Langmuir 1991, 7, 964-974
Interaction of Procaine with Stearic Acid Monolayers at the Air/Water Interface Maria Tomoaia-CotigelfJ and D. Allan Cadenhead'ts Department of Physical Chemistry, University of Marburg, 0-3550 Marburg, FRG, and Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14214 Received May 21, 1990. In Final Form: October 19, 1990 The adsorption characteristics of procaine have typically been studied by measuring the surface tension of aqueous procaine solutions and by recording compressional isotherms, i.e., surface pressure as a function of molecular area, for stearic acid monolayers in the presence of various procaine subphase concentrations at both pH 2 and pH 8. The presence of the stearic acid monolayers promotes the enhanced adsorption of procaine in liquid or fluid states of the stearic acid monolayers. On compression of the stearic acid monolayer the procaine adsorption increases and, after attaining a maximum value, decreasesand vanishes near the collapse of the stearic acid monolayer. The surface characteristics derived from the compressional isotherms, as well as the area increase recorded at constant surface pressure, are interpreted by taking into account models both for the mechanism of procaine penetration into stearic acid monolayers and for the protolytic equilibria occurring in the system. The penetration numbers, i.e., the ratio of procaine molecules to stearic acid molecules in the mixed penetrated films, are derived from the molecular area incrementa and are in good agreement with the values obtained by using the Gibbs' adsorption equation. Molecular species of procaine are initially thought to be adsorbed at the air/water interface in a horizontal orientation. On compression of the spread stearic acid film, the procaine is gradually forced to adopt a vertical position. A t higher surface pressure values procaine is squeezed out from the monolayer and is accumulated in an adjacent layer, thus causing a significant increase in the collapse pressure of the stearic acid monolayers. At pH 2 this latter effect has been interpreted in terms of ion-dipole interactions between the positively charged procaine molecules and the uncharged carboxyl groups of the stearic acid monolayers and in terms of hydrogen bonding between the carboxyl group and primary amino group of the procaine monocation. At pH 8 electrostatic interactions between the stearate anions and the procaine monocation are also taken into account. Procaine penetration occurred preferentially in the liquid or fluid phase of stearic acid monolayers and was found to be dependent on the surface pressure. Maximum penetration was established at around 10 mN/m for monolayers on pH 2 and at 5 mN/m for stearic acid monolayers at pH 8. The maximum values are much higher at pH 8 than at pH 2 due to the protolytic equilibria in which both the stearic acid and the procaine participate. The physical insertion of procaine between the stearic acid molecules is presumably primarily responsible for the expansion effect found. At the sametime, electrostaticinteractionsor ion-dipoleinteractionswillmodify such interchain interactions, although the latter are increasingly important in the solid state of the monolayer as collapse is approached.
Introduction Local anesthetics are known to exert their action by closing the sodium channels' of nerve membranes, thus blocking nerve signal propagation. The molecular mechanism of anesthetic action has been the subject of extensive studies, but it is still unclear whether this blocking is the result of a direct anesthetic-protein interaction2 or a perturbation by anesthetic of the lipid matrix changing some physicochemical property of the bilayer surrounding the channel^.^ Studies performed using different experimental techniques showed that some anesthetics increase the fluidity of lipid bilayers4and/ or decrease the order in hydrocarbon chains- and either increase the surface area of mono+ University of Marburg. t Permanent address: Department of Physical Chemistry, University of Cluj-Napoca, 3400 Cluj-Napoca, Romania. 1 State University of New York at Buffalo. (1)Trudell, J. R.In Molecular Mechanisms of Anesthesia: Progress in Anesthesiology; Fink, B. R., Ed.; Raven Press: New York, 1980:Vol.
2, pp 261-270. (2) Bogga, J. M.; Roth, S.M.; Yoong, T.; Wong, E.; Hsia, J. C. Mol. Pharmacol. 1976,12,136-143. (3)Seeman, P. h o g . Anesthesiol. 1975,I, 243-251. (4) Trudell, J. R.; Hubbell, W. L.: Cohen, E. N. Fed. Proc. 1972.31. 549. (5) Turner, G. L.; Oldfield, E.Nature (London) 1979,277,669-670. (6) Boulnnger, Y.; Schreier, S.;Smith, I. C. P. Biochemistry 1981,20, 68244830. (7) Kelusky, E.C.; Smith, I. C. P. Biochemistry 1983,22,6011-6017.
,~ - ,
layers maintained at constant surface pressureloor increase the surface pressure of lipid films maintained at constant area.11 Although these experiments involve different physical parameters, the molecular origin of the effects observed is presumably the same, viz. the modification of the structural and dynamic properties of ordered acyl chains due to the binding and penetration of anesthetics into oriented lipid systems. However, there is also the very real possibility that various anesthetics act by different mechanisms. The quantification of the interaction with, or the binding to and penetration of anesthetics into, a membrane model system is clearly a fundamental problem in biophysics. Very few studies of the effects of tertiary amine anesthetics such as procaine on membrane models have been carried out, and their interpretation at a molecular level is not always completely obvious. Thus, it appears that procaine is bound by egg phosphatidylethanolaine~ but is only weakly bound by the more ordered acyl chains in identical acyl chain egg phosphatidylcholine8 aqueous lamellar dispersions. While in this case, the differing results must be attributed to the differing polar head(8)Kelusky, E. C.; Smith, I. C. P. Can. J. Biochem. Cell Biol. 1984, 62,178-184. (9)Auger,M., Jarrell,H. C.,Smith, I. C.P.,Siminovitch,D. J., Mnntach, H.H.; Wong, P. T. T. Biochemistry 1988,27,6086-6093. (IO) Seelig, A. Biochim. Biophys. Acta 1987,699,196-204. (11)Vilallonga,F.A.;Phillips, E. W. J.Pharm. Sci. 1979,6C,314-316.
0 1991 American
Chemical Society
Interaction of Procaine with Stearic Acid groups, it is not immediately clear precisely how the headgroup controls penetration. A simple approach to a detailed study of effects of procaine on highly ordered acyl chains would appear to be to use stearic acid monolayers spread a t the air/aqueous interface. While stearic acid is not a typical membrane lipid, it does permit hydrophobic, polar, and electrostatic interactions to be examined. A key parameter to be assessed here is the molar ratio of penetrated procaine to stearic acid present. These data are essential for any full understanding of the anesthetic-membrane interaction. A t pH 2 our study is restricted to the liquid condensed (LC) and solid (S)states only, while at pH 8 much more fluid states are examined. Generally, monolayer techniques provide controlled conditions for the investigation of ordered biosurfactant molecules.12 Thus monolayers can offer a suitable structural arrangement, ranging from partially to highly ordered acyl chains, for the experimental investigation of physicochemical parameters including the quantitative examination of interactions between film-forming molecules and subphase components such as anesthetics. Monolayer experiments have often been performed at a constant monolayer area, by the injection of anesthetic solutions into the subphase of an already spread film and by observing either the surface pressure increase a t a constant surface area or the surface area increase at a constant surface pressure. Such an approach, particularly the latter, provides the best resemblance to membrane penetration. For closely packed film molecules, however, great care must be taken to ensure that equilibrium or near equilibrium is reached. The present experiments were carried in a different way. The monolayers were directly spread on an anesthetic containing subphase of a selected concentration. After the attainment of spreading equilibrium, the compressional isotherms were recorded under near equilibrium conditions. While any one of the techniques described could yield equilibrium surface pressure values, the advantage of this approach is that equilibrium or near equilibrium between the stearic acid monolayer and the subphase Components can be followed over a very large surface pressure range, from very low surface pressures (spreading equilibrium surface pressures) to the collapse pressure of the monolayer. The aim of this paper is to measure the effects of procaine on stearic acid monolayers spread under various conditions and to derive the extent of procaine penetration or retention and its variation under different conditions. Because both stearic acid and procaine participate in known protolytic equilibria, our investigations have been performed under controlled pH conditions, viz. at pH 2 and pH 8. Since procaine has the effect of expanding the stearic acid monolayer, attempts have been made to derive the amount of procaine present in the monolayer by using the Gibbs' adsorption equation and by means of molecular models for the different proposed mechanisms of penetration. Experimental Section Materials. Stearic acid (SAH) was purchased from Schuchardt. Procaine hydrochloride (PR-HCl) was obtained from Hoechst. Benzene used as spreadingsolvent was purchased from Merck. Water used for buffers was doubly distilled. The buffers used were HCl (pH 2) and KHzPOd and NazHP01.2Hz0 (pH 8). The concentration range of procaine solutions measured was (12) Kozarac, Z.;Dhathathreyan, A.; MBbius, D. Colloid Pdlym. Sci. 1989,267,122-129.
Langmuir, Vol. 7, No. 5, 1991 965 0.001-10 mM. All substances were analytical grade and were used without further purification. Methods. Surfacetensions of procaine solutions not covered by stearic acid monolayers were measured at 20 O C by using du Noiiy's ring method. Stearicacid monolayers were spread at the air/aqueous solution interface at both pH 2 and pH 8, using benzene as a spreading solvent. Known amounts of stearic acid were dissolved in benzene and were delivered to the aqueous subphase by a Hamilton syringe. The stearic acid monolayer was left (15-30 min) to attain equilibrium and to allow for evaporation of the benzene. The resultant monolayer was then compressed. Equilibrium between the monolayer and the subphase was established rapidly, allowing us to record typical compression isotherms (surface pressure versus molecular area) in about 30 min. Compressional speeds between 0.005 and 0.025 nm2/ (molecule min) ensured good isotherm reprodu~ibility.~~J~ In penetration and adsorption studies, the stearic acid monolayer was spread onto a procaine aqueous solution of given concentration at a chosen pH. Equilibrium between the stearic acid monolayer and procaine subphase was rapidly established, as was evidenced by the constancy of the surface pressure values. With this procedure a mixed monolayer is initially formed at the air/water interface, containing water (designated component 1) procaine (designatedcomponent 2), and stearic acid (designated component3). Duringcompression,from time to time, the mixed monolayers were kept at the air/aqueous solution interface for periods ranging from 60 min to 2 h, and the results were always the same within experimental error. This means that our measurements can be regarded as being obtained under nearequilibriumor steady-stateconditions. Compression of the monolayers was performed discontinuously while using the Wilhelmy plate method of recording the surface pressure. The latter was measured with an accuracy of 0.1 mN/m. In all cases at least 10 compressional isotherms were recorded under identical experimental conditions. All measurements were performed at room temperature (-22 "C). We will designate UO, UPR, and u respectively, as the surface tensions of pure buffer, of aqueous procaine solution, and of subphases covered by a stearic acid monolayer. The surface pressure (r)is defined in the absence of stearic acid monolayer as r p =~ a0 - U ~ R and , in its presence simply as ?r = a0 - u. The surface pressure measured directly by means of the Wilhelmy method is then ?r = U ~ -R u for mixed stearicacid-procaine monolayers.
Results and Discussion 1. Molecular Species Involved. Since both stearic acid and procaine can participate in protolytic equilibria, diagrams have been constructed, giving the fraction a of the different molecular species as function of pH (Figure 1). In the case of stearic or octadecanoic acid, CH3(CH2)16COOH, calculations have been performed by using the surface acidity constant reported earlier pKa = 5.63.13 As seen from Figure la, a t pH 2 stearic acid is completely un-ionized (SAH) and forms an uncharged film. At pH 8 stearic acid molecules are completely ionized and give a charged film, containing only stearate anions (SA-). Procaine or novocaine (4-aminobenzoic acid, [2-(diethylamino)ethyl]ester; 4-HzNC~H~COz[CHzCHzN(CzHs)z1; PR) is a tertiary amine, containing a primary amino group linked to an aromatic ring. Consequently, it may exist as a neutral molecule (PR), a monocation (4-H2NCeH,COZ[CHZCHZNH+(C~H~)Z] (PRH+)),or a dication (4-HzNH+C~H~COZ[CHZCHZNH+(CZHS)Z] (PRHP)). We presumed that the pK, of the monocation is practically the same as that of dibucaine, for which a pK, = 8.5 has already been reported.'" For the primary amine group the basicity (13)Tomoaia-Cotieel, M.;Zsako, J.; Mocanu, A.; Lupea, M.; Chifu, E. J. Colloid Interface Sci. 1987, 117, 464-476. (14) Tomoaia-Cotieel, M.; Chifu, E.; Mocanu, A.; Zsako, J.; Salajan, M.; Frangopol, P. T. Rev. Roum.Biochim. 1988,25, 221.
966 Langmuir, Vol. 7, No. 5, 1991
Tomoaia- Cotigel and Cadenhead Table I. Maximum Procaine Adsorption (r-') and Its Limiting Molecular Area (A,,oo) in the Absence of Stearic Acid b l U
A2,0°,
PH Figure 1. Fraction of molecular species as a function of p H (a) SA- (- - -); (b) procaine: PR -), stearic acid, SAH (-), PRH+ (-), PRH*2+(- --). (-a
x 10-13, molecules/cmZ
nm2/molecule
I+
PH 2
PH 8
4.831
11.017 0.91
2.01
/
I
+
75
Figure 3. Extreme conformations of procaine: HE, horizontal extended;VE, vertical extended;VF, vertical forced. The dashed line accompanying the latter two conformations indicates the air/water interface.
70
E
t
E 65 a a.
t) 60
55
-6
-5
-4
-3 log c,
-2
-1
-0
Figure 2. Surface tension of procaine solutions at pH 2 (curve 1) and at pH 8 (curve 2) as a function of the logarithm of the procaine molar concentration (cp) in the absence of a stearic acid monolayer. constant Kb = 1.7 X of p-aminobenzoic acidl5 was taken. Our calculations show (see Figure lb) that at pH 2,60 % of the procaine is PRHz2+and the remaining 40 % is PRH+. At pH 8, PRH+ is the predominant species, but there are also neutral molecules present (about 24%). 2. Adsorption of Procaine in Absence of Stearic Acid. The surface tension of procaine solutions, U ~ in R mN/m, as a function of the logarithm of the procaine molar concentration (cp)is presented in Figure 2 for pH 2 (curve 1) and pH 8 (curve 2). From Figure 2, the procaine adsorption ( I ' p O ) can easily be obtained for both pH values. For every UPR value according to the Gibbs' equation
where c2 is the bulk procaine concentration and k and T are the Boltzmann constant and the absolute temperature, respectively. In Figure 2, at higher c2 values, the UPR versus log c2 plots become linear, indicating saturation of the interface. With increasing pH, the concentration c2 diminishes until I'2O attains its limiting value (I'p,mqo), corresponding to the quasi-saturation in the adsorption monolayer at the (15) Sprauotchnik Khimika, T.III; Goskhimizdat: Moscow, 1952; p 606.
air/water interface. From the slope of these linear portions, values of the maximum procaine adsorption ( 1 - 2 , ~ ~ were ) calculated and consequently the limiting molecular area values (A2,0°) in the adsorbed procaine monolayer a t the air/water interface were obtained (Table I). As is seen in Table I, with increasing pH, the limiting (I'z,-O) adsorption value increases, indicating that the surface activity of the molecular species involved increases in the order PRHz2+< PRH+ < PR. The mean molecular area of procaine calculated from the value of the maximum procaine adsorption for an alkaline phase (pH 8) is much less than that found at pH 2 (Table I). This large difference is presumably due to the protolytic equilibria in which procaine participates. In acidic media only cationic species are present, but at pH 8 neutral procaine molecules also play an important role (Figure 1)and, therefore, the electrostatic repulsion between the adsorbed procaine molecules is reduced, allowing for closer molecular packing. In order to calculate the procaine area/molecule, A2, molecular models have been constructed by taking into account the bond lengths, bond angles, and van der Waals radii of the atoms. Areas required have been calculated for three extreme conformations as visualized in Figure 3, in accordance with the intrinsic mobility of the ethyl groups. Conformation HE is a horizontal extended one, with the molecule portrayed as lying flat in the air/water interface. The horizontal linear dimensions of the molecules are denoted by a and b, with a > b. The conformations VE and VF are vertical ones, presented in Figure 3 as perpendicular to the air/water interface. The VE conformation may be considered as being a vertical extended conformation with the ethyl groups extending along the air/water interface. With the VF conformation the headgroup is forced underneath the vertically oriented aromatic ring. The linear dimensions a and b, derived from these models, are presented in Table 11, together with the molecular areas Ad and A,. The Ad value is defined as a2 and corresponds to a tetragonal close packing of rotating or
Langmuir, Vol. 7, No. 5, 1991 967
Interaction of Procaine with Stearic Acid Table 11. Linear Dimensions and Areas Occupied by Procaine in Its Extreme Conformations dimensions areas' conformation a, nm b, nm Ad, nmz A., nm*
HE VE VF a
1.23 0.85 0.52
Ad = a2,nm2. A, = a
X
0.52 0.47 0.47
1.513 0.722 0.270
I
I
0.640 0.400 0.244
b, nm2.
A3, nm*/molec. Figure 5. Compressional isotherms of stearic acid spread on aqueous procaine solutions at pH 8 and indicated procaine concentrations (cz). The area/molecule As,oOis indicated for zero procaine concentration. The arrows indicate the changing film collapse with changing procaine concentration.
nm*/molec. Figure 4. Compressional isotherms of stearic acid spread on aqueous solutions at pH 2 and indicated procaine concentrations (cz). The arem/molecule As,o0 and As,o'O are indicated for zero procaine concentration. The arrow indicate the changing film collapse with changing procaine concentration. A3,
randomly oriented molecules. The A , value is calculated as a 4 and represents the close-packed area of the oriented molecules, i.e. their cross-sectional area. According to our molecular model calculations the area occupied by a procaine molecule in a horizontal orientation, at the air/water interface is about 1.5 nm2/molecule, if the molecules are randomly oriented or are freely rotating. In the case of closely packed, oriented molecules, the area occupied should only be 0.64 nm2. By comparison of these calculated area values for different packing degrees in procaine surface lattices (Table 11) with the experimental molecular area of procaine in a pure adsorbed monolayer at both pH 2 and pH 8 (Table I), it can be seen that A2,0° determined at pH 8 lies between the area values Ad and A , for a horizontal extended conformation while A2p0 at pH 2 is even higher than the calculated area for the molecular conformation in a randomly oriented network; Le., presumably both procaine cations and procaine free base molecules are adsorbed at the air/water interface in a horizontal orientation. 3. Compressional Isotherms and Surface Characteristics of Stearic Acid Monolayers. Compressional isotherms, i.e. surface pressure (?r) versus mean molecular area (A31 curves, of stearic acid monolayers recorded on subphases of pH 2 and pH 8 for several procaine concentrations are presented in Figure 4 and Figure 5, respectively. The stearic acid monolayers were spread at the same molecular area both in the presence and in the absence of procaine in the subphase to avoid the influence of kinetic spreading effects.16 As a general feature, one may observe that all isotherms at pH 2 contain two linear portions (Figure 4) corresponding to the liquid condensed (LC) and solid (S)states, (16) Gaines, G.L.,Jr. Insoluble Monolayers at Liquid-GasInterfaces; Wiley-Interecience: New York, 1966.
respectively. In other words, the procaine does not abolish the phase transition from liquid condensed to solid phase that occurs in pure stearic acid monolayers at pH 2. It does, however, broaden it somewhat. The surface properties of stearic acid monolayers depend on the subphase pH due to the protolytic equilibrium between the neutral stearic acid molecules and the stearate anions (see Figure 1). On alkaline subphases the LC S phase transition in pure stearic acid monolayer is not observed (Figure 5, curve 1). In addition in the pH range between 2 and 8the collapse pressure of the pure monolayers exhibits a significant increase (see curve 1in Figure 4 and curve 1 in Figure 5). Obviously, the shape of all curves at pH 8 (Figure 5 ) differs from those recorded at pH 2 (Figure 4). For the former a single linear portion at high surface pressures is observed with the charged stearic acid film showing no transition and remaining in a fluid state all the way to collapse (Figure 5 ) . For both pH 2 and 8 subphases, procaine produces an expansion of the stearic acid monolayer and also increases its collapse pressure. The expansion effect of procaine can be characterized by the increase in surface pressure recorded at different constant A3 values, as well as by the increase of the molecular area corresponding to different ?r values. This expansion effect increases with increasing procaine concentration. From the mixed film compressional isotherms (Figures 4 and 5 ) the following surface characteristics of the stearic acid monolayer were derived: As,()', the limiting molecular area for the liquid condensed state at pH 2 (by extrapolating to ?r = 0 the first linear portion of the isotherm (Figure 4) obtained at intermediate ?r values); A3,o, the limiting molecular area for the solid film at pH 2 (by extrapolation of the second linear portion of the isotherm at high ?r values (Figure 4) or the limiting molecular area for the very compact liquid condensed monoand ?rc, the collapse area and layer at pH 8 (Figure 5 ) ) ; collapse pressure, respectively, corresponding to the sudden slope change observed at high ?r values (indicated by arrows in Figures 4 and 5 ) ; A3,t and At, the molecular area and surface pressure at the phase transition (the intersection of the two linear portions in Figure 4); (Cmq1)',
-
968 Langmuir, Vol. 7, No. 5, 1991
Tomoaia-Cotigel and Cadenhead
Table 111. Surface Characteristics of Stearic Acid Monolayers on Procaine Containing Subphases at pH 2 c2 = 0 10-6 M 1od M lo-' M 10-3 M 10-2 M nm2/molec A3,t, nm2/molec A3,o, nm2/molec A'3.0, nm2/molec
uc,mN/m ut,
mN/m
Cdl,mN/m (&-I)',
mN/m
0.180 0.188 0.200 0.255 40.8 26 408.0 99.0
0.180 0.190 0.205 0.280 41 26 336.2 80.9
0.180 0.200 0.226 0.310 44 26 209.0 73.3
0.178 0.206 0.245 0.317 46 26 168.2 74.3
0.175 0.210 0.260 0.340 47 26 143.8 68.0
0.180 0.220 0.268 0.370 47.5 26 144.7 64.1
Table IV. Surface Characteristics of Stearic Acid Monolayers on Procaine Containing Aqueous Subphases at pH 8 ca = 0 10-6 M M 104 M 10-3 M 10-2 M As,c,nm2/molec 0.176 0.178 0.180 0.180 0.180 0.180 A3,o, nm*/molec rC,mN/m Cd-1,mN/m
0.220 51 255
0.226 51 240
the surface compressional modulus for the liquid condensed state given by the relation
CW-l,the surface compressional modulus for solid-state or a very compact two-dimensional phase given by
The surface characteristics of stearic acid monolayers derived from compressionalisotherms presented in Figures 4 and 5 are given in Tables I11 and IV, respectively. Inspection of Tables I11 and IV shows that A3,c is not affected by the presence of procaine in the subphase, i.e. A3,c has practically the same value irrespective of the presence or absence of procaine in the subphase. In contrast with A3,c values, all the other characteristic areas, A3,t, A3,0, and A3,o' increase with increasing procaine concentration, indicating that procaine penetrates into stearic acid monolayers but is subsequently expelled as collapse is approached; Le., in the final portion of the compression isotherm, procaine is squeezed out of the monolayer. The collapse pressure, uC,increases systematically with increasing procaine concentration revealing a stabilizing effect of procaine upon the stearic acid monolayer. This finding suggests that the expelled procaine molecules remain in an adjacent layer and the uc value increases because of a vertical specificinteraction between the stearic acid and procaine molecules. This expulsion of procaine can be considered as a transformation of the mixed stearic acid-procaine monolayer into a "bilayer", the upper layer being the insoluble stearic acid film including the hydrated headgroups, the lower one an adsorbed film of procaine. The stabilizing effect of procaine upon the stearic acid monolayers at pH 2 thus seems to be due to specificdipoleion interactions between the neutral stearic acid monolayers and the adjacent adsorbed layer of procainium ions and/or to hydrogen bonding between the uncharged carboxyl group of stearic acid molecule and the primary amino group of procaine monocations (PRH+). This stabilizing effect is reduced at pH 8, since under these conditions the charged, pure stearic acid film has a much higher stability than that at pH 2, due to the electric double layer that is formed under close-packed conditions near to collapse, even in the absence of procaine. Thus, the effect of procaine on the collapse of the stearic acid monolayers on alkaline media probably consists of the replacement of Na+ or K+ ions by PRH+ ions.
0.232 52 232
0.236 52.5 221
0.240 53 212
0.260 53.5 174
At pH 2, it is obvious that the liquid condensed to solid phase transition occurs at the same ut value, irrespective of the procaine concentration in the subphase (pH 2). The procaine penetrating into the monolayer has an expanding and fluidizingeffect upon the stearic acid monolayers both at pH 2 and 8 as is indicated by the enhanced compressibility of the monolayers, Le., by the decrease of the surface compressional moduli with increasing procaine concentration (Tables I11 and IV, respectively). 4. Extent of Procaine Penetration in Stearic Acid Monolayers. As a first step in elucidating the mechanism of penetration, we have estimated the amount of procaine inserted into the stearic acid monolayers by using the thermodynamic theory of monolayer In our experiments the surface of the aqueous procaine buffer is initially covered by an expanded monolayer of stearic acid and interactions can occur between both the polar headgroups and the hydrocarbon chains of stearic acid and procaine. Due to these interactions the adsorption of procaine may increase in the presence of stearic acid, and this phenomenon is frequently called monolayer p e n e t r a t i ~ n . l ~ JThis ~ - ~results ~ in the formation of a mixed monolayer containing both water molecules and molecules of stearic acid and procaine. Thermodynamically, the equilibrium between the bulk subphase and the mixed stearic acid-procaine monolayer has been treated by using Gibbs' e q ~ a t i o n , l ~which - ~ at constant temperature and mean molecular area of stearic acid, A3, has the following form:
where r2' stands for the number of procaine molecules adsorbed per unit area of the "free" interface, not covered by stearic acid molecules. By use of this approach plots of surface pressure versus log c2 were obtained for different constant A3 values, by using the compressional isotherms illustrated in Figures 4 and 5, at pH 2 and 8, respectively. A set of curves constructed according to this procedure is presented in ~
~~~
(17) Pethica, B. A. Trans. Faraday SOC. 1955,51, 1402-1411. (18)Nakagaki, M.; Okamura, E. Bull. Chem. SOC.Jpn. 1982,55,13521356. (19) Nakagaki, M.; Okamura,E. Bull. Chem. SOC.Jpn. 1982,66,33813385. (20) Nakagaki, M.; Okamura, E. Bull. Chem. SOC.Jpn. 1983,56,16071611. (21) Daviee, J. T.; Rideal, E. K. Interfacial Phenomena, 2nd ed.; Academic Press: New York, 1963; pp 295-298. (22) Panaiotov, I. I.; Ter-Minaesian-Saraga, L.; Albrecht, G. Longmuir 1985,1, 395-399. (23) Ter-Minassian-Saraga,L. Langmuir 1986, 1, 391-394.
Langmuir, Vol. 7, No. 5, 1991 969
Interaction of Procaine with Stearic Acid
2E E
"1 10
t
0.30
1
/
X
-6
-5
-4
-3
I
-2
c, Figure 6. Surface pressure versus the logarithm of the procaine concentration at constant areas per molecule of stearic acid on pH 2 subphases. The values cited for each curve indicate the area per molecule of stearic acid expressed in nmz. log
50
I
40
E 2
0.2
0.3
0.4
0.5
A,, nm2/molec.
Figure 8. (a) Adsorption of procaine per unit area of the 'free" interface (rd)for stated values of cz as a function of AS and calculated by means of eq 2, at pH 2. The horizontal dashed lime represents the 6bserved procaine adsorption in the absence of a stearic acid monolayer (I'zo) for cz = 1 mM. (b) Adsorption of procaine per unit area of the mixed monolayer (rz)at pH 2, for stated values of c2, as a function of Aa and calculated by means of eq 4.
30
4
E 3
r" 20
2
. 5
N
10
-v P)
0
-6
-5
-3
-4
log
-2
E
1
I I
I
I
I
I
I
c2
Figure 7. Surface pressure versus log cz at constant mean molecular areas Aa of stearic acid at pH 8. The values cited for each curve indicate the value of As expressed in nm*/molecule.
Figure 6 for pH 2 and another one in Figure 7 for pH 8. The general picture is similar to that of amino acid penetration into lecithin monolayers.lem The slope of the curves increases with decreasing A3 values, attaining a maximum at about 0.25 nm2 for pH 2 (Figure 6) and at about 0.35 nm2for pH 8 (Figure 7). After this the amount decreases, indicating that with further compression of the monolayer, expulsion of the penetrated molecules occurs. The c w e s given in Figure 6 and Figure 7 allowed us to obtain the derivative @r/alog Cp)T,& of the Gibbs equation (2) for different cp and A3 values. By use of these derivatives the procaine adsorption per unit area of the residual'free" interface of the stearic acid monolayer (I'z') was calculated by means of eq 2. Plots of'z'I as a function of A3 for different cp values are given in Figure 8a (pH 2) and Figure 9a (pH 8). In Figures 8a and 9a, the horizontal straight linea indicate the amount of procaine adsorption in the absence of the stearic acid monolayers ( I ' p O ) , from lb3M procaine buffer solutions, as obtained by means of eq 1, with the surface tension derivative being taken from the curves given in Figure 1. From Figure 8a and Figure 9a it is obviousthat I'2'values are inexcess of the I'2O values, indicating that, for
0.2
I
0.4 0.6 0.8 A,, nm*/molec.
Figure 9. Procaine adsorption per unit area of the "free" interface (a)into a stearic acid monolayer (Pi) and (b)a mixed procainestearic acid monolayer (I"& as a function of AB at pH 8 for stated values of the subphase procaine concentration (cz). The horizontal dashed line indicates the procaine adsorption at the air water interface for cz = 1 mM, in the absence of stearic acid.
both pH 2 and 8, an important interaction exists between stearic acid and procaine molecules at the air/water interface, producing an enhanced adsorption of procaine at the interface. The relative increase is greater at lower procaine concentrations. On the other hand the procaine adsorption also depends on As, i.e. on the surface density of stearic acid molecules. On compression of the monolayer the procaine adsorption I'2' increases and passes through a maximum in the range 0.25 < A3 < 0.3 nm2/ molecule for pH 2 (Figure 8a) and 0.3 < A3 < 0.4 nm2/ molecule for pH 8 (Figure 9a). With further compression it decreases, as expected on the basis of both the
Tomoaia-Cotigel and Cadenhead
970 Langmuir, Vol. 7, No. 5, 1991 compressional isotherms (Figures 4 and 5 ) and the surface pressure increments as a function of AB. The adsorption of procaine per unit area of the mixed monolayer (r2) can be calculated from r2' (Figures 8a and 9a) by taking into account the actual area occupied by the stearic acid molecules, viz., the partial molecular area of stearic acid, denoted as As, the area of the interface made inaccessible for the adsorption of the procaine by the presence of a single stearic acid molecule. One obtains17J8
x , mN/m
I
10
20
I
I
40
30
r2= (1 - 5)r;
(3) A3 Attempts were made to approximate A3 by A3', i.e. by taking the mean molecular area of component 3, at the same ?r value but in the absence of component 2.'' This approximation, however, is not satisfactory since stearic acid is anchored at the interface by its carboxyl group and the area of the carboxyl group is less than A3', especially at low surface pressures. A more reasonable approachjs to take the collapse area in a pure monolayer A3,c0for A3 in eq 3. This leads to
r2= (1
-%)r;
np I'2/I'3 = r2A3 (5) With eq 5 we can easily obtain from Figures 8b and 9b the penetration number of procaine into stearic acid monolayers as a function of As, c2, and pH. Alternatively, by combining eqs 2,4, and 5, one obtains the following expression for deriving penetration numbers directly from the experimental curves presented in Figures 6 and 7: =
Penetration numbers calculated by means of eq 5 or eq 6 are visualized in Figure 10 (pH 2) and in Figure 11(pH 8) in two different plots. In Figure 10a and Figure l l a , np values are given as a function of A3, corresponding to the I'z' and I'z curves in Figures 8 and 9. In Figures 10b and l l b , npvalues are shown as a function of the surface pressure measured for differing procaine concentrations in the subphase. The npvalues exhibit a maximum at a surface pressure of about 5-10 mN/m. The maximum values are much higher for subphases at pH 8 (Figure 11) than at pH 2
0.5
0.3 0.4 A,, nm2/molec.
Figure 10. Penetration numbers np calculated by means of eq 6 (a) as a function of A3 and (b) as a function of ?r at pH 2. x , mNlm
(4)
Procaine adsorption per unit area of the stearic acid monolayer (rz)was calculated by means of eq 4, by taking = 0.18 nm2. The results are shown in Figure 8b (pH 2) and Figure 9b (pH 8). The curves I'2 versus A3 exhibit a maximum, shifted slightly toward higher molecular areas compared to r2' versus A3 curves. Inspection of Figures 8 and 9 further reveals that, for a procaine concentration M, there is a large A3 interval in which r2 exceeds of rZo;i.e., the number of procaine molecules per unit area of the mixed monolayer is larger than that (I'2') in the adsorbed procaine monolayer in the absence of a stearic acid film. r2 values are, however, much less than those of rz', especially at low molecular areas, i.e. at high surface densities of stearic acid molecules, where the stearic acid monolayer becomes somewhat insensitive to subphase procaine concentrations. The extent of procaine penetration is characterized by the penetration number, np, defined as the ratio of the number of procaine molecules (r2)to stearic acid molecules (r3)per unit area of the mixed monolayer
nP
0.2
10
20
30
40
50
"P O . j O
I,' 0.2
I
I
I
0.4 0.6 0.8 A,, nm*/molec.
Figure 11. Penetration numbers calculated by means of eq 6 (a) as a function of A3 and (b) as a function of surface pressure at pH 8. Symbols are the same as those given in Figure 9.
(Figure 10) and are obviously also dependent on the procaine concentration. The important increase of npwith increasing pH results from a cooperative effect of the protolytic equilibria in which both stearic acid and procaine participate. On the one hand, the successivedeprotonations PRHz2+ PRH+ PR lead to the formation of molecular species with higher surface activity. On the other hand, deprotonation of the neutral stearic acid molecules and the formation of the stearate anions entail the appearance of important electrostatic attractions perpendicular to the interface between the oppositely charged surfactant ions which in turn could alter chain-chain interactions. From equation of state studies, it appears that it is the uncharged form of procaine that is inserted into the stearic acid monolayers at pH 8, instead of the charged form (unpublished results). The amino acid adsorption (r2) values reported for dimyristoylphosphatidylcholine (DMPC) monolayers,l*m especially with norleucine, are similar to those obtained in the present paper. The penetration number, estimated from the np diagram, for norleucine penetration into
-
-.
Langmuir, Vol. 7, No.5, 1991 971
Interaction of Procaine with Stearic Acid
DMPC monolayers should be about 0.23 at a norleucine concentration of 10-l M. This value is about 1.5 times larger than that expected for procaine penetration into stearic acid monolayers on the basis of Figure 11 at pH 8. Sincethe collapsearea of DMPC is higher than the collapse area of stearic acid, this estimate is consistent with the adsorption data. The small n pvalues obtained for the penetration of procaine into stearic acid monolayers are a consequence of the high molecular density, since each stearic acid molecule has only one chain, and the chain's cross-sectional area determines the close-packed area. The effect of the molecular area (A31 values of the lipid component on the penetration number (eq 5 ) can also be illustrated with the following example. For dibucaine penetration into l-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), penetration numbers up to 0.4 have been found for a subphase dibucaine concentration of 4 X M.l0 By taking into account the fact that the molecular area of POPC is almost 4 times greater than that of stearic acid, the adsorption r2 value of dibucaine in the presence of POPC can be estimated to be about twice the adsorption r2 of procaine in the presence of stearic acid, under similar conditions of anesthetic concentration and surface pressure. These findings are consistent with the molecular structures of these surfactants, indicating a stronger interaction between dibucaine (with its disubstituted quinoline ring) and POPC than that between procaine and stearic acid. Correlations between the various penetration numbers of anesthetics into the lipid monolayers can be extended by making the following quantitative comparison on a per chain basis. In the case of M procaine solutions the maximum penetration into stearic acid monolayers corresponds to a ratio of one procaine molecule to 20 stearic acid molecules at pH 2 and to a ratio of 1.3 procaine moleculesto 10stearic acid moleculesat pH 8. This means 1 procaine molecule for 20 chains, i.e 1 procaine molecule for 10 phospholipid molecules, at pH 2, and 13 procaine molecules per 100 fatty acyl chains, or 13 procaine molecules for 50 phospholipid molecules, i.e. 1 procaine molecule for about 4 phospholipid molecules. Taking into account the fact that two fatty acyl chains are attached to each phospholipid molecule, from our data the penetration number estimated for phospholipid monolayers or bilayers would be 0.1for pH 2 and 0.26 on pH 8. These estimated data are in excellent agreement with the values reported for 0.1and 0.2mol of bound anesthetic (tetracaine) per mole of lipid (1,2-dipalmitoyl-sn-glycero3-phosphatidylcholine) in lamellar aqueous dispersions.6 5. Procaine Interactions with Stearic Acid Monolayers. To obtain a clear image of the penetration mechanism of procaine into stearic acid monolayers in yet another way, plots have been constructed, for both pH 2 and pH 8 at a constant procaine concentration, of the molecular area increase (AA) corresponding to different constant surface pressure values. We chose a M procaine concentration because the procaine effect upon stearic acid monolayers, as indicated by the increase of the film collapse pressure with increasing procaine concentration (Figures 4 and 5; Tables I11 and IV), is increasinglysignificant up to this concentration value and then diminishes. This concentration of procaine is also of physiological significance. The AA value represents the difference between the A3 values recorded in the presence (As,curve 5 ) and in the absence (A3 = As0,curve 1) of procaine, at the same surface pressure value (Figures 4 and 5 ) . The AA versus A curves obtained on the basis
5 0.1 t
\ I
0
20
10
30 n, mN/m
40
50
Figure 12. Molecular area increases (AA) due to the procaine penetration (curve 1) at pH 2 and (curve 2) at pH 8 on a constant procaine concentration c2 = 1 mM.
of the compressional isotherms (Figures 4 and 5 ) are given in Figure 12 for both pH 2 and 8. In Figure 12, AA exhibits a maximum at low surface pressures and rapidly diminishes with increasing A. This shape indicates procaine penetration at low surface densities of stearic acid, followed by expulsion of the incorporated procaine as the compression of the film is increased. The expansion effect becomes very important in alkaline media. On the basis of L 4 vs A curves (Figure 12)the amount of penetrated procaine can be evaluated by a quantitative description of the change in. surface area as follows. The simplest approach considers the pure stearic acid monolayer consisting of fatty acid molecules only, with their molecular area, denoted by A3", being given by A," = n3A/n3= A
(7)
where n3 is the constant number of fatty acid molecules and A denotes the molecular surface area of stearic acid. In the presence of procaine, which penetrates into the monolayer, the mixed penetrated monolayer is treated as a binary solution, and the area per molecule of stearic acid, now denoted by As, may be expressed as A, =
n3A + n2A2
n3 where n2 stands for the number of procaine molecules and A2 for the area they occupy. In this expression both A and A2 can depend on A. On the presumption that A is a unique function of A and with AA = A3 - A3" as the mean molecular area difference measured at a given A value, the penetration number, n,,, is given by n2
AA
np=n,=Aa
The penetration number can also be derived in a more sophisticated manner, by taking into account the presence of subphase liquid (water, component 1)molecules in the monolayer, i.e. by considering the Upure"monolayer to be a binary solution and the penetrated mixed monolayer a ternary one. In this approach eqs 7 and 8 become A," =
nlAl + n,A n3
(10)
and A, =
n,'A,
+ n3A + n2/A2
(11) n3 respectively, where nl, nl', and A1 stand for the number of water molecules from the monolayer in the absence and presence of procaine and the area occupied by a water molecule, respectively.
Tomoaia- Cotigel and Cadenhead
972 Langmuir, Vol. 7, No. 5,1991 The equilibrium between the bulk subphase (B)and the monolayer (M) implies the equality of the chemical potentials of water in both phases, i.e.
Table V. Molecular Areas A, (in nm') of Procaine Located in Stearic Acid Monolayers as a Function of I variant 0 3mN/m 10mN/m 26mN/m xC ~~
~~
+ kT In xlB + kT In flB = ploM+ kT In x I M +
ploB
kT In flM
+ *A,
(12)
where xlB, xl', f l B ,andfiMstand for the mole fraction and activity coefficient of water in the B and M phases, respectively. In the case of pure water one has x l B = x l M = fiB = fiM = 1and A = 0. Consequently, p l o B = ploM,and from eq 12 one obtains In xIM = In x:
fIB AAl + In -fIM
+ xlAl
(14)
+ x{Al + x;A2 (15) Taking into account that x 1 + x 3 = 1, eq 14 yields x3/A3= x3/A
XI
=
A,' - A A,' A, - A
+
Since at constant surface pressure X I = x i and XI' = 1, from eqs 15 and 16 one obtains
+ xg'
n2/ n P = - =n3/
8 9 10 11 12 13 14
0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
0.25 0.40
1.00 1.00 1.00 1.00 1.30 1.30 1.50 1.50 1.50 1.50 1.50 1.50
0.40
0.64
0.40 0.40 0.40 0.40 0.40 0.64 0.40
0.64 0.64
kT
On the right-hand side of eq 13 the first term can be neglected in comparison with the last. If the subphase is M procaine pure water In x l B = 0 and if it is a concentration solution, one has In x l B = -1.8 X Obviously, if the subphase contains a buffer solution, the modification of In xlB due to the introduction of the procaine, will be even less. The second right hand side term, even if it differs from zero, as a first approximation may be presumed to be negligibly small. Therefore, at a given A, xlM may be presumed to have the same value, irrespective of the presence or absence of the procaine. By multiplying eqs. 10 and 11 by the mole fraction of stearic acid in the monolayer, i.e. byx3 and x i , respectively, for the sake of simplicity the superscript (M) is omitted, one obtains x3A30= x3A
1 2 3 4 5 6 7
A3-A30 A30-A+A2
(16)
+ x2' (17)
Obviously, for As0 = A, eq 17 turns into eq 9; Le. in the case of condensed films both equations practically give the same results. If the monolayer is an expanded one, eq 17 is to be preferred. was used, Le., In our calculations A = 0.18 nm2 = the collapse area of stearic acid, since this value approximates the actual area occupied by the carboxyl group in the monolayer. The experimental AA vs 17 curves presented in Figure 12 were processed according to eqs 9 and 17 by using different hypotheses concerning the dependence of A2 upon A. The variants tested are summarized in Table V. In this table the A2 values are indicated, for several values-characteristic of a stearic acid monolayer at pH 2. These A values are those at the spreading of the monolayer ( A = O), a t the appearance of the liquid condensed state (3 mN/m), a t the beginning of the linear portion of the compression isotherm a t (10 mN/m), at the LC S phase transition (26 mN/m), and at monolayer collapse (ac).Between the two constant A2 values indicated in Table V,A2 was presumed to be a linear function of A. As an example, let us take variant 10. It was presumed that, on spreading the stearic acid monolayer, the procaine
-
0.10
1
I 0
\
4
10
I
I
20
n, mNlm
Figure 13. Derivation of penetration number (n,)values at pH 2 by means of eq 17. The figures indicate the variant used for A2 (see Table V). The circles represent np values obtained by using Gibbs' equation (see Figure lob).
molecules adsorbed at the airlwater interface adopt a horizontal position, corresponding to A2 = 1.50 nm2 (see Table 11). This value is in accord with the adsorption data for a pure procaine monolayer. On compression the molecules gradually are forced to adopt a vertical position, corresponding to A2 = 0.40 nm2. This process is thought to be achieved a t A = 26 mN/m. Therefore, in the range 0 5 A I26 mN/m, for A2 the following expression is used A, = 1.50 - la50- 0'40,i= 1.50 - 0.042s
(18) 26 - 0.0 Beginning a t 26 mN/m, the compression of the monolayer is presumed to further reduce A2, the latter reaching 0.25 nm2 at collapse, obeying the following linear law: A, = 0.40 -
0.40 - 0.25 ( A - 26) T , - 26
As an example, the several np versus s curves, calculated for subphases of pH 2 by eq 17 and the function A2 = Az(7r) given by eq 18, are represented in Figure 13.
In order to choose the best variant, the np values calculated from AA were compared with the np values obtained by applying Gibbs' equation to the adsorption of procaine in the presence of a stearic acid monolayer (Figure 10). The latter results are illustrated in Figure 13 as the individual points. It is obvious that the best agreement with adsorption measuremente was obtained by means of variant 10. This finding suggests that procaine is adsorbed in a horizontal position prior to the spreading of the stearic acid monolayer (Table I). On compression of the monolayer the procaine molecules gradually adopt a vertical position (VE conformation, Table 11)and this process is completed a t about 26 mN/ m.
Interaction of Procaine with Stearic Acid ".''I 0.10 np
I n 1 \
Langmuir, Vol. 7, No. 5, 1991 973 I
0.08
I
0.061
I
I
\
t
n, mN/m
Figure 14.
values derived by means of eq 17 according to variant 10 in?I'able V, from the L\A versus T curves given in Figure 1 2 (1) pH 2, (2) pH 8; circles (0,O)represent values obtained by using the Gibbs equation for pH 2 an? pH 8, respectively (Figures 10b and llb).
A similar result was also obtained at pH 8. Thus, in Figure 14, the curve of npversus r was obtained from AA in the same way as shown above for pH 2. Once again variant 10 describes the n pvalues obtained by the Gibbs equation. In Figure 14, the penetration numbers versus r a t pH 2 are also represented for the entire range of surface pressure, up to the collapse of the stearic acid monolayer. In the range 26 mN/m I?r Ir c ,the function Az(r) was obtained by eq 19 for both pH 2 and 8. The data, in Figure 14, show that there is a good agreement between npvs ?r obtained by both methods on pH 2 and pH 8 even at high surface pressure. This finding suggests that residual procaine molecules are present in monolayers at very high surface pressure in a vertically forced conformation (VFconformation, Figure 3). It seems likely that, in this VF conformation, procaine molecules will penetrate deeper into the monolayer at high surface pressure than at the lower surface pressure, leading to enhanced interactions. Upon compression of the stearic acid monolayer spread on subphases of pH 2 and pH 8, the penetration number initially increases, attains a maximum value at about 10 and 5 mN/m, respectively (Figure 14), and then begins to decrease, indicating a squeezing out of procaine molecules from the stearic acid monolayer. The use of eq 9 instead of eq 17 does not modify the general picture. As the amount of intercalated procaine depends critically on the monolayer pressure (Figure 14), it seems that procaine penetrates preferentially into the fluid expanded and liquid condensed states of the stearic acid film. At monolayer collapse procaine is completely squeezed out from the stearic acid monolayer but remains associated with the solid state of stearic acid monolayer producing an enhanced surface collapse pressure. This result can be interpreted in terms of dipole-ion interactions between the uncharged carboxyl group of stearic acid monolayer on pH 2 and the positively charged procaine molecules as well as to hydrogen bonding, especially between the carboxyl group and primary amino group of the procaine monocation (PRH+). Clearly at pH 8, electrostatic interactions are very important, particularly at monolayer collapse. Our results are consistent with data obtained for deuterated procaine in lamellar aqueous dispersions of phos-
pholipids by spectroscopictechniquess indicating that the procaine has difficulty penetrating into highly ordered, tightly packed acyl chains at high surface pressures. The present results show that procaine both expands the packing of ordered acyl chains of stearic acid monolayers and increases the fluidity of the monolayers, especially in their liquid condensed and expanded states. Comparable effects should also occur in bilayers having similarly ordered acyl chains in that the two model membrane systems behave in a similar way.= Application of the monolayer technique reported here to more complex systems, including phospholipids and to mixed lipid-protein systems, in interaction with procaine and other anesthetics, should add significantly to the understanding of the mechanism of anesthesia.
Conclusions Adsorption measurements in the absence of stearic acid quite clearly demonstrate that all three forms of procaine are surface active, the non-charged form exhibiting a stronger surface activity than the charged (PRHz2+ < PRH+ < PR). This is paralleled by the hydrophobicity of the three species, since water solubility for the neutral form is low and increases for the charged forms. Data obtained from stearic acid compressionalisotherms and the resultant molecular area increases in the presence of various procaine subphase concentrations at pH 2 and pH 8 and constant surface pressure are interpreted by using molecular models and by taking into account different variants for the mechanism of procaine penetration into the stearic acid monolayers and the protolytic equilibria occurring in the system. The penetration number, i.e., the ratio of procaine molecules to stearic acid molecules in the mixed penetrated film, derived from molecular area increments are in good agreement with values obtained by using the Gibbs adsorption equation. Procaine is proposed to be adsorbed at the air/water interface in a horizontal position, in the absence of stearic acid monolayers. The spreading of stearic acid monolayers promotes the enhanced adsorption of procaine, due to both polar and nonpolar interactions between the stearic acid and procaine. On compression of the stearic acid monolayer the procaine adsorption initially increases and the horizontally oriented procaine molecules are gradually forced to adopt a vertical orientation in which the hydrophobic interactions with the fatty acyl chains increase and increased amounts of procaine moleculesmay penetrate into the monolayer. Subsequently, the penetration numbers attain maximum values, after which they decrease and vanish near to the point of collapse of the monolayer. At higher surface pressures procaine molecular species are squeezed out of the monolayer and appear to be accumulated in an adjacent layer, producing a significant increase in the collapse pressure of the stearic acid monolayers. This effect has been interpreted at pH 2 in terms of both ion-dipole interactions between the positively charged procaine molecules and the uncharged carboxyl group of stearic acid monolayer and through hydrogen bonding, especially between the carboxyl group and primary amino group of the procaine monocation. At pH 8 electrostatic interactions between the stearate anions and the procaine monocation are also taken into account. Procaine penetration occurs preferentially in the fluid expanded and liquid condensed states of stearic acid monolayers and was found to be dependent of the surface (24) Cadenhead,D.A. InStructuresandProperties of CellMembranes; CRC Press: Boca Raton, FL,1986; Vol. 111, Chapter 2, pp 2 1 4 2 .
974 Langmuir, Vol. 7, No.5, 1991 pressure of the film. Maximum penetration was established to occur at about 10 mN/m for monolayers on pH 2 and at 5 mN/m for monolayers on pH 8. The amount of procaine penetrating at pH 8 was greater than at pH 2. This latter phenomenon is interpreted in terms of protolytic equilibria in which both stearic acid and procaine participate.
Acknowledgment. Maria Tomoaia-Cotisel acknowledges the financial support in the form of an Alexander von Humboldt Foundation Fellowship in the completion of this work. D. Allan Cadenhead would similarly like to acknowledge the financial support of New York State Science and Technology Foundation through Grant CAT887. We would also like to express our thanks to Professors W. Luck, E. Chifu, J. Zsako, P. T. Frangopol, and P. J. Quinn for their useful suggestions and comments during the writing of this manuscript. Nomenclature rzo,the number of procaine molecules adsorbed per unit area of the interface in the absence of stearic acid monolayers; rZ0= f ( ~ ) l'2,mar0, the maximum value of I'2', i.e. the value at saturation of the interface, l'2,mar0 # f ( ~ ) rl),the number of procaine molecules adsorbed per unit area of the "freen interface, i.e. the area not covered by the stearic acid molecules in mixed procaine-stearic acid monolayers rz, the number of procaine molecules adsorbed per unit area of the mixed monolayer
Tomoaia-Cotigel and Cadenhead A
= uo -
monolayer
u,
the surface pressure of the pure stearic acid
A = UPR - u, the surface pressure of the mixed procaine stearic acid monolayer uo, the surface tension of pure buffers u, the surface tension of the interface covered with pure stearic acid monolayers or with mixed procaine-stearic acid monolayers. UPR, the surface tension at the airlaqueous procaine solution interface in the absence of stearic acid As, th_epartial molecular area of stearicacid in mixedmonolayers, A3 taken as A3,c0 = 0.18 nm2/molecule A2,0°,the limiting adsorbed molecular area in a pure procaine monolayer; A2,0° # f ( ~ ) A2, the molecular area occupied by procaine in a mixed procaine-stearic acid monolayer A3', the area per molecule of component 3, in the absence of the component 2 As, the mean area per stearic acid molecule in a mixed monolayer A3,0', the limiting area per molecule of stearic acid in a pure stearic acid film A$0, the limiting molecular area of stearic acid for the liquid condensed state of pure stearic acid at pH 2 the area per molecule of stearic acid in a pure stearic acid film at the point of collapse the area per molecule of stearic acid in a pure stearic acid film at the liquid condensed/solid transition As,?, A3$,.A3,0 and are the corresponding areas for stearic acid in a mixed procaine-stearic acid film. n the ratio of the number of procaine molecules to stearic acid'molecules per unit area of the mixed monolayer, the penetration number
