4777
J. Phys. Chem. 1991, 95,4171-4782 in Figure 4b, and values for v4 of 646.3 cm-I, 605.2 cm-’, and 627.9 cm-’ were determined for HCOBr, DCOBr, and H W O B r , respectively, from the frequency of the central Q branch maximum. C-Br Bending ( u s ) . By analogy with CH3COBr,’* the C-Br bending band of HCOBr is expected near 300 cm-I, outside the range of these measurements. For DCOBr, us can be estimated from the difference between u4 (605.2 cm-I) and (v4 + us) (963 cm-I, observed in Fermi resonance with v 3 ) . This difference is equal to us x4,$,where is an anharmonic coupling constant Therefore the difference between and is small compared to (v4 + vs) and v4 gives an approximate value for us of 358 cm-I. Out-ofPlane Bending ( v 6 ) . By analogy with HCOCI,’O the out-of-plane bending vibrations of HCOBr and HI3COBr are expected near 932 cm-’ with very weak intensity. Bands were observed with Q branch maxima a t 893.4 cm-’ and 88 1.6 cm-’ for HCOBr and H”COBr, respectively, while for DCOBr the frequency of this band was shifted down to 746.8 cm-I. As expected for an out of plane (A”) vibration, the band contours were typical of a type C band, as shown for HI3COBr in Figure 4c. Structure of HCOBr. From the rotational contours of the five fundamental bands of HCOBr observed in this study, it can be concluded that the vibrations vl-v4 are in plane (A’), while v6 is out of plane (A”). Although vs was not observed, it can be concluded that it is an in-plane vibration because the combination
+
+
level (v, us) must have overall A’ symmetry in order to be in Fermi resonance with v3 in DCOBr. Therefore, the six fundamentals are divided into five in-plane vibrations and one outof-plane vibration, consistent with the structure of HCOBr being planar.’s 4. Conclusions From an FTIR analysis of the products of the visible photolysis of Br2-HCHCbN2 mixtures, it has been shown that the only major product channel of the reaction of HCO radical with Br2 is that leading to HCOBr Br. It was found that HCOBr is highly susceptible to heterogeneous decomposition to HBr CO, and an upper limit was determined for the rate constant of the corresponding homogeneous reaction of k4 = 0.0025 &. An upper limit has been placed on the rate constant for the reaction of Br cm3molecule-’ s-I. Infrared atoms with HCOBr of kS = 4 X spectra of the fundamental bands vI-v4 and v6 of HCOBr, DCOBr, and H13COBr have been observed and are consistent with the structure of the molecule being planar.
+
+
Acknowledgment. The authors thank Mr. L. P.Breitenbach and Mr. C. M. Savage (Ford Motor Co.) and Dr. M. Green, Mr. N. Peng, and Ms. M. I. Freedhoff (York University) for their assistance during the experimental stages of this work. Financial support for this work was provided by NSERC and AES.
Direct Osmotic Stress Measurements of Hydration and Electrostatic Double-Layer Forces between Bilayers of Double-Chained Ammonium Acetate Surfactants V. A. Parsegian, National Institutes of Health, Bethesda, Maryland 20892
R. P. Rand,* and N. L. Fuller Biological Sciences, Brock University, St. Catharines, Ontario L2S 3A1, Canada (Received: May 24, 1990)
Using the osmotic stress method, we have directly measured forces between bilayers of the long-chain substituted amine dihexadecyldimethylammonium acetate in 5-500 mM acetate solutions. Including this molecule with such a small polar group, every lipid system yet investigated shows hydration repulsion at short range. For these frozen hydrocarbon chain bilayers brought together in solutions of low salt concentration down to separations of 15-17 A, the repulsive force is well described by electrostatic double-layer interactions. Because the layers are stiff, there is no amplifying action of undulatory thermal fluctuations. Below 15-17-A separations, there is a clear break and a transition to a region with an exponentially varying force with a 2.7-3.3-A decay constant. No oscillatory forces are seen. That these shorter range interactions were not seen in previous measurements with lipids adsorbed onto the crossed cylindrical mica sheets of a “surface force apparatus” Brady, J.; Evans, D. F. J. Phys. Chem. 1986,90, 1637) can be explained (Pashley, R. M.; McGuiggan, P. M.; Ninham, B. W.; by the opposite bilayer curvatures enforced in that system and the stress limitations caused by mica bending. Force measurements between oppositely curved surfaces, even of dimensions of centimeters in radius, systematicallysmother short-range and emphasize long-range forces. Therefore critical examination of forces between contact and 20-A separations should, where possible, be made between parallel rather than oppositely curved surfaces.
Introduction Ever since the delineation of hydration forces between phospholipid there has been an open question as to whether these interactions, which appear to dominate all other forces at separations less than 20-30 A, will be seen in other systems. Indeed, solvation forces had been seen between charged soap films4 (1) LeNeveu, D. M.; Rand, R. P.; Parsegian, V. A. Nature 1976, 259, 601-603. (2) LeNeveu, D. M.; Rand, R. P.; Parsegian, V. A.; Gingell, D. Biophys. J . 1977. 18. 209-230. (3) Parse&m V . A.; Fuller, N. L.; Rand, R. P. Proc. Nail. Acad. Sci. W.S.A. 1979. 76, 2750-2754. (4) Clunic, J. S.; Goodman, J. F.; Symons, P. C. Nature 1967, 216, 1 203- 1 204.
0022-3654/91/2095-4777$02.50/0
and were reported for several nonbiological materials;$ exponentially decaying hydration forces were later measured between DNA double helices: neutral and charged polysaccharides? some mica surfaces in solutions of adsorbing cations,8v9clay particles,IO (5) Churaev, N. V.; Deryagin, B. V. J . Colloid Interface Sci. 1985, 103, 542-553. (6) Rau, D. C.; Lee, B. K.; Parscgian, V. A. Proc. Nail. Acad. Sei. W.S.A. 1984.81, 2621-2625. (7) Rau, D. C.;Parsegian, V. A. Biophys. J . 1987, 51, 503a; Science, in press. ( 8 ) Pashlcy, R. M.; Israelachvili. J. N . J . Colloid Interface Sci. 1981,80, 153-162. (9) Pashley, R. M. J . Colloid Interface Sci. 1983, 83. 531-546. (IO) Viani, B. E.; Low, P. F.; Roth, C. B. J . Colloid Interface Sei. 1984, 96, 229-244.
0 1991 American Chemical Society
4778 The Journal of Physical Chemistry, Vol. 95, NO. 12, 1991
and many neutral and charged lipid bilayers."-" One puzzling exception to this general behavior is the force reported to have been measured between bilayers of dihexadecyldimethylammonium acetate (DHDAA) that had been immobilized onto crossed mica cylinders.2I In that case, forces down to a separation of 5 A (where surface force apparatus measurements ceased)were explained in terms of electrostatic double-layer interactions. We show here that this conclusion is incorrect. We have used the osmotic stress (OS)technique22to measure forces between DHDAA bilayers directly in the multilayer arrays that can form spontaneously in acetate solutions.23 Between bilayers in solutions of low ionic strength, electrostatic double-layer interactions are seen down to a separation of about 15 A. At separations less than this, we measure forces quite similar to those observed between bilayers of neutral phospholipids in distilled water, i.e. forces reminiscent of hydration forces. Reconciliation of the results here and those in the mica system is easily established once one recognizes that imposing opposite curvatures on the interacting surfaces by mica cylinders so weights the contribution of the longer range electrostatic double-layer forces that the hydration force contribution is largely submerged. Materials and Methods The DHDAA salt, a generous gift from D. F. Evans, was prepared as described earlier.2'J3 Dihexadecyldimethylammonium hydroxide (from the bromide salt (Fisher) by ion exchange on Rexyn 201 (Fisher)) was mixed with a stoichiometric amount of acetic acid. The precipitated acid was filtered, washed, recrystallized, and dried. Sodium acetate (NaOAc) was of analytical reagent grade (BDH Analar). The water in all solutions was doubly distilled. Flame tests showed less than 1% residual bromide after this exchange. Since we worked with effectively infiniteexcess acetate solution, there was no need to worry about significant action of bromide. All dimensions of the lamellar lattice structure were determined by X-ray diffraction. To determine the relation between repeat spacing, bilayer thickness, and bilayer separation, we measured the lamellar repeat spacing in stoichiometric mixtures of amphiphile and 0.3 mM NaOAc in 2 mM TES buffer solution, pH 7.3. The repeat spacings, d, so determined were divided into two layers, the bilayer of thickness d, and the separation between bilayers d,. The value of each was computed from the volume fraction, 4, of the lipid by
d, = 9d
d, = (1 - f#J)d
The volume fraction 4 was computed from the stoichiometrically set weight fraction by using specific volumes of 1.O cm3/g for aqueous solutions, 1.OS cm3/g for frozen hydrocarbon chains,12 and 1.47 cm3/g for dimethylamines, and 0.953 cm3/g for acetic acid,24which must be included with each lipid. (1 1) Rand, R. P. Annu. Rev. Biophys. Bioeng. 1981, I O , 277-3 14. (12) Rand, R. P.; Parsegian, V. A. Biochim. Biophys. Acta 1989, 988, 351-376. (13) Lis, L. J.; McAlister, M.; Fuller, N . L.; Rand, R. P.; Parscgian, V. A. Biophys. J . 1982, 37, 657-666. (14) Lis, L. J.; Lis, W. T.;Parsegian, V. A,; Rand, R. P. Biochemistry 1981,20, 1771-1777. (15) Marra, J.; Israelachvili, J. N. Biochemistry 1985, 24, 4608-4618. (16) McIntosh, T. J.; Simon, S.A. Biochcmisfry 1986, 25, 4058-4066. (17) McIntosh, T. J.; Magid, A. D.; Simon, S.A. Biochemistry 1987, 26, 7235-7332. (18) McIntosh, T.J.; Simon, S.A. Biochemistry 1986, 25, 4948-4952. (19) Cowley, A. C.; Fuller, N . L.; Rand, R. P.; Parsegian, V. A. Biochemistry 1978, 17, 3163-3168. (20) Loosley-Millman, M. E.; Rand, R. P.; Parsegian, V. A. Biophys. J . 1982, 40, 221-232. (21) Pashley, R. M.; McGuiggan, P. M.; Ninham, B.W.; Brady, J.; Evans, D. F. J . Phys. Chrm. 1986,90, 1637-1642. (22) Parsegian, V. A.; Rand, R. P.; Rau, D. C. Methods Enzymol. 1986, 127, 400-416. (23) Brad, J . E.; Evans, D. F.; Kachar, B.;Ninham, B. W. J . Am. SOC. 1984, 106,4278. (24) Handbook of Chemistry and Physics; CRC Press: Boca Raton, FL, 1967; Vol. 47, p 47.
Parsegian et al. 200
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Figure 1. Calibration for lipid layer thickness dl (squares) and separation d, (circles) as a function of weight fraction of lipid. Repeat spacings for the lamellar lattice (triangles) are also shown. Note the virtually constant lipid thickness and a separation that varies with repeat spacing. These measurements were made in 10 m M NaOAc solution with 2 mM TES buffer, but the structural parameters are insensitive to these ionic conditions.
The projected area per molecule, A, parallel to the bilayer plane was computed from Ad, = 2v, where VI is the volume of the lipid molecule. The DHDAA multilayer system was particularly well-behaved. Accuracy in measurement of repeat spacings was typically quite high, fO. 1 A. Reproducibility of measurements, including sample-to-sample variation evident in the data, was within 1 A at lower water contents and about 2% at very high water contents. Forces between bilayers in the multilayer array were directly measured through the application of osmotic stress to the multilamellar lattice as described e l s e ~ h e r e . The ~ - ~ multilayers ~ were equilibrated against polyethylene glycol (PEG) or dextran solutions of known osmotic pressure or against vapors of saturated salt solutions. PEG (Sigma Chemicals) was of M W 15 000-20000; it was dialysed extensively against distilled water to remove any residual salts. Dextran T2000 (Pharmacia) of mean M W 2000000 was used as purchased. Polymer solutions of the desired concentration were made up from dextran or PEG in NaOAc solutions. The relevant osmotic pressure was the part due to the addition of polymer that was not able to exchange with the lamellar lattice, while the exchangeable NaOAc solution acted as a reservoir of fixed activity. Osmotic pressures were measured on a membrane osmometer.22 The resulting repeat spacing of DHDAA at each PEG or dextran osmotic pressure was determined by X-ray diffraction; the corresponding bilayer separation was taken from the earlier stoichiometric calibration. Experiments were conducted at room temperature, between 20 and 25 O C . The melting temperature of DHDAA is greater than 25 OC (but less than 40 "C).Unless otherwise stated, solutions were buffered to pH 7.3 in 2 mM TES solution. The omission of this TES buffer did not cause detectable changes in the measured forces. Bathing solutions were in sufficient excess to ensure equilibration of lamellar phases with reservoirs of well-defined salt and water activity. To apply very high osmotic pressures (greater than 100 atm), we exposed samples to vapors of known relative humidity, also as described in detail earlier.3-'2*22These measurements were effectively in salt-free solutions. To ensure fair comparison with earlier studies,2' the same nonlinear Poisson-Boltzmann double-layer equation used there was used here. Actual computations were performed by the courtesy of Dr. Roger Horn. The program is in fact written for the plane parallel geometry of the multilayers observed here rather than the crossed cylinder configuration of the surface force apparatus (SFA). (25) Simon, S. A.; McIntosh, T.J. Biochim. Biophys. Acta 1984, 773,
169-1 72.
The Journal of Physical Chemistry, Vol. 95, No. 12, I991 4779
Double-Chained Ammonium Acetate Surfactants
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Figure 2. Repulsive pressure vs separation of DHDAA layers in 5, 10, 15, and 500 mM NaOAc solutions as well as of DHDAA exposed to
vapors of controlled humidity (symbol V). Forces are sensitive to ionic strength log P < 7 (pressures < l o atm) and separations >-17 A. In this long-distance regime, the exponential decay rate is close to that predicted from electrostatic double-layer theory.
Results Graoimetric Mixtures. The plot of lamellar repeat spacing d, bilayer thickness 4, and separation d, in Figure 1 shows that there is an 1 S-A systematic change in bilayer thickness measured over all water contents. The average measured thickness, 25 A, is reasonably close to the 27 f 2 A thickness estimated by Pashley et a1.21 The data of Figure 1 allow one to relate repeat spacings measured under osmotic stress to bilayer separations. The structure of the interacting bilayers is well-defined in this method. On the one hand the actual 25-A bilayer thickness is itself of some interest. The sharp wide-angle X-ray line from the hydrocarbon chains shows that they are frozen and perpendicular to the bilayer surface. Its value of 4.12 A is characteristic of chains packed into a hexagonal net and yields a chain area of 20.3 A2. On the other hand, from the X-ray data of Figure 1 , and the known densities and the formula described under Materials and Methods, the area A per molecule at separations in the electrostatic region is 80.2 AZ.Together, these measurements give a value of 4 chains per molecule, showing their complete interdigitation. Curiously, the observation that effectively the same bilayer thickness is obtained by SFA measurements of two DHDAA monolayers independently stuck onto mica sheets suggest that each half of an interdigitated bilayer can be independently structured. In any case, such frozen biiayers are likeiy to be stiff against bending and, we expect, unlikely to show undulatory steric forces of the type suggested for meltedchain bilayers.26J’ The molecular cross section is far greater than the cross section of the methylated amine polar group, so a significant fraction of the lipid-water interface will necessarily be occupied by the nonpolar hydrocarbon groups. Osmotic Stress Measurements. In Figure 2 we have plotted the repulsive pressure P between DHDAA bilayers. Data here are from five sources, four sets of measurements from polymer osmotic stress in 5, 10, 15, and 500 mM NaOAc (in 2 mM TES) solutions and a set of points at high stress from exposure of DHDAA to vapors of saturated salt solutions. Forces measured at separations >- 17 A all show approximately exponential decays. For the 5, 10, 15, and 500 mM solutions, the best-fit exponential decay rates are 48.4, 37.4, 29.3, and 3.7 A, respectively. These are close to the theoretical 42.4-, 30.3-, 24.7-, and 4.2-A decay lengths calculated from DebytHuckel (DH) theory; lines fitted with the theoretical DH length also give a visually good fit to the
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(26) Helfrich, W. Z . Narurforsch. 1978, 33A, 305-315. (27) Evans, E.; Panegian, V. A. Proc. Natl. Acad. Sci. U.S.A. 1986,83, 7 132-7 136. (28) Ninham, B. W.; Parsegian, V. A. J . Theor. Biol. 1971,31,405-428. (29) Ninham, B. W.; Evans, D. F. Foraday Discuss. Chem. Soc. 1986, No. 81, 1-17. (30) Israelachvili, J.; Wennerstrom, H. Longmuir 1990, 6, 873-876. (31) de Gennes, P. G.; Pincus, P. C. R. Acad. Sci., Ser.2 1990,310,697. (32) Deryagin, B. V. Kolloid-Z. 1934, 69, 155.
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separation (Angstroms) Figure 3. Ma nified view of the region where all curves converge to a common -3- exponentially decaying force for all ionic conditions, from the salt-free vapor measurements (V) to those in 0.5 M solution. Such
1
insensitivity to ionic strength is taken as evidence for the action of nonelectrostatic double-layer forces.
data. We defer further consideration of electrostatic double-layer theory to the Discussion. The 15 and 500 mM solution data show limited swelling, suggesting the onset of attractive forces. This limited swelling is consistent with what is seen in other charged multilayer systems in solutions of sufficiently high ionic strength (e.g. 20). The data at short range are shown in greatly magnified detail in Figure 3. It shows that forces measured at separations
