phosphatidylcholine hydro-
Fig. 3. The surface concentration of e-PC as a function of subphase pH.
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phobic chains. As it was written above, these value are calculated as a difference of the experimental values for monolayer and bilayer composed of the same egg lecithin [7]. The calculated values obtained from Eq. (19) are denoted in the same Figure by the solid line. It is worth emphasising that for the hydrophobic chains /air interface this run is dependent on pH. The latter interface is dominating as for as the values of the interfacial tension are concerned. When the pH value approaches the isoelectric point we obtain the minimum of the interfacial tension values. It is equal $/40 mN/m at pH 4.15. With the changes of subphase pH, the interfacial tension values increase until the pH reaches $/9. The interfacial tension value of phosphatidylcholine monolayer on the basic subphase is then approximately 48 mN/m. In similar investigations concerning polylysine a minimum in the interfacial tension values was observed at pH near the pK value for polylysine. The surface tension values change with temperature and pH in poly-lysine aqueous solution. It was concluded that at this pH value, the concentration of hydrophobic side chains at the surface was maximal [14]. The structure and organisation of the lipid layer play an important role in the ionisation processes. It is necessary to examine the layer at a molecular level. Larger molecular areas are obtained at acidic pH than at pH neutral or basic. It is confirmed by means other measurements that the structure of the monolayer is altered by the changes of the pH subphase [15]. Employing on the assumed model, one can calculate the area of LH form, using the Eq. ˚ 2/molec.). (10). Its value is 3.53 /105 m2/mol (59 A However, since the extrapolation from only a few experimental points can produce unreliable results, we have proceeded differently. In order to confirm the obtained result, we calculated the value of LH phosphatidylcholine form by fitting the experimental curve using the algorithm for least square estimation of parameters. Then, it is equal to 3.28 /105 mol/m2. It is worth noting that we can use the extrapolated LOH value of surface concentration for such calculations. From Fig. 1 one can see that in the pH range of 9.5 /12 we can
treat the experimental points as reliable. The respective surface concentration value of LOH ˚ 2/molec.) form is equal to 3.34 /105 m2/mol (54 A We proceed in the same way in order to calculate the interfacial tension values. Then, interfacial tension values of respective forms are: 43.8 and 48.6 mN/m. The calculated values of interfacial tension for pH less than 2 are calculated on the basis of Eq. (17). The results are presented in Fig. 3. Remarkably, the experimental values are in good agreement with the calculated ones presented in Figure as a solid line. The verification of the assumed model is presented in the form of the Eq. (12) for the molecular areas of phosphatidylcholine forms and as Eq. (19) for the interfacial tension values. As can be seen from the Fig. 3 the obtained values (solid line) are very close to the experimental results represented by points. The good agreement between points and the solid lines (representing the calculated values) means that the proposed model is well derived and the obtained values are correct. The results confirm the existence of four phosphatidylcholine forms, no matter whether a phosphatidylcholine exists as a monolayer or a bilayer. A head group of zwitterionic phosphatidylcholine contains two separated oppositely charged moieties. Then, there is a possibility of strong electrostatic attraction between hydrophobic parts and appropriate local charge [16]. The negative charge of the phosphate group is distributed among four oxygen atoms, while the positive charge of ammonium group is concentrated on a single nitrogen atom, which is favourable for electrostatic interaction with anions [16]. The P end of the phosphatidylcholine head group is anchored at the air /water interface, while the hydrocarbon chains are driven towards air, thus the hydrophobic effects drives the methyl and methylene groups around the N charge towards the hydrocarbon. In order to reduce hydrocarbon /water contact, the polar head groups are packed closely. This restricts the freedom of the lipid chains and results in them exerting a lateral pressure on the surroundings. The area of hydrophilic heads of lipids determines the whole value of the area per molecule.
I. Brzozowska, Z.A. Figaszewski / Colloids and Surfaces B: Biointerfaces 27 (2003) 303 /309
The surface charge is dependent on pH and thus loosing protons can easily modify it. As we can see, the influence of pH of subphase results in the molecular packing of the phosphatidylcholine headgroup, and */directly */on the area taken up by chains at the air /chains interface. One can see as comparing Figs. 2 and 3 that the interfacial tension values for air /water interface is no so strongly dependent on pH as it is in the case of air /phospholipid chains. The packing properties can depend significantly on the degree of ionisation of the charged polar headgroups [17]. No changes in surface pressure are observed during changes in the pH range from 2.5 to 11.5. The differences between obtained values presented in Fig. 1 are quite small; however, the agreement between experimental and theoretical values is good. Eibl and Blume studied negatively charged phospholipids, which show changes in the transitions temperature in the region of pH 3/5. It was interpreted as the effect of increasing ionisation on the phase transition temperature of phospholipids [18]. They found that at pH /4, the maximum of transition temperature for negatively charged phospholipids is observed [18]. The differences at the air/hydrophobic parts of molecules are quite small comparing to those observed at the head group level, as the results presented above indicate.
5. Conclusions The assumed model is based on the additivity of the interfacial tension values and molecular area values of phosphatidylcholine forms. The contribution of following phosphatidylcholine forms: LH , L, LHOH, LOH depend on pH of subphase. The value of the molecular area of hydrophilic heads of phosphatidylcholine determines the surface concentration of phosphatidylcholine and, as
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the result, the interfacial tension values at the air/ hydrophobic chains interface. The difference between monolayer interfacial tension obtained experimentally and the interfacial tension of bilayer equals the interfacial tension of hydrophobic layer /air interface. The assumed model agreed well with the experimental values.
Acknowledgements This work was supported by a grant from Polish Commitee of Scientific Research No 4T09A 153 22.
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