J. Phys. Chem. 1988, 92, 4416-4420
4416
The X-ray scattering data indicate, as in the other systems, that the middle-phase microemulsions are bicontinuous, but improvement of the theories is needed to fully interpret the data.
Acknowledgment. We are indebted to Dr. T. Zemb for helpful discussions. Dr. B. P. Binks would like to thank the Royal Society
for the award of a Postdoctoral Fellowship. We have benefited from partial financial support from CNRS-PIRSEM (Greco Microemulsions) and CEE (contract EN3C-0018F). Registry No. DTAB, 1119-94-4; CTAB, 57-09-0; NaC1, 7647-14-5; NaBr, 7647-15-6; toluene, 108-88-3; butanol, 71-36-3; dodecane, 11240-3.
pH-Dependent Fusion of Didodecyl Phosphate Vesicles. Role of Hydrogen-Bond Formation and Membrane Fluidity Leo A. M. Rupert,? Jan F. L. van Breemen,t Dick Hoekstra,*gs and Jan B. F. N. Engberts*,+ Departments of Organic Chemistry and Biochemistry, University of Groningen, Nijenborgh 16, 9747 AG Groningen, The Netherlands, and Department of Physiological Chemistry, University of Groningen, Bloemsingel 10, 9712 K Z Groningen, The Netherlands (Received: August 4 , 1987; In Final Form: January 8, 1988)
The Caz+-inducedfusion activity of didodecyl phosphate (DDP) vesicles, both in terms of the initial fusion rate and the extent of fusion, has been studied as a function of pH. Using a resonance energy transfer (RET) fusion assay, it was found that fusion is minimal around the effective pK, (5.2) of the phosphate head groups. Fluorescence polarization studies indicate that this low fusion activity is caused by a diminished membrane fluidity. The latter, in turn, presumably arises from the formation of hydrogen bonds between head groups as indicated by 31PNMR spectroscopy. Below pH 5.2, the head groups cluster and, concomitantly, the hydrocarbon chains bulge, leading to an increased membrane fluidity and an enhanced fusion activity. At low pH the bilayer seems to possess an organization intermediate between a lamellar and a hexagonal HI[ phase, as supported by 31PNMR spectroscopic and electron microscopic evidence. Cryoelectron microscopic measurements revealed that after Ca2+-induced fusion at low pH, two types of tubular structures are formed which both possess a hexagonal HII packing of DDP molecules. One type of hexagonally arranged cylinders consists of DDP molecules with almost completely extended hydrocarbon chains and contains a high concentration of Ca2+. In the other type, the hydrocarbon chains are melted and the concentration of Ca2+is low. The shape concept provides a rationale for the interpretation of the present results.
Introduction Recent years have witnessed a wedding between physical chemistry and biochemistry. The abundant offspring includes membrane mimetic chemistry' as a rapidly expanding and versatile area of research. It is highlighted by the successful application of a large variety of model systems to investigate various aspects related to the chemistry of complex cell membranes. The fact that the structure of simple, synthetic amphiphiles can be readily varied in a controllable manner has greatly contributed to the usefulness of membrane mimetic systems composed of these In particular, emphasis has been placed on studies of relations between amphiphile structure and aggregate structure,14 dynamical behavior of amphiphiled and their aggregates,e6 and stability of bilayer membra ne^.^^^,' Generally, the main driving force for aggregation is provided by hydrophobic interactions3g but the stability of the aggregates is governed by a delicate balance of attractive and repulsive forces.3g The latter involve electrostatic repulsions between head groups as well as unfavorable overlap of hydration shells of the head groups. Changes in aggregate morphology are often analyzed in terms of the shape concepts which considers conical, truncated conical, and cylindrical shapes of the amphiphiles as governing factors in the formation of micelles, inverted phases, and bilayers, respectively. Recently we have shown that a change in head-group structure from R2N+Me2Br-to (R0)2P02-Na+ ( R = n-CI2H2,) has farreaching consequences for such bila er properties as molecular packing and membrane ~tability.~,"*~ These factors were probed in terms of fusogenic activity of didodecyldimethylammonium
Y
* Authors to whom correspondence should be addressed. 'Department of Organic Chemistry. I Department of Biochemistry. Department of Physiological Chemistry.
bromide (DDAB) and didodecyl phosphate (DDP) vesicles. Herein we describe the changes in DDP bilayer properties that result from a reduction of electrostatic head-group interactions by protonation of the phosphate moiety. It is shown that the head-group mobility and the bilayer fluidity are strongly affected by protonation. These results are related to the dramatic changes (1) Fendler, J. H. Membrane Mimetic Chemistry; Wiley: New York, 1982. (2) (a) Fuhrhop, J.-H.; Mathieu, J. Angew. Chem. 1984, 96, 124. (b) Paleos, C. M. Chem. SOC.Rev. 1985, 14, 45.
(3) (a) Kunitake, T.; Okahata, Y.; Tamaki, K.; Kumamamu, F.; Takayanagi, M. Chem. Lett. 1977, 387. (b) Kunitake, T.; Okahata, Y. Bull. Chem. Soc. Jpn. 1978,51, 1877. (c) Sudhiilter, E.J.R.; Engberts, J.B.F.N.; Hoekstra, D. J. Am. Chem. SOC.1980, 102, 2467. (d) Kunitake, T.; Okahata, Y.; Shimomura, M.; Yasunami, S.-i.; Takarabe, J. Am. Chem. SOC.1981, 103, 5401. (e) Hundscheid, F.J.A.; Engberts, J.B.F.N. J. Org. Chem. 1984, 49, 3088. (f) Yamada, K.; Ihara, H.; Ide, T.; Fukumoto, T.; Hirayama, C. Chem. Lett. 1984, 1713. (g) Evans, D. F.; Ninham, B. W. J. Phys. Chem. 1986,90, 226. (h) Murakami, Y.; Kikuchi, J.-i.; Takaki, T.; Uchimura, K. J. Am. Chem. SOC.1985, 107, 3373. (i) Miller, D. D.; Evans, D. F.; Warr, G. G.; Bellare, J. R.; Ninham, B. W. J. Colloid Interface Sci. 1987, 116, 598. (4) Rupert, L.A.M.; van Breemen, J.F.L.; van Bruggen, E.F.J.; Engberts, J.B.F.N.; Hoekstra, D. J. Membr. Biol. 1987, 95, 255. ( 5 ) Rupert, L.A.M. J. Colloid Interface Sei., in press. (6) (a) Brady, J. E.; Evans, D. F.; Kachar, B.; Ninham, B. W. J. Am. Chem. SOC.1984,106,4279. (b) Murakami, Y.; Nakano, A,; Yoshimatsu, A,; Uchitomi, K.; Matsuda, Y. J. Am. Chem. SOC.1984, 106, 3613. (c) Rupert, L.A.M.; Hoekstra, D.; Engberts, J.B.F.N. J. Am. Chem. SOC.1985, 107,2628. (d) Murakami, Y.; Kikuchi, J.4; Takaki, T.; Uchimura, K. Chem. Lett. 1986, 325. (e) Rupert, L.A.M.; Engberts, J.B.F.N.; Hoekstra, D. J. Am. Chem. SOC.1986, 108, 3920. (f) Rupert, L.A.M.; Hoekstra, D.; Engberts, J.B.F.N. J. Colloid Interface Sci. 1987, 120, 125. (g) Siegel, D. P. Biophys. J. 1986, 49, 1155. (h) Siegel, D. P. Biophys. J . 1986, 49, 1171. (7) (a) Carmona-Ribeiro, A. M.; Yoshida, L. S.; Chaimovich, H. J. Phys. Chem. 1985,89, 2928. (b) Carmona-Ribeiro, A. M.; Chaimovich, H. Biophys. J. 1986, 50,621. (c) Johnson, N. W.; Kaler, E. W. J. Colloid Interface Sci. 1987, 116, 4440. (8) Israelachvili, J. M.; Marcelja, A. S.; Horn, R. C. Q. Reu. Biophys. 1980, 13, 121.
0022-3654/88/2092-4416$01.50/0 0 1988 American Chemical Society
The Journal of Physical Chemistry, Vol. 92, No. 15, 1988 4411
Fusion of Didodecyl Phosphate Vesicles in fusion activity of DDP vesicles observed upon alteration of the PH.
Experimental Section Materials. Didodecyl phosphate (mp 59.1-60.2 "C, lite958-59 "C) was obtained from Alpha Chemicals and was used as supplied. N-(7-Nitrobenz-2-oxa- 1,3-diazol-4-yl)phosphatidylethanolamine (N-NBD-PE) and N-(lissamine Rhodamine B sulfony1)phosphatidylethanolamine (N-Rh-PE) were purchased from Avanti Polar Lipids Inc. Diphenylhexatriene (DPH) was obtained from Aldrich, 4-( 2-hydroxyethy1)- 1-piperazineethanesulfonicacid (HEPES) was from Sigma, and calcium and magnesium chloride were from Merck. Vesicle Preparation. Sodium didodecyl phosphate (DDP) vesicles were prepared by the ethanol injection procedure as reported previously.& For the 31PN M R line-shape measurements, a lamellar dispersion of DDP was prepared by vortexing a film of DDP in 5 mM HEPES/5 mM sodium acetate buffer (pH 7.4, and containing 20% (v/v) D 2 0 ) above the phase transition temperature. The film was obtained by dissolving DDP (90 mg) in chloroform and subsequently evaporating the solvent in a stream of nitrogen. The film was vacuum-dried overnight. DPH-labeled vesicles were prepared by adding small aliquots of the DPH stock solution in tetrahydrofuran (THF) to the vesicle solution, followed by equilibration for 1 h at 50 OC. The DPH to DDP ratio was 1:lOOO. The effect of T H F (0.05%) on the vesicles can be neglected.1° Fusion Measurements. DDP vesicle fusion was monitored continuously by using a resonance energy transfer (RET) fusion assay."" Thus, vesicles containing 0.8 mol % each of N-NBD-PE and N-Rh-PE were prepared by solubilizing appropriate amounts of DDP and the fluorophores in ethanol/NaOH. Fusion measurements were performed in HEPES/sodium acetate buffers adjusted to the desired pH by adding aliquots of a 1 M HC1 stock solution. Equimolar amounts of labeled and nonlabeled DDP vesicles were employed. The pH of the vesicle solutions was controlled with a Corning 130 pH instrument. The total amphiphile concentration was 59 pM. After equilibrating the vesicle suspensions at 40 OC, fusion was initiated by injecting a CaC12 solution into the suspension. Then, NBD fluorescence (Aex = 475 nm, &, = 530 nm) was monitored continuously by using a Perkin Elmer MPF 43 spectrofluorometer equipped with a thermostated cell holder and a magnetic stirring device. The fluorescence scale was calibrated such that the residual NBD fluorescence of the vesicles is taken as the zero level and the value obtained after the addition of cetyltrimethylammonium bromide (final concentration 1% (w/v) and corrected for sample dilution as 100% (infinite dilution). Fluorescence Polarization Measurements. These measurements were carried out in the spectrofluorometer equipped with a polarization accessory. DPH was excited at 360 nm whereas the emitted light was measured at 428 nm. The degree of fluorescence polarization ( P ) was calculated according to eq 1 in which IIIand I , are the fluorescence intensities detected with the polarizers parallel and perpendicular, respectively, to the direction of the polarization of the excitation light.12 The value of I , was corrected for the intrinsic polarization of the i n s t r ~ m e n t . ' ~ p = (11,- I d / ( I , , + 1.L)
(1)
31PN M R Measurements. 'H-decoupled 31PN M R measurements were performed with a 10-mm tube at 81 MHz on a Nicolet NT200 instrument equipped with a temperature controller and a deuterium lock. Chemical shifts (ppm) are relative to the external reference of hexachlorocyclotriphosphazene in CDC13 (+19.9 ppm downfield from 85% H3P0,). Accumulated free (9) Czarniecki, M. F.; Breslow, R. J . A m . Chem. SOC.1979, 101, 3675. (10) Lentz, B. R.; Barenholz, Y.; Thompson, T. E. Biochemistry 1976, IS, 4521. (1 1) Struck, D. K.; Hoekstra, D.; Pagano, R. E. Biochemistry 1981, 20, 4093. (12) Shinitzky, M.; Barenholz, Y . J. Biol. Chem. 1974, 249, 2652. (13) Chen, R. F.; Bowman, R. L.Science 1965, 147, 729.
3.0
I
- 60
4
Initial
I
I
p
fusion I
rate (
01.
I I
lsec)
(0-0)
2.0 .
1.0
Extent of
.
fusion ( '1.
I
ID--O)
i
2o
0 2
3
L
5
6
7
0
PH
Figure 1. Effect of pH on the initial fusion rate (0)and the extent of fusion (0)of DDP vesicles. After equilibration a t the appropriate pH, fusion was induced by injecting Ca2+into the medium. The incubation temperature was 40 "C, the ratio of labeled to nonlabeled vesicles 1:1, total [DDP]= 58 X 10" M, and [Ca*+] = 3.85 X M.
induction decays were obtained from 400 to 10000 transients (see legends) with an interpulse time of 2.3 s and a pulse time of 19 ps, which corresponds to a pulse angle of ca. 90". The vesicle solutions contained 20 vol % of D 2 0 to lock the signal. Electron Microscopy. Aliquots of DDP vesicle dispersions before or after the pH change or the addition of a CaCl, solution were stained with a 1% (w/v) solution of uranyl acetate as described previously.& The carbon-coated Formvar grids were pretreated by glow discharge in air. The samples were examined in a JEM 1200 EX electron microscope, operating at 80 kV. The cry0 EM experiments were carried out as described previ0us1y.l~ Briefly, a film of the vesicle suspensions, placed on carbon-coated grids, was quenched frozen in liquid ethane. The latter was used to avoid vapor film formation around the specimen during freezing. The grid was transferred to liquid nitrogen, mounted in a Philips cooling holder (PW 6591), and rapidly introduced into a Philips E M 400 electron microscope. The temperature was kept below -140 OC to avoid crystallization of the vitrified buffer.
Results and Discussion The effect of the pH on the initial fusion rate (% s-l) and on the extent of fusion (%) is portrayed in Figure 1. To analyze the origin of these peculiar correlations, the influence of pH on the physical properties of the hydrocarbon region of the vesicles as well as on the water-bilayer interface was further examined since it is known that the fusion process is strongly affected by changes in the bilayer f l ~ i d i t y . ~ ~First * ~ Jthe ~ fluorescence polarization ( P ) of diphenylhexatriene (DPH)-labeled vesicles was measured as a function of the pH (Figure 2). The magnitude of P increases when lowering the pH from 7.4 to 5.2, but decreases abruptly below pH 5.2 until around pH 3 a bilayer fluidity is reached which is even higher than that at pH 7.4. These pHinduced alterations in membrane fluidity are reversible since readjustment of the pH to 7.4 restores the original polarization. The curve in Figure 2 is reminiscent of the relation between the phase transition temperature and the pH as reported for 1,2dimyristoylalkylphosphatidic acids in which the alkyl group is methyl, ethyl, or n-propyl.16 It was found that the phase transition (14) Rupert, L.A.M.; Engberts, J.B.F.N.; Hoekstra, D.; van Breemen,
J.F.L.;van Bruggen, E.F.J., manuscript in preparation. (15) Wilschut, J.; Duzgunes, N.; Hoekstra, D.; Papahadjopoulos, D. Biochemistry 1985, 24, 8 . (16) (a) Eibl, H.; Woolley, P. Biophys. Chem. 1979, 10, 261. (b) Eibl, H. Angew. Chem. 1984, 96, 247.
4418 The Journal of Physical Chemistry, Vol. 92, No. 15, 1988
Rupert et al. 09
0.25
I
A
P
&,Ip
0E
'El I
( P P ~ )
(%I
Dl \
IO--O I
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os 0.15
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80
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/ /
03
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0.10
0
0 L
3
0.05 2
3
I
I
6
5
I
1
I
6
7
8
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Figure 2. pH dependence of the membrane fluidity of D D P vesicles as reflected by the D P H fluorescence polarization P. Conditions: [DDP] = 58 X IO" M, [DPH] = 5 X M, 40 "C.
5
8
7
6
PH
Figure 3. pH dependence of the relative intensity (Ircl) and the upfield shift of the chemical shift (A631p)of DDP vesicles. The effect of pH on the upfield shift of the isotropic signal of multilamellar DDP vesicles (A)is also indicated. The measurements were performed a! 40 OC using DDP concentrations of 5.8 X lo-) and 15 X M for the vesicles (400 transients) and multilamellar vesicles (10.000 transients), respectively.
temperature is at a maximum at the effective pKa of the phosphate moieties.16 The effective pKa of dihexadecyl phosphate (DHP)," a longer chain analogue of DDP, is ca. 5.2 and we assume that this value also holds for DDP. Thus we suggest that the maximum in fluorescence polarization (Figure 2 ) corresponds with the effective pKa of the bilayer-forming amphiphile. In addition, it is most likely that the changes in fluorescence polarization with pH are caused by changes in the phase transition temperature, the increase being indicative of a stabilization of the gel state, very similar as reported for phospholipids. The observed changes in membrane fluidity, as reflected by the pH dependence of P, are consistent with results obtained from leakage experiments. It has been shown that the permeability of methylviologen across a D H P bilayer is minimal around pH 5.8, which corresponds closely with the pKa of the head group.18 Taking into account that leakage is related to membrane fluidity,Ig it is likely that the D H P bilayer possesses also a reduced fluidity around the effective pK, of its phosphate group. An increased permeability below pH 4 has been reported for nylon capsules coated with DDP bilayers.*O This pH range coincides with the high fluidity region in Figure 2. On the basis of this increased permeability, the erroneous conclusion has been drawnZothat the pKa of the DDP head group is 3.2. It will be shown below that, at pH values corresponding to the pKa, the bilayer is in a highly ordered state rather than in a disordered one. It has been suggested that the shift in the phase transition temperature with decreasing p H is caused by formation of hydrogen bonds of the type (RO)2P(0)O--.HO(O)P(OR)2 following (partial) protonation of the head groups.I6 This suggestion accounts for the observation that the structuring of the bilayer is maximal at the effective pKa of t h e h e a d groups, since a t these conditions the number of these hydrogen-bond interactions will be maximal. This theory provides a rationale for the results shown
in Figure 2 as well as for those obtained for phospholipid systems.I6 However, we emphasize that nonionized dialkyl phosphates are excellent hydrogen-bond acceptors themselves.2ia22 In bilayer vesicles, the phosphoric acid head group is not only hydrogen bonded to water but also faces adjacent head groups suitably arranged for the formation of hydrogen bonds.23 Since the latter hydrogen-bonding interactions occur between preoriented components, the entropy loss involved in these interactions will be minimized. Therefore, we submit that effective structuring of the vesicle surface is also expected below the effective pKa of the DDP head groups. This structuring may be enforced by the polarizing effect of PO, groups on the hydrogen-bond network at the surface area of the bilayer.% Further evidence for head-group structuring at low pH is provided by the 31P N M R spectral data given in Figure 3. The line broadening at lower pH values indicates an increased contribution of the chemical shift anisotropy to the line width, presumably as a result of a reduced head-group mobility.@ The signal completely vanishes in the noise below pH 4.5 and, still more importantly, it does not reappear at further lowering
(17) Tricot, Y.-M.; Furlong, D. N.; Sasse, W.H.F.; Daivis, P.; Snook, I. Ausr. J . Chem. 1983, 36, 609. (18) Tricot, Y.-M.; Furlong, D. N.; Sasse, W.H.F.; Daivis, P.; Snook, I.; van Megen, W. J . Colloid Interface Sci. 1984, 97, 380. (19) (a) Kano, K.; Romero, A,; Djermouni, B.; Ache, H. J.; Fendler, J. H. J . Am. Chem. SOC.1979, 101, 4030. (b) Rossignol, M.; Uso, T.; Thomas, P. J . Membr. Biol. 1985, 87, 269. (20) (a) Okahata, Y.; Seki, T. J . Am. Chem. SOC.1984, 106, 8065. (b) Okahata, Y. Ace. Chem. Res. 1986, 19, 57. The amphiphiles in these bilayer-corked capsule membranes are arranged in well-oriented,multilamellar bilayers, which behave similarly to multilamellar vesicles in terms of phase transitions and permeability to enclosed solutes.
(21) (a) Peppard, D. F.; Ferraro, J. R.; Mason, G. W. J . Inorg. Nucl. Chem. 1958, 7, 231. (b) Ferraro, J. R.; Mason, G. W.; Peppard, D. F. J . Inorg. Nucl. Chem. 1961, 22,285. (c) Ferraro, J. R.: Peppard, D. F. J . Phys. Chem. 1963, 67, 2639. (22) Murphy, A.S.M.; Rao, C.N.R. Appl. Spectrosc. Reu. 1968, 2, 69. (23) An analysis of the line shape of the 'P NMR signal of multilamellar DDP vesiclesSrevealed that the conformation of the head groups is favorable for hydrogen-bond interactions of this type. (24) The effective pK, of the head groups in DDP vesicles is ca. 4 pK, units higher than that of low molecular weight counterparts of DDP, as a result of the negative surface charge of the vesicles. However, the surface potential will decrease upon protonation, and PO4- groups will still remain present at rather low pH values.
I
I
~ K ~ 2 5 5 . 2
I R
I R
Figure 4. Schematic representation of the effect of pH on DDP bilayer packing.
The Journal of Physical Chemistry, Vol. 92, No. 15, 1988 4419
Fusion of Didodecyl Phosphate Vesicles
30
20
I
,
IO
0
,
-10
,
-20
,
-30 ppm
I
!
5.1
-
I
45
15
10
5
0
truncated conical. The thus perturbed bilayer structure will, in fact, be a precursor for a hexagonal HII phase. In such an HII phase the amphiphiles are arranged such that their head groups surround an aqueous channel while the hydrocarbon chains extend radially from this channel. This notion is supported by 31PN M R data and by electron microscopy on multilamellar DDP dispersions. At 50 “ C the 31PN M R spectrum shows two signals: (i) a narrow, isotropic signal a t 0.5 ppm and (ii) a broad, asymmetric signal between ca. 14 and -8 ppm (Figure 5 ) . On the basis of its characteristic temperature dependence, the isotropic signal has been assigned to an “isotropic phase”.I4 Corresponding isotropic signals have been reported for lipidic systems and probably originate from intermediate stages during a lamellar to hexagonal HIIphase tran~ition.~’,~~ The membrane structural changes have been interpreted in various ways, Le., in terms of a honeycomb model or by assuming rapid reorientation (on the NMR time scale) of the lipid via lipidic particles, e.g., inverted micelle^.^^^^^^ The broad signal should be assigned to DDP molecules arranged in multilamellar vesicles (diameter ca. 5000 A).30 Lowering of the pH results in an increase of the isotropic signal3I and a decrease of the broad signal (Figure 5 ) , indicating a more pronounced bias for the “isotropic phase”. These findings are in accord with the electron microscopic observation of the presence of tubular structures in a multilamellar DDP dispersion at pH 3.5 but not at pH 7.4. These tubular structures are characteristic for a ~ context, we note that phosphatidic hexagonal HIIp h a ~ e . ~InJ this acid and phosphatidylserine also adopt a hexagonal HIIphase upon p r o t o n a t i o ~ i ,while ~ ~ the La HII transition temperature for didodecylphosphatidylethanolamine is pH-dependent.28d We now return to the pH effects on the fusion process as shown in Figure 1. It is evident that the decrease in fusion activity on lowering the pH from 7.4 to 5.2 parallels the decrease in membrane fluidity (Figure 2). As discussed previously,6flowering of the membrane fluidity hampers the reorientation of hydrocarbon chains necessary for formation of the fusion ir~termediate~,~~ which is thought to resemble an unstable, inverted micellar intermediate (IMI). Below pH 5.2, the membrane fluidity increases again but now the increase of fusion activity is clearly retarded, most likely because protonation of the head groups decreases the number of binding sites for Ca2+. Below pH 4, this effect is presumably overshadowed by the effect of high membrane fluidity which speeds fusion. The steep increase in initial fusion rate is clearly accompanied by an increase in the extent of fusion relative to the measurements at pH 7.4. Two factors may be responsible for these observations. First, clustering of the head groups below pH 5.2 results in the formation of precursors for nonbilayer structures (vide supra) which presumably resemble the unstable inverted micellar structure which has been proposed to act as an intermediate in the fusion p r o ~ e s s . ~ - ~The ‘ - ~ second effect may well involve increased binding of Ca2+ by DDP vesicles in the aggregated state, as proposed by Ekerdt and Papahadjopo~los~~ for negatively charged phospholipid vesicles. Protonation of DDP
5
IO
IS ppm
Figure 5. ,IP NMR spectra (50 “ C ) of multilamellar DDP dispersions ([DDP] = 15 X M) a t different pH values.
of the pH. Thus, at those low pH values the head-group regions remain strongly structured, presumably because of the hydrogen-bond interactions between head groups. These interactions will repel water from the surface area, consistent with the observed upfield shift of the 31PN M R signal (Figure 3).6f The structuring process is fast, since under all experimental conditions the upfield shift upon adding acid was already complete in the minimal time needed for the N M R experiment (3 min).25 Interestingly, the changes in the head-group region as indicated by the 31PN M R data (Figure 3) occur already between pH 7.0 and 7.4 whereas changes in the mobility of the hydrocarbon chains, as monitored by fluorescence polarization measurements (Figure 2 ) , take place only below pH 7.0. Therefore, the latter alterations are most likely the result of changes in the head-group region. If we combine the results of fluorescence polarization and 31P N M R measurements for the pH region below 5.2, the following picture emerges. The head groups are clustered, presumably by hydrogen bonding. Since the phosphate head-group area is smaller than the cross section of the hydrocarbon chains,“ clustering of the head groups forces the hydrocarbon chains to bulge with a concomitant increase of chain mobility (Figure 4).26 In addition, the shape of the amphiphile tends to change from cylindrical to ~~~
(25) The fact that no distinct spectra were observed for the phosphate head groups in the inner and outer leaflets of the vesicle bilayers indicates a fast transport of Ht across the bilayer. Similar results have been obtained for bilayers of dioctadecyldimethylammonium chloride by using fluorescent probes: Tran, C. D.; Klahn, P. L.; Ramero, A.; Fendler, J. H. J . Am. Chem. SOC.1978, 100, 1622. (26) The resulting defects in the bilayer packing are, presumably, responsible for the increased leakage of a fluorescent probe over the DDP b i l a ~ e r . ’In ~ addition, a similar or anization of clustered lipidic head groups has recently been reported for a C>+-dioleoylphosphatidate complex: Smaal, E. B.; Nicolay, K.; Mandersloot, J. G.; De Gier, J.; De Kruijff, B. Biochim. Biophys. Acta 1987, 897, 453.
(27) Brown, P. M.; Steers, J.; Hui, S. W.; Yeagle, P. L.; Silvius, J. R. Biochemistry 1986, 25, 4259. (28) (a) Ellens, H.; Bentz, J.; Szoka, F. Biochemisrry 1986,25, 4141. (b) Gagne, J.; Stamatatos, L.; Diavoco, T.; Hui, S. W.; Yeagle, P. L.; Silvius, J. R. Biochemistry 1985, 24,4400. (c) Tilcock, C.P.S.; Cullis, P. R.; Gruner, S. M. Chem. Phys. Lipids 1987,40,47. (d) Seddon, J. M.; Cevc, G.; Marsh, D. Biochemistry 1983, 22, 1280. (29) Cullis, P. R.; de Kruijff, B.; Hope, M. J.; Nagar, R.; Schmid, S. L. Can. J . Biochem. 1986.58, 1091. (30) As indicated by electron microscopy. (31) (a) The fact that the upfield shift of the isotropic signal closely follows the upfield shift assigned to the 900-.& vesicles indicates that both membranes respond in a similar manner to a change in pH (Figure 3). (b) The isotropic pattern cannot be explained by spontaneous vesiculation as judged by electron microscopic measurements; the size distribution of the MLV preparation was not affected. (32) (a) Hope, M. J.; Cullis, P. R. Biochem. Biophys. Res. Commun. 1980, 92, 846. (b) Farren, S. B.; Hope, M. J.; Cullis, P. R. Biochem. Biophys. Res. Commun. 1983, 111, 675. (c) Cullis, P. R.; de Kruijff, B.; Hope, M. J.; Verkleij, A. J.; Nagar, R.; Farren, S. B.; Tilcock, C.; Madden, T. D.; Bally, M. B. In Membrane Fluidity in Biology; Aloia, R. C., Ed.; Academic: New York, 1983; Vol. 1. (33) Ekerdt, R.; Papahadjopoulos, D. Proc. Natl. Acad. Sci. USA 1982, 79, 2273.
4420 The Journal ojPhysica1 Chemistry. Vol. 92, No. I S . 1988
Rupert et al.
Figure 6. Electron micrographs of negatively stained DDP vaicla at pH 3.7 (a) and of tubular structures farmed after addition of Ca” (b). Cryoelectron microscopy indicates the hexagonal character of the tubes having diameters of 23 A (c) and 17 A (d) of the hexagonally arranged cylinders.
head g r o u p will reduce interbilayer elmrostatic repulsions and, therefore, promote aggregation. Concomitantly, the availability of Ca2+ for the formation of interbilayer “trans” complexes necessary for fusion will he enhanced. However, below pH 3.6 the number of binding sites for Calf apparently reaches a threshold value, and a further decrease of the pH precludes Cazc-mediated fusion. The above conclusions, which hinge on results obtained by the RET assay, are supported by electron microscopic observations. Figure 6a shows electron micrographs of negatively stained DDP vesicles (diameter ca. 900 A) at pH 3.7. These micrographs are similar to those of DDP vesicles a t pH 7.4.4,6rThe insensitiveness of the overall vesicle structure to this change in pH agrees with a similar observation for vesicles formed from dihexadecyl phosphate based upon light-scattering measurements.18 When Ca’+ is added to DDP vesicles a t pH 3.7, long tubular structures are observed which originate from large, fused vesicles (Figure 6b)?.14 The mean length of these tubes (ca. 1 pm) is shorter than that of the tubes formed a t pH 7.4 (several micrometers), and the outer shape is less sharply defined. In addition, a significant number of the tubes is curved, which was only observed for tubes a t pH 7.4 when the Ca’+ concentration was close to the fusion threshold concentration. Cryoelectron microscopic studies revealed a further interesting difference between the tubes formed a t pH 7.4 and 3.7. At pH 7.4, dark striations on the tubes indicate that the DDP molecules are organized in hexagonally arranged cylinders,“ in which the phosphate head groups surround a narrow aqueous channel containing Ca*+ ions.’J4 Whereas at pH 7.4 only a repeat distance of 23 A (4and its associated distance of 40 A ( 3 W J is ~bserved’~ (Figure 6c), the cryomicrographs of the tubes a t pH 3.7 exhibit an additional smaller repeat distance of 17 A (plus 29 A. which corresponds to 3 % f ) (Figure 6d). These repeat distances correspond with the center to center distance in the two-dimensional projection of two hexagonally arranged cylinders. Therefore, the tubes at pH 3.7 also contain cylinders with a smaller radius of 17 A. It is unlikely that this decrease in radius is caused by a reduction of the radius of the aqueous channel. Since Calf effectively dehydrates the phosphate head groups,’6f the channel in the hexagonal cylinder contains only a small amount of water. Thus,the decrease in cylinder diameter from 46 to 34 8, is difficult to reconcile solely with an increased dehydration of the head group hy protonation compared to that caused by Caz+ions. We assume, therefore, that the reduction in cylinder radius originates from a decrease in the distance spanned by the hydrocarbon chains.
As argued previously. these chains in the cylinders formed at pH 7.4 are (almost) completclv cxtcndcd. taking into account that Ca2’ induccc an isothermal phase transition in DDP vcsiclci 31 subthreshold concentration^.^^ In contrsst, DDP vesicles at pH 3.7 p r e s s a highly fluid core. and it is likely that the hydrocarbon chains in the carraponding tubes are highly disordered. This will result in a smaller cylindcr radius. Thcreiore. u c conclude that at pH 3.7. large. fused vesicles of DDP arc trancformcd into two types of hexagonal tubcc: (i) tuber with a high Ca” content and DDP molcculeF with extended hydrocarbon chains and (ii) tubes with a low Ca” content and containing melted hydrocarbon chains. The second type of t u k s will contain a decreased number o i binding sites for Caz* on protonation of thc head groups. However, the presence of Ca’* IS crucial for the transformation of a fused vesicle to a tubular S~NCIUW; multilsmellsr DDP vnicles form only a small number of t u b a at pH 3.7 while hexagonal tubes arc readily in the prescnce of Ca” at pH 7.4. These findings reinforce the idca (vide supra) that the molecular organization of DDP in vesicles at lower pH values resembles that of an organi7ation intermediate between a bilayer and a hexagonal HI, phase Clearly, Ca2‘ IS ncccncary for the final transformation into the hexagonal Hi, arrangement. Conclusion We find that the fusion activity of DDP bilayer vesicles, as dctermined by the initial rate of fusion and the extent of fusion. is strongl) affected by changes in pH. The effects originate from pll-induced alterations in the head-group region which influence the complete bilayer organization. Protonation of the head group leads to a diminished hcnd-group repulsion but also to interhead-group hldrugen-bonding interactions The rcsulting change in the shape factor of thc DDP amphiphile is then responsible for changes in head-group structuring and bilayer fluidity. In addition. changes in pH also exert an important influence on the Ca”induced trancformation of fused vesicle\ to a hexagonal I I , , phase. Remarkable similarities arc notcd betueen the pH-dependent behavior of phospholipid vesicles and DDP vesicles, which add further rclevance to membrane mimetic chemistry carried out using simple, synthetic amphiphilcs.
Acknowledgment. The investigations were Supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organiiation for the Advancement of Pure Research (ZWO). Regislr> No. DDP. 11026-45-8; Ca. 7440-70-2
