surface potential at surfactant and phospholipid vesicles as determined by amphiphilic ph indicators

J. Phys. Chem. 1983, 87, 5045-5050 5045

describe the pressure dependence of the molecular mobility in the glass state, it may be necessary to take into account the pressure dependence of both B and To. t i s

A 1 bar V 750 bar v 250 bar 0 1000 bar A 5QObar 0 1250 bar ,

:/ : 1 o-2 Summary and Conclusion

We have studied the temperature and pressure dependence of the photon correlation spectra of o-terphenyl in the supercooled liquid state as a function of temperature and pressure. At all temperatures and pressures the mean correlation times obtained from VV and VH spectra are identical.

The temperature and pressure dependence of the mean lo-'l

Flgure 6. Master curve constructed for the mean relaxatlon time as a function of X (8 + a'P) / (T - T o - b P ) for o-terphenyl at varlous temperatures and pressures. T o = 219 K, b = 0.02 K/bar. Circles and triangles represent the experimental data.

close to the value computed from the slope of the curves in Figure 5.

It is now possible to represent all of the pressure- and temperature-dependent data on a master curve. According to eq 8, we plot in Figure 6 ?(T,P) vs. (B + aP)/(T - To - bP). The quality of this master curve is good and the resulting error is less than a factor of 2 as compared to the fit of the data in accordance with eq 7 which assumes a pressure-independent To. We thus feel justified in representing the experimental relaxation times of o-terphenyl by eq 8. However, it should be pointed out that if the activation volume AV' is calculated according to eq 8, the result gives a small pressure dependence for AV' (eq 6); i.e., if the r vs. P plot given in Figure 3 is extended to higher pressure, it should show a slight curvature. This is difficult to verify experimentally in the present pressure range used, but it may be possible to verify the pressure dependence of AV' in measurements covering a wider pressure range. Our result nevertheless suggests that, to

extended Vogel-Fulcher equation in which the activation parameter B and the limiting temperature To are consid- ered to depend linearly on pressure. This empirical approach reproduces the experimental data within experimental accuracy. The physical model behind this is based upon the phenyl-group motion determined by free volume requirements. With increasing pressure, the height of the activation barrier increases and the free volume appears to decrease. When both effects are incorporated into eq 8, it predicts a slowing down of the process as the pressure is increased in the supercooled liquid state. This treatment is based on the assumption that the high temperature limiting value ro is pressure independent. Although this is not a necessary assumption, it seems to be supported by our data analysis and is justified to a certain extent by the quality of the fits as well as the physically plausible values of the parameters obtained.

Acknowledgment. Acknowledgments for financial sup- port are made to the Deutsche Forschangsgemeinschaft (DFG) and the Fonds der Chemischen Industrie (to Th.D.) and to the NSF Polymer Program, Grant No. DMR 79- 12457 (to C.H.W.).

Registry No. o-Terphenyl, 84-15-1.

Surface Potential at Surfactant and Phospholipid Vesicles As Determined by Amphiphilic pH Indicators

Sava Lukac

Xerox Research Centre of Canada, Mississauga, Ontario, L5L 1J9 Canada (Received: November 16, 1982; I n Final Form: March 25, 1983)

The surface potential (+) of negatively (dihexadecyl phosphate (DHP)) and positively (didodecyldimethyl- ammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DODAB)) charged surfactant vesicles was determined through the pK shifts of the surfactant coumarin pH indicators incorporated in the vesicles. The obtained values of (-127 and +156 mV, respectively) are very close to those found in micelles and indicate similar surface charge densities of both surfactant aggregates. However, the polarity of the vesicle interface is significantly more polar (eeff - 45) than in the case of micelles (eeff - 32). When the pH indicators were solubilized into the zwitterionic phospholipid vesicles, either the phosphate or the choline groups were surface active, depending on the type of indicator (hydroxy- or aminocoumarin). Such behavior prevented us from using the phospholipid vesicles as a neutral standard system for the surfactant vesicles.

Introduction Surfactant vesicles formed from the double-chain sur-

factant molecules have been stable alternative to phopholipid vesicles as model systems for

mimicking various functions of biological membranes. One of the most important features of these organized assem- blies is the existence of the surface charge as the conse- quence of the dissociation of the head groups of the surfactant molecules. These fixed charges in turn result in the creation of a potential between the bilayer interface and the bulk aqueous phase. Such a potential, usually

as a

(1) J. H. Fendler, Acc. Chern. Res. , 13, 7 (1980).

0022-3854/83/2087-5045$0 1.50/0 0 1983 American Chemical Society

5046 The Journal of Physical Chemistry, Vol. 87, No. 24, 1983

addressed as the surface, interfacial, or "outer" potential, plays a crucial role in various catalytic applications of surfactant vesicles including solar energy conversi~n.~.~ In this application the charged surface is beneficial to the charge separation in the photosensitized ionization or charge-transfer processes which is of paramount importance for a successful solar energy converting ~ y s t e m . ~ Therefore, the determination of the surface potential of vesicles, as well as monitoring changes with varying experimental conditions, is of great interest. This is even more evident in view of the recent successful experiments on the improvement of the colloidal and structural stability of vesicles through the polymerization of their bilayer^.^

Since the size of vesicles precludes the use of any direct electrode methods for the determination of the surface potential, the dye pH indicators610 and/or spin-labels'l have been frequently applied in various aggregated systems. In the case of relatively large multilamellar phospholipid vesicles, the determination of the electrokinetic ({) potential is also possible.12

The use of a dye pH indicator is based on the fact that the charged vesicular surface (interface) represented as the Gouy-Chapman diffuse double layer13 creates a concentration gradient of protons between the interface and the bulk water. Such a difference in the proton concentration will be reflected through the pK shift of the pH indicator located a t the interface.6

The distribution of protons is related to the electrostatic surface potential by Boltzmann's law and in combination with the definition of pK of an acid-base indicator it leads to eq l.697 Here, pKa stands for the apparent pK of the

(1)

indicator in the presence of a charged interface and pK' refers to the "intrinsic" interfacial contribution to the pKa. The surface potential is represented by Ji. F, R, and T stand for the Faraday constant, the universal gas constant, and the absolute temperature, respectively.

When a water-soluble pH indicator is used for the surface potential determination, an uncertainty about its location at the aggregate's interface as well as its distribution between the interface and the bulk aqueous phase may cause undesirable complications. The introduction of amphiphilic pH indicatorsg (i.e., an indicator molecule containing one or two long hydrocarbon chains) simplifies the situation because such an indicator is practically water insoluble and mainly solubilized in the aggregate. Fur- thermore, the amphiphilic character of the indicator en- sures well-defined orientations of the indicator molecules in the aggregate. The long chain is presumed to become aligned along the tails of the surfactant molecules with the

pK" - pK' = -FJi/(2.3RT)

Lukac

(2) W. E. Ford, J. W. Otvos, and M. Calvin, Nature (London), 274,507 (1978).

(3) M. S. Tunuli and J. H. Fendler, Adu. Chem. Ser., 177, 53 (1982). (4) (a) J. H. Fendler, J . Phys. Chem., 84, 1485 (1980); (b) S. S. Atik

and J. K. Thomas, ibid., 103, 3550 (1981). (5) (a) S. L. Regen, B. Czech, and A. Singh, J. Am. Chem. SOC., 102,

6638 (1980); (b) E. Lopez, D. F. O'Brien, and T. H. Whitesides, ibid., 104, 305 (1982); (c ) P. Tundo, D. J. Kippenberger, P. L. Klahn, N. E. Prieto, T.-C. Jao, and J. H. Fendler, ibid., 104, 456 (1982).

(E) G. S. Hartley and J. W. Roe, Trans. Faraday SOC., 36,101 (1940). (7) P. Mukerjee and K. Banerjee, J . Phys. Chem., 68, 3567 (1964). (8) M. Montal and C. Gitler, Bioenergetics, 4, 363 (1973). (9) P. Fromherz, Biochim. Biophys. Acta, 323, 326 (1973). (10) N. Funasaki, J. Colloid Interface Sci., 60, 54 (1977). (11) D. S. Cdiso and W. L. Hubbell, Annu. Rev. Biophys. Bioeng., 10,

(12) S. McLaughlin, A. Bruder, S. Chem, and C. Moser, Biochim.

(13) P. C. Hiemenz, "Principles of Colloid and Surface Chemistry",

217 (198l), and references therein.

Biophys. Acta, 394, 304 (1975).

Marcel Dekker, New York, 1977, Chapter 9, p 352.

polar, pH-sensitive chromophore located in the interface. It was previously shown14 that the intrinsic interfacial

contribution, pK', to the apparent pKa of the pH indicator located at the micelle interface consists mainly of the effect of the polarity change on the indicator's acid-base equilibrium, represented by eq 2. Such a change is the result

A + B- + H+ ( 2 4

A + + B + H+ (2b)

of the transfer of the indicator from the pure water into the aggregate's interface. From eq 2 it is obvious that, depending on the type of pH indicator, the reduction in the effective dielectric constant of the interface, relative to that of pure water, causes a shift of the acid-base equilibrium to the left (eq 2a) or to the right (eq 2b). For practical reasons it is more convenient to use eq 1 in the following form:

(3)

where the ApK" is the apparent shift of the pK of the indicator located at the interface and in pure water, i.e., ApKa = pK" - p P and the ApK' is the polarity contribution to the ApKa (ApK' = pKi - pK", the pK' is the intrinsic interfacial pK of the indicator, however, not di- rectly measurable). The value and the "sign" of the latter contribution depend on the type of pH indicator (viz., eq 2).

In the case of the amphiphilic (surfactant) pH indicator, the problem of the surface potential determination in micelles14 or vesicles is reduced to the estimation of the interface polarity contribution to the ApKa (the boundary, the dipole, and Donnan potentials are normally ignored in charged systems with high surface charge den~ities'~).

Fernandez and Fromherz previously dem~nstratedl~ two plausible ways of such a determination in micellar systems. The prerequisite of the first method is the availability of a neutral system with an interface having the same properties as the charged system in question. Then, the ApK" of the indicator in the neutral aggregates can be taken as the Ap@ for the charged interface (eq 3).

The second approach requires the determination of ApKa of two conjugate pH indicator^'^ (the acid-base equilibria of which are represented by eq 2) in the same aggregate. In such a case, the Ji of the system can be calculated from eq 4.14 The ApK'I and ApK'II are the

(4)

respective apparent shifts of the indicators I and I1 incorporated in the charged interface of the aggregate under investigation. The values of ApK.1 and ApK'II were also used to obtain the value of ApK' for the micellar interface from eq 5.14 Based upon a solvent polarity scale estab-

(5)

lished for the coumarin pH indicators, the ApK' served also as an indicator of the micelle interfacial p01arity.l~

In this work we describe the measurement of the surface potentials as well as the polarity of the interface of positively and negatively charged surfactant vesicles determined by using two surfactant coumarin pH indicators.

ApK" - ApK' = -FJi/(2.3RT)

-FJi/(2.3RT) = '/2[ApKaI + ApKaII]

IApK'I = '/2[ApKaI - ApK'II]

(14) M. S. Fernandez and P. Fromherz, J. Phys. Chem., 81, 1755 (1977).

(15) S. McLaughlin in 'Current Topics in Membrane and Transport", Vol. 9, F. Bronner and A. Kleinzeller, Eds., Academic Press, New York, 1977, p 71.

Surfactant and Phospholipid Vesicles

An attempt was also made to make use of the zwitterionic phospholipid vesicles as a neutral reference for the ionic surfactant vesicles.

Experimental Section Materials. Double-chained surfactants, dioctadecyldi-

methylammonium bromide (DODAB), didodecyldi- methylammonium bromide (DDAB), and dihexadecyl phosphate (DHP), were purchased from Eastman and ICN Pharmaceutical. The ammonium salts were recrystallized twice from acetone and DHP was crystallized from methanol. Dipalmitoyl-L-a-phosphatidylcholine (DPPC) and distearoyl-L-a-phosphatidylcholine (DSPC) were used as received from Sigma. The long-chain hydroxycoumarin and aminocoumarin (referred to as indicators I and 11,

C15H31 C17H35

The Journal of Physical Chemistry, Vol. 87, No. 24, 7983 5047

Indicator I Indicator II respectively) were generous gifts from Dr. P. Fromherz (University of Ulm, West Germany). Sephadex G-50-80 was obtained from Sigma. The buffer solutions were prepared from sodium tetraborate, sodium acetate, and potassium dihydrogen orthophosphate, all purchased from BDH Chemicals.

Sample Preparation. Surfactant as well as phospholipid vesicles were prepared by sonication. An aliquot of M solution of indicator I in methanol or that of indicator 11 in methylene chloride was added to the methylene chloride solution of surfactant. After evaporation of the solvent under N2 stream, a corresponding amount of water was added (in all cases the surfactant concentration was 5 mg/mL) and a sonication was carried out at elevated temperatures (between 50 and 75 OC) with a Sonicator 350 (Heat System Ultrasonic). The preparation of phospholipid vesicles containing the indicator was analogous to that of the surfactant vesicles except that 2.5 mg of phospholipids per milliliter of 5 X M phosphate buffer was taken into the vesicle preparation. The molar ratio of the indicator to the amphiphile was between 1:300 and 1:450.

All vesicular dispersions were passed through a Sepha- dex column (15 X 1.5 cm) and the transport of the vesicles containing the fluorescent pH indicator was followed by a UV lamp. In a typical experiment the sample was prepared by adding 0.3 mL of a vesicle solution into 2 mL of 5 x M buffer solution adjusted a t the required pH. First, the bulk pH of the sample was determined by using a combined electrode (Radiometer GK23 21C) connected to a Radiometer Model PHM 64 followed by the fluorescence intensity measurement with a Perkin-Elmer fluorescence spectrophotometer MPF-4. The acid-base titrations of the pH indicators in vesicles were performed in the 1.8-12.5 pH range.

Results and Discussion The basic forms of both coumarin indicators are fluor-

escent with the emission maxima at 453 nm when excited a t about 370 nm. Since the absorption spectrum of the acid form is blue shifted (Amm = 328 nm), the titration of the basic form can be easily followed through the decrease of the fluorescence intensity, IF Dividing IF at a given pH by the IF at the pH corresponding to the fully dissociated indicator (Le., at the highest bulk pH), one obtains the dissociation degree of the indicator, a. When the titration curves are constructed in this way, the apparent pKa is

bulk pH

Figure 1. Acid-base titrations of indicator I in DHP (D) and DDAB (A) and indicator I1 in DHP (0) vesicles. The dash curves correspond to the tltrations of the nonsurfactant coumarln indicators in water.I4 a = dissociation degree (see text).

TABLE I : Different Vesicles

pK Shifts of Surfactant pH Indicators in

~

system A P K ~ ~ Q A ~ K ~ I I ~ o , b m V

1.55 -127 DHP 2.75 DDAB (DODAB) -2.05 + 1 5 6

DPPC + DHP DPPC (DSPC) 2.75 --1.85

3 .25 --0.25 DPPC + DDAB -1 .35

a In pH units; ApKa = pKa - p K W ; I and I1 refer t o indicators I and 11, respectively. t o r I and /o r I1 in water and the values of 7.15 and 2.35, r e ~ p e c t i v e l y , ' ~ were taken for t he calculations. surface potential .

pKW is t h e pK of indica-

The

equal to the bulk pH at a = 0.5. Surfactant Vesicles. Figure 1 shows the comparison of

the titration curves of both indicators in the positively (DDAB) and negatively (DHP) charged surfactant vesicles and in water (the values of CY for the indicators in water were taken from the literature14). Because the same pK, was found for indicator I in both DDAB and DODAB vesicles, only the titration in DDAB vesicles is presented in Figure 1. Indicator I1 was incorporated only in DHP vesicles since its pK in the positively charged DDAB vesicles is shifted toward immeasurably low pH values. All vesicle samples used in the titration experiments were those passed through the Sephadex column. When indicator I or I1 was dispersed in H 2 0 and introduced on the column, the fluorescent zone representing the indicator was retained on the top of the column and no transport of the zone was visible. This observation leads to the conclusion that all our results refer to the behavior of the surfactant pH indicator located in the vesicle bilayer.

A significant pK shift of both coumarin indicators toward a higher bulk pH when located in DHP vesicles, as evident from Figure 1, signifies a considerably higher proton concentration at the vesicle interface than in the bulk aqueous phase. This result is expected for the negatively charged surface of DHP vesicles. However, the observed pK shifts are obviously asymmetric. In the case of indicator I, the ApKal is equal to 2.75 whereas that of indicator I1 is only 1.55 pH units. On the other hand, the shift of the pK of indicator I in DDAB vesicles in the opposite direction (i.e., toward the lower pH as compared with the pK in pure water) corresponds to a lower proton concentration at the positively charged interface relative to the bulk aqueous phase. Interestingly, the ApK"1 in DDAB vesicles is smaller than that in DHP vesicles (compare 2.05 with 2.75, respectively, Table I). Unless there is a significant difference in the charge densities of the negatively and positively charged vesicles, such an asymmetry in the ApK" of the same indicator in the vesicles suggests, by analogy with micellar systems,14 the presence of the interface polarity contribution to the ApKa.

5040 The Journal of Physical Chemistry, Vol. 87, NO. 24, 1983 Lukac

polarity scale.14 According to this scale the ApK' = 0.6 corresponds to the effective dielectric constant, teff, of about 45. Such a relatively high polarity is quite acceptable keeping in mind the orientation of the amphiphilic pH indicator in the bilayer, i.e., with the pH-sensitive group located at the bilayer-water interface. Interestingly, this value of teff is significantly lower than that reported for the micelles of similar surfactants.14 This may be an indication that the micelle interface has a different structure than that of vesicles.

Examination of eq 2 makes it evident that underesti- mation of the ApK' for the positively charged vesicles results in a smaller J , (with the unchanged ApK") which would be more comparable with that of DHP vesicles. However, we do not have any obvious reason to believe that the molecules of indicator I in DDAB vesicles are extended more into the water phase (a higher polarity corresponds to a smaller value of ApK') or that the DDAB vesicles' interface is more loosely organized than in the case of the DHP vesicles. The solubilization experiments with fluorescence probes" showed the same or lower effective polarity in DODAB vesicles with respect to DHP vesicles.

The experimental conditions used here for the preparation of surfactant vesicles yielded DHP vesicles of a significantly larger average size than the DODAB and/or DDAB vesicles (RH of 700 and 300 A, respectively).18 Because a change in the size of surfactant aggregates af- fects the aggregation number,16 and perhaps also the packing of the amphiphilies in the mesophase which results, in turn, in an alteration of the surface charge density of the aggregate, it is appropriate to study the effect of the surface curvature on J,. It was previously shown18 that by variation of the amount of a buffer in the DHP vesicles preparation a change in the vesicle size is achieved. The higher the buffer concentration, the smaller the DHP vesicles formed at the same DHP concentration.18 Indi- cator I was incorporated into DHP vesicles at four different buffer concentrations: 5 X lo4, 5 X and M. Regardless of the difference in the size of the resulting vesicles, the same pKaI = 10.5 was obtained in all cases from the titration experiments.

Perhaps the most reasonable rationalization of the asymmetry in J , of the positively and negatively charged surfactant vesicles can be seen in the different chemical nature of the head groups of DHP and DDAB surfactants as well in the effect of unlike counterions on the effective charge density of the interface. This view is supported by the similar difference reported for the positively (tri- methylammonium head group) and negatively charged mi~e1les.l~ In the case of micelles, the identical polarity contributions, ApK', in both micellar systems were confirmed by using both methods for the rl, cal~ulation'~ (see Introduction).

I t should be mentioned here that the relatively close values of J , in DDAB vesicles and CTAB micelles (+157 and +148 mV,14 respectively) are in agreement with the observation that the rl, is independent of the size of vesicles (see above). Note that there is nearly 1 order of magnitude difference in the size of micelles (about 100 Al9) and vesicles (about 800 A18p20g21). These results may also indicate that the surface charge densities determining the surface potential15 are similar at the micelle and vesicle interfaces.

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 bulk pH

Flgure 2. Acid-base titrations of indicator I in DPPC (O), DSPC (m), DPPC + DDAB (A), and DPPC + DHP (V) and indicator I1 in DPPC (V) and DPPC + DHP (0) vesicles. The dash curves correspond to the tiations of the nonsurfactant coumarin indicators in water. cy = dissociation degree (see text).

As mentioned in the Introduction, there are two methods for surface potential determination in molecular aggregates. So far, there has not been described in the literature a neutral surfactant vesicular system suitable for the requirements of the first method so we have tried to use synthetic phospholipid vesicles in that role.

The phospholipids taken into the experiments (DPPC and DSPC) have zwitterionic head groups and it is reasonable to assume that the positive (the choline group) and negative (the phosphate moiety) charges are mutually neutralized yielding a net neutral vesicle surface. There- fore, there should be no proton concentration gradient established between the interface and the bulk water and the pH indicator located at the interface should reveal the pK shift, if any, due only to the polarity contribution.

The titrations of the solubilized indicator I in both phospholipid vesicles showed the same and very significant pK shifts (Figure 2) very close to that found in DHP vesicles. Indicator 11, in phospholipid vesicles, however, manifested a very insignificant decrease of IF when titrated in the range of 1.8-9 pH. Thew results suggest that DPPC and DSPC vesicles cannot serve as a neutral standard system for the ionic surfactant vesicles when the amphiphilic coumarin pH indicators are used.

Therefore, the second procedure mentioned in the In- troduction ought to be used. When the pK shifts of indicators I and 11, ApKaI and ApKan, in DHP vesicles (Table I) were inserted into eq 4, the surface potential of the vesicles was found to be -127 mV.

In the case of DDAB and DODAB vesicles where the ApKan is not available, the surface potential can be estimated only when the polarity contribution, ApK' (eq 3), to the apparent ApKa is known. This can be obtained from eq 5 when the ApKf and ApKfI found in DHP vesicles are used. The value of 0.6 pH unit resulted from such a calculation. When this value, together with the ApKal in DDAB vesicles (Table I), was substituted into eq 2, the surface potential of +156 mV was obtained for the positively charged vesicles. The values of J , of both surfactant vesicles are of the same order of magnitude as expected from the model of surfactant vesicles.le However, there is an apparent asymmetry in the J , of the systems (+156 mV compared with -127 mV) which may be attributed to several reasons.

Because the Aplc used for the calculation of J , in DDAB vesicles was determined in DHP vesicles, there may be a reasonable doubt as to whether the two vesicle systems consisting of different surfactant molecules have an identical polarity in their interfaces. The polarity of the indicator's environment can be estimated from the empirical

(16) D. J. Mitchell and B. W. Ninham, J. Chem. SOC., Faraday Trans. 2, 77, 601 (1981).

(17) S Lukac, submitted for publication. (18) S. Lukac, S. Desgreniers, and D. Landheer, in preparation. (19) K. Shinoda, T. Nakagawa, B. Tamamushi, and T. Isemura,

"Colloidal Surfactants", Academic Press, New York, 1963. (20) U. Hermann and J. H. Fendler, Chem. Phys. Lett., 64,270 (1979). (21) M.-P. Pileni, Chem. Phys. Lett., 71, 317 (1980).

Surfactant and Phospholipid Vesicles

TABLE 11: Effect o f Salt (NaCl) o n Surface Potential of DHP Vesiclesa

The Journal of Physlcal Chemlstty, Vol. 87, No. 24, 1983 5049

concentration available with DHP vesicles (2 or 3 orders of magnitude are typically explored in similar experi- m e n t ~ ' ~ ~ ~ ~ ~ ~ ~ ) . Thus, the interface of surfactant vesicles is adequately represented by the Gouy-Chapman diffused double layer model.

Phospholipid Vesicles. The significant opposite shifts in pK of indicators I and I1 (Figure 2) solubilized in the same system (DPPC) can be accounted for either by the dominant polarity contribution to the shifts or by the association of each indicator with an opposite charge of high density. The electroneutrality, Le., zero surface potential, of the vesicles formed from the phospholipids possessing the zwitterionic head group (DPPC) confirmed by { potential mea~urements, '~~~' would favor the former explanation. The effect of opposite charges on pK of the indicators might be, however, achieved by assuming a different location of each indicator in the bilayer interface, i.e., indicator I in the vicinity of the negatively charged phosphate moiety and indicator I1 close to the positively charged choline group.

The polarity contribution can be determined by using eq 5 when both ApKaI and ApKaII are available for the system. When the value of ApK'II estimated from Figure 2 to be about 0.5 is taken into the calculation, eq 5 yields ApK' = 2.3 and a rather small negative potential of -26 mV is obtained from eq 3 or 4. These results seem to be reasonable in view of the demonstrated identical locationB of both indicators in the vicinity of the phosphate moiety. Of course, such an observation rules out any strong association of the indicators with opposite charges.

A rather high value of the polarity contribution (ApK') indicates in turn a low polarity of the indicator sites in the bilayer (e - 15). This seems to contradict the interfacial location of the indicators. Recently, we have that the polarity of the phospholipid bilayers (e - 25) is significantly lower than that of surfactant vesicles ( e - 40). A plausible explanation for the lower polarity value found in this work may be seen in an inaccuracy of the estimation of ApKaII. A greater error in the titration process is expected in the region of very low pH than at higher pH values. Interestingly, the polarity of e - 25 found by a fluorescence probe in DPPC17 is very close to the polarity estimated from ApK' of indicator I in neutral phosphati- dylglycerol vesicles.28

Any significant contribution to the pK shifts from the dipole potential possibly established in the zwitterionic phospholipid vesicles like DPPCZ9 can be rejected on the basis of the dominant opposite pKa shifts. The dipole membrane potential should affect pKs of both indicators only in one direction on the bulk pH scale.

Interestingly, results very similar to those presented in this work have been published for indicator I in the DPPC monolayersg together with a strong effect of salts on the ApKa. In view of the good observed correlation between the surface potentials of monolayers and the corresponding bilayers30 the above similarity and the salt effect suggest that there is a real surface potential manifestation present when indicator I is incorporated into the DPPC bilayer. We believe that the negative potential revealed by the lipoid pH indicators is of a local character suggesting a nonuniform proton distribution in the DPPC interface. This is caused probably by the presence of positive and

~

1OZ[NaC1], M A p K a l ~ $ , mV 1.0 2.70 -124 .0 2.5 2.35 -103 .0 5.0 2.05 -85.6 7.5 1.75 -67.9

a The slope d$ /d log [NaCl] is 6 2 mV.

It is now worth comparing our results with those obtained with nonsurfactant pH indicators. Using bromo- thymol blue8 and pyranineZ2 as pH indicators we have previously determined considerably lower values of J , in the same vesicles, Le., DHP and DODAB (-36 and +64 mV, respec t i~e ly) .~~ Such a discrepancy may be rationalized through the pH effect on the binding properties of the indicators. Both indicators are in the anionic forms at high pH and in the neutral ones at the pH below their pK. Therefore, the adsorption of the indicators a t the charged interface varies with pH and, consequently, in- fluences the distribution of the indicators between the bulk aqueous phase and the vesicle interface in the course of the experiment. This should obviously have a great effect on the surface potential determination, especially in the case of the negatively charged DHP vesicles. Another problem encountered with the nonsurfactant pH indicators, and also possibly contributing to the observed discrepancy in J,, is the ambiguity of their location at the interface. The different pK shifts were found when bro- mothymol blue was present during sonication of DODAB vesicles.23 The use of the surfactant pH indicators obviously avoids such problems.

Salt Effect. The procedure of the surface potential determination presented above is based upon the Gouy- Chapman theory of the diffuse double layer.13 Regardless of the fact that this theory, originally derived for flat surfaces, was shown several times14p24-26 to describe satis- factorily the situation on the curved charged surfaces as well, it is appropriate to test separately this assumption for any new system under the study. A typical test of the validity of the theory is the effect of a salt (a symmetrical monovalent ele~trolyte'~) on the surface potential. Ex- perimental results obtained from such a study are of significant importance for most catalytic applications of surfactant vesicles including solar energy con~ersion.~ The experiment was carried out with a typical sample of DHP vesicle with solubilized indicator I. The salt (NaC1) concentration was varied from 5 x to 7.5 x M. A t a higher salt concentration the screening of the surface charge lead to destabilization of the vesicles and consequently resulted in their coagulation. A measurable change in J , was found only above M concentration of NaC1. The surface potentials of DHP vesicle determined at several salt concentrations are summarized in Table 11. The slope dJ,/d log [NaCl] was obtained by the least- squares treatment of the data in Table 11. The resulting value of 62 mV is in very reasonable agreement with 59 mV predicted by the Gouy-Chapman theory15 especially keeping in mind the intrinsic experimental error of the pH indicator method and a relatively small range of the salt

(22) K. Kano and J. H. Fendler, Biochim. Biophys. Acta, 509, 289

(23) S. Lukac and J. H. Fendler, unpublished results. (24) S. G. A. McLaughlin, G. Szabo, G. Eisenman, and S. M. Ciani,

(25) P. Fromherz and B. Masters, Biochim. Biophys. Acto, 356, 270

(26) M. S. Fernandez, Biochin. Biophys. Acta, 646, 23 (1981).

(1978).

h o c . Natl. Acad. Sci. U.S.A., 67, 1268 (1970).

(1974).

(27) D. A. Haydon and V. B. Myers, Biochim. Biophys. Acta, 307,429

(28) A. Haase, Ph.D. Thesis, University of Giessen, 1980. (29) S. B. Hladky and D. A. Haydon, Biochim. Biophys. Acta, 318,464

(30) R. C. MacDonald and A. D. Bangham, J. Membr. B i d , 7, 29

(1973).

(1973).

(1972).

5050 The Journal of Physical Chemistry, Vol. 87, No. 24, 1983

negative charges of the zwitterionic head groups and their significant f le~ib i l i ty .~~ The above-mentioned salt effect could be accounted for by, e.g., a specific binding of the counterions to the phosphate moiety. In this context, it should be noted that nonsurfactant pH indicators recorded practically zero surface potential in similar systems.8,22

Preliminary experiments with the mixed vesicles (1:l mole ratio of DPPC and DDAB or DHP), the results of which aree also shown in Figure 2, may suggest that the incorporation of the surfactant molecules into the phospholipid bilayer establishes a net surface charge of a con- siderable density but also simultaneously modifies the polarity of the bilayer periphery.

Conclusion Surfactant pH indicators were shown to be reliable

probes for the determination of the surface potential of surfactant vesicles. The unambiguous location of the indicator a t the vesicle interface allows us to estimate the

Additions and Corrections

effective polarity of the interface. A significant difference in the effective dielectric constant of the vesicle interface as compared with that of micelles14 together with very similar values of $ for both aggregate systems suggests that the micellar interfaces are quite different from those of vesicles.

The zwitterionic phospholipid vesicles “failed” to serve as a neutral standard system for the charged surfactant vesicles when the surfactant pH indicators were used. This was rationalized through a significantly lower polarity of the phospholipid bilayer than that of surfactant vesicles together with a specific location of the indicators in the former bilayers. Such a phenomenon is likely to be ena- bled by a significant flexibility of the phospholipid head groups.

Acknowledgment. I thank one of the referees for drawing ref 28 to my attention. Registry No. Dihexadecyl phosphate, 2197-63-9; didodecyl-

dimethylammonium bromide, 3282-73-3; dioctyldecyldimethyl- ammonium bromide, 3700-67-2; dipalmitoyl L-a-phosphatidylcholine, 63-89-8; distearoyl L-a-phosphatidylcholine, 816-94-4. (31) P. L. Yeagle, Acc. Chem. Res., 11, 321 (1978).

ADD IT IONS AND CORRECTIONS

1983, Volume 87

J. M. Bennett, C. S. Blackwell, and D. E. Cox*: High-Resolution Silicon-29 Nuclear Magnetic Resonance and Neutron Powder Diffraction Study of Na-A Zeolite. Lowenstein’s Rule Vindicated.

Page 3785. The caption for Figure 2 should be corrected to read as follows:

Theoretical curves for Si(A1) distribution with observed points superimposed as follows: (0) Si(OAl), (0) Si(lAl), (0) Si(SAl), (X) Si(3A1), (+) Si(4A1). The observed points are based on the Si:Al ratios calculated from the %i NMR data. The numbers to the right refer to the theoretical number of Si-0-A1 linkages for a calculated curve.

Table I1 presents the data in numerical form and is correct as printed.

surface potential at surfactant and phospholipid vesicles as determined by amphiphilic ph indicators

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