Langmuir 1987, 3, 855-857 off from the monolayer to give a discrete ribbon or platelet resting on but independent of the underlying monolayer. Undoubtedly, in some cases the folding and bending structure may simply move to a horizontal position and the double-layer ribbon may remain attached to the monolayer as in the bending state. However, as indicated in Figure 1A and lB, the double-layer fold in rigid type films may break its attachment to the monolayer and deposit an independent fragment (ribbon or platelet) on the monolayer. Support for this breaking off is now provided by electron microscopy. A representative electron micrograph of a collapsing monolayer of cerebronic acid is shown in Figure 2A. The micrograph provides remarkable support for the tall folds or thin-ridge stage.13 Figure 2B is representative of a great many collapsed monolayers in which long narrow platelets or ribbons have been observed. Such ribbons are generally two molecules thick as indicated in the schematic drawings of Figure 1. Whether or not one edge of the ribbon remains attached to the monolayer is difficult to determine in such cases, as the attachment could be in the form of a sharp
855
bend that would produce a somewhat ambiguous micrograph. Striking evidence, however, for the separation of the collapsed ribbons from the monolayer is presented in the micrographs of Figure 3. Twisting of the ribbons with subsequent flattening is dramatically shown in Figure 3A and at a higher magnification in Figure 3B. Clearly the ribbons must be free of the underlying monolayer in order to exhibit such twisted fragments. Thus for films of high rigidity (low compressibility) that collapse abruptly after reaching their maximum pressures, such detachment of the collapsed ribbons or platelets can certainly take place.
Acknowledgment. We are indebted to Professor P. G. deGennes of the College de France for stimulating comments. We thank Dr. Christopher Chou for valuable technical assistance. This work was supported in part by a grant from the United States Public Health Services (Ca-14599). Registry No. Cerebronic acid, 544-57-0; cholesterol, 57-88-5; isoleucine-gramicidin B, 6311-07-1.
Reanalysis of the Acid-Base Dissociation Behavior of Dimyristoyldansylcephalin in Dimyristoylmethylphosphatidic Acid Membranes Calum J. Drummond* and Franz Grieser Department of Physical Chemistry, The University of Melbourne, Parkville, Victoria, 3052 Australia Received March 30, 1987 The work of Vaz et al. (Yaz,W. L. C.; Nicksch, A,; Jahnig, F. Eur. J. Biochem. 1978,83,299)concerning the acid-base dissociation behavior of dimyristoyldansylcephalin in dimyristoylmethylphosphatidicacid (DMPA) membranes has been reanalyzed. The reanalysis has established that the acid-base behavior of dimyristoyldansylcephalin in DMPA membranes is entirely consistent with the prototropic moiety of the dansyl chromophore residing, on average, in the plane of the charged head groups of the DMPA membranes and thus responding to induced changes in the electrostatic surface potential of the membranes. 1. Introduction It is generally considered that the apparent pKa, pK,"b"d, of a prototropic moiety residing at or in the vicinity of a charged interface can be separated into two components, i.e.
where pK,O is the intrinsic interfacial pKa of the prototropic moiety, e is the elementary electrostatic charge, \k is the mean field potential at the average site of residence for the prototropic moiety, k is the Boltzmann constant, and T i s the absolute temperature. For lipoidal acid-base indicators, it is usually assumed that it is the surface potential which affects the pK,"b*l values.'-14 In other words, (1)Fromherz, P.Biochim. Biophys. Acta 1973,323,326. . 1974,356,270. (2)Fromherz. P.:Masters. B. Biochim. B i o ~ h v sActa (3) Fernandez, M. S.; Fromherz, P. J. Ph$s.-Chem. 1977,81, 1755. (4)Frahm, J.; Diekmann, S.; Haase, A. Ber. Bunsenges. Phys. Chem. 1980,84,566. ( 5 ) Fromherz, P.; Arden, W. J. Am. Chem. SOC.1980,102, 6211. (6)Fromherz, P.In Electron Microscopy at Molecular Dimensions; Baumeister, W., Vogell, W., Eds.; Springer: Berlin, 1980;p 338. (7)Lukac, S. J.Phys. Chem. 1983,87,5045. (8)Kramer, R.Biochim. Biophys. Acta 1983,735,145.
0743-7463/81/2403-0855$01.50/0
it is usually assumed that the prototropic moiety of a lipoidal indicator resides, on average, in the plane of charge. Trauble et al.I5 have shown that the Gouy-Chapman theory can be employed to obtain the surface potential of dimyristoylmethylphosphatidic acid (DMPA) membranes in aqueous NaCl solution. Vaz et a1.16 have investigated the acid-base dissociation behavior of the lipoidal indicator dimyristoyldansylcephalin in DMPA membranes. In particular, Vaz et al. determined the apparent pKa value of dimyristoyldansylcephalin, or more precisely the pH at (9)Fromherz, P.;Kotulla, R. Ber. Bunsenges. Phys. Chem. 1984,88, 1106. (10)Lovelock, B.;Grieser, F.; Healy, T. W. J. Phys. Chem. 1985,89, 501. (11)Lovelock, B.; Grieser, F.; Healy, T. W. Langmuir 1986,2, 443. (12)Drummond, C. J.; Grieser, F.; Healy, T. W. Faraday Discuss. Chem. SOC.1986,81,95. (13)Drummond, C.J.; Grieser, F. Photochem. Photobiol. 1987,45,19. (14)Hartland, G. V.; Grieser, F.; White, L. R. J . Chem. SOC.,Faraday Trans. 1 1987,83,591. (15)Trauble, H.; Teubner, M.; Woolley, P.; Eibl, H. Biophys. Chem. 1976,4,319. (16)V u , W. L. C.; Nicksch, A.; Jahnig, F. Eur. J. Biochem. 1978,83, 299.
0 1987 American Chemical Society
Letters
856 Langmuir, Vol. 3, No. 5, 1987 R-gH2
membranes. The second part uses this information to reanalyze the pH-titration data of Vaz et a1.16 for dimyristoyldansylcephalin in DMPA membranes.
!
R-CH-CH2-O-P-OH I
0-CH3
R-CH2
l?
R- CH-CH2-0- P-0O-CH3
H3C\
H
AcC H3
@
R-CHZ
I 0II R-CH-CH2-O-P-O-CH2-CH2-NH
so2
I
0-
ib)
H3C\N/CH3 +HA I H +
R-CH2 I R-CH-CH2-0-
B
2. Results and Discussion 2.1. Ionization Behavior of DMPA Membranes. The acid-base equilibrium for DMPA is shown in Figure 1. The degree of ionization of DMPA membranes was shown by Trauble et al.15 to be a function of the pH and electrolyte concentration of the surrounding aqueous solution. Freeze fracture experiments demonstrated that the lipid lamellae in dispersions of DMPA do not form closed vesicles but instead are organized into stacks of lamellae. Therefore, as indicated by Trauble et al., the majority of the ionizable polar head groups should be readily accessible to externally added electrolyte, and the Gouy-Chapman theory may be able to describe the experimentally observed ionization behavior of DMPA membranes. The ionization process for self-assembled DMPA molecules can be represented as (Figure l)
Q$ so2
P-O-CHz-CH2-NH
0Figure 1. Acid-base equilibria for (a) dimyristoylmethylphosphatidic acid (DMPA) and (b) dimyristoyldansylcephalin. R denotes CI3H.&O2.
which 50% of the dansyl chromophores were protonated, in several DMPA membrane solutions which differed by their concentration of added NaC1. Vaz et al. quantitatively interpreted their results by utilizing the GuoyChapman theory and concluded that the prototropic moiety of dimyristoyldansylcephalin was, on average, located out from the plane of the charged DMPA head groups in the diffuse part of the electrical double layer. Since this finding of Vaz et al. is at odds with the usual assumption that the potential experienced by the interfacially located prototropic moieties of lipoidal acid-base indicators is the surface potential,'-'* we were prompted to reexamine the pH-titration data of Vaz et al. for dimyristoyldansylcephalin in DMPA membranes. The reexamination was also motivated by the fact that dimyristoyldansylcephalin and closely related homologues and analogues are increasingly being employed to probe the physico-chemical properties of model lipid memb r a n e ~ . " - ~Therefore ~ it is imperative that the time-averaged position of the "reporter" dansyl chromophore in model lipid membranes, such as those formed by DMPA, is correctly established. The reexamination of the data of Vaz et al. revealed that an erroneous assumption of complete ionization of the DMPA membranes resulted in the incorrect assignment of the average position of the prototropic moiety of the dansyl probe in the DMPA membranes. The details which led us to this conclusion are discussed in the present report in two parts. The first part presents the findings of Trauble et al.15 on the ionization behavior of DMPA (17) Waggoner, A. S.; Stryer, L. Proc. Natl. Acad. Sci. U.S.A. 1970, 67, 579. (18)Faucon, J. F.; Lussan, C. Biochim. Biophys. Acta 1973,307,459. (19) Lussan, C.; Faucon, J. F. Biochim. Biophys. Acta 1974,345, 83. (20) Teissie, J. Biochim. Biophys. Acta 1979, 555, 553. (21) Teissie, J. Chem. Phys. Lipids 1979, 25, 357. (22) Teissie, J. Biochemistry 1981, 20, 1554. (23) Ghiggino, K. P.; Lee, A. G.; Meech, S. R.; OConnor, D. V.; Phillips, D. Biochemistry 1981, 20, 5381. (24) Iwamoto, K.; Sumamoto, J. Bull. Chem. SOC. Jpn. 1981,54,399. (25) Kimura, Y.; Ikegami, A. J.Membr. Biol. 1985, 85, 225.
where H$ and 4-denote the unionized and ionized forms of the self-assembled DMPA molecules and HE+denotes a free proton at the membrane surface. The degree of ionization of the DMPA membranes is defined as (3)
where square brackets denote concentrations. The degree of ionization can also be given by15 CY=
K,O(DMPA) K,O(DMPA) + [H+]b exp(eqo/kT)
, .
(4)
where K,O (DMPA) is the equilibrium ionization constant for process 2, [H+Ibis the proton concentration in the bulk aqueous solution, and qois the surface potential of the DMPA membranes. In the Gouy-Chapman theory the relationship between the surface potential and the surface charge density, go, of a planar charged surface in a supporting uni-univalent electrolyte solution is given by 'ko=
~
2kT argsinh e
[
]
(8no~tokT)~/~
(5)
where no is the number concentration of ions in the bulk solution, e is the dielectric constant of the solution, and to is the permittivity of a vacuum. In the DMPA systems studied by Trauble et al. the high potential approximation (sinh x E 'I2 exp x ) can be employed for the GouyChapman equation so that 2kT e
\ko = -In
[
2g0
]
(8~1ottokT)~/~
(6)
The surface charge density can be expressed in terms of the molecular area of the lipid head group, Ao, which in the case of the DMPA molecules is reasonably approximated by 50 A2, and the electrostatic charge per head group, ea, i.e. ea 'To = (7) A0
Substituting the relationships given in eq 6 and 7 into eq 4 one obtains eq 8 for the experimental conditions employed by Trauble et al. and also by Vaz et al. (vide
Langmuir 1987,3,857-858 Table I. Reanalysis of the pH-Titration Data of Vaz et a1.I6 for Dimyristoyldansylcephalin in DMPA Membranes at 20 O C [NaCII, mol dm" 5 x 10-4
pK.OM(dansyl) 6.10 5.15 3.86 3.45
5 x 10-3 7 x 10-2 5 x 10-1
' a 0.555 0.570 0.527 0.662
pKaO(dansyl)
W0,mV -247 -191 -120 -82
1.85 1.87 1.80 2.04
Oa is defined as the degree of ionization of the DMPA membranes.
infra). In eq 8, c denotes the concentration of added uni-univalent electrolyte. a =
K,O(DMPA) K,O(DMPA) + 29.15[H ' ]
/
ba2 C
(8)
Trauble et al. demonstrated that eq 8 was able to provide a very good quantitative description of the experimentally observed ionization behavior of DMPA membranes in aqueous NaCl solution when the pK,O(DMPA) value was set equal to 1.75. 2.2. Acid-Base Dissociation Behavior of Dimyristoyldansylcephalinin DMPA Membranes. The acid-base equilibrium for dimyristoyldansylcephalin is depicted in Figure 1. As mentioned in the introduction, Vaz et al.16 investigated the acid-base dissociation behavior of dimyristoyldansylcephalin in DMPA membranes. The details of their analysis will not be restated herein because there is a major flaw incorporated into their treatment of the data. In analyzing the pH-titration data for dimyristoyldansylcephalin in DMPA membranes, Vaz et al. erroneously assumed that under their experimental conditions the DMPA membranes were fully ionized (i.e., a = 1). Table I contains the actual values for the degree of ionization of the DMPA membranes in each system investigated by Vaz et al. at 20 "C when the bulk pH is the same as the apparent pK, value found for dimyristoyldansylcephalin, pKaobsd(dansyl),in the system. The pK,OM(dansyl) value in each DMPA/NaCl system was ascertained from Figure 4 in ref 16. The a values were calculated by using eq 8 with pK,O(DMPA) set equal to 1.75. The \ko values of the charged DMPA membranes, which are given in Table I, were obtained by substituting the a values into eq 4. The pK:(dansyl) values in the various DMPA/NaCl systems were then determined with
857
the aid of eq 1, the calculated \ko values, and the pK,"bBd(dansyl)values. The concordancy of the pK,O(dansyl)values determined by the reanalysis of the pH-titration data obtained by Vaz et al. clearly indicates that the acid-base dissociation behavior of the dansyl chromophore in the DMPA membranes can be rationalized if the prototropic moiety of the dansyl chromophore (i) resides, on average, in the plane of the charged head groups, thereby experiencing the surface potential of the DMPA membranes, and (ii) has an intrinsic interfacial pK,O value of 1.9 f 0.1. Fernandez and Fromherz3 have developed a procedure whereby reference pK,O values can be obtained from the pK, values of indicators in 1,4-dioxane/water mixtures. This procedure assumes that (i) a l,Cdioxane/water mixture can mimic the mean solvent properties of a particular interfacial microenvironment, (ii) there is no specific molecular interaction between the acid-base indicators and the lipid head groups, and (iii) the "salt-effect" on the intrinsic acid-base equilibria of interfacially located indicators is negligibly small. The pK, value of the dansyl chromophore in a 45% l,&dioxane/water mixture with no added electrolyte and a dielectric constant of 40 was found by Vaz et al. to be 2.5. If one employs the procedure of Fernandez and Fromherz? a reference pK,O(dansyl)value of 2.0 is obtained for this l,Cdioxane/water mixture. Thus, the pK,O(dansyl) values of Table I are consistent with the interfacial region of the DMPA membranes possessing an effective dielectric constant, teff, of ca. 40. The results of many previous determinations of the effective dielectric constants of lipid/ water interfaces3J2@p2' suggest that this is a reasonable interfacial eeff value. In conclusion,we reiterate that the self-consistentresults of the reanalysis indicate that the prototropic moiety of dimyristoyldansylcephalinresides, on average, in the plane of the charged DMPA head groups.
Acknowledgment. This work was supported by grants from the Australian Research Grants Scheme. C.J.D. is the recipient of a Commonwealth Postgraduate Research Award. We gratefully acknowledge Prof. Thomas W. Healy for his suggestions regarding this work. Registry No. DMPA, 54672-40-1; dimyristoyldansylcephalin, 66091-40-5. (26) Zachariasse, K. A.; Van Phuc, N.; Kozankiewicz, B. J. Phys. Chem. 1981,85, 2676. (27) Law, K. Y. Photochem. Photobiol. 1981, 33, 799.
An Unusual Gel without a Gelling Agent V. Bergeron and F. Sebba* Department of Chemical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received February 24, 1987. In Final Form: June 11, 1987 Gels have been produced by using kerosine and water but no gelling agent, the stability being created by the foam matrix characteristic of polyaphrons. The aphrons are submicron in sue, a necessary condition for gel formation. The generally accepted model for a gel is that it is composed of two interlocking phases, one of which is made up of solids such as polymer chains or bentonite, the other being a continuous aqueous phase. This paper is to report that we have succeeded in making gels of remarkable 0743-746318712403-0857$01.50/0
stability out of two very mobile liquids, kerosene and water. The basic structure is that of a polyaphron, i.e., a biliquid foam. Biliquid foams are akin to foams except that, instead of having gas in the bubbles that comprise the foam, there is a nonpolar liquid, itself encapsulated in a soap fii. 0 1987 American Chemical Society
