Ca2+-mediated fusion of didodecyl phosphate vesicles

Ca2+-Mediated Fusion of Didodecyl Phosphate Vesicles LEO

A. M .

RUPERT,*

DICK

HOEKSTRA,t

'1 AND J A N

B. F. N. E N G B E R T S *

*Department of Organic Chemistry, University of Groningen, Nijenborgh 16, 9747 AG Groningen, The Netherlands; and ~The Laboratory of Physiological Chemistry, University of Groningen, Bloemsinge110, 9712 KZ Groningen, The Netherlands Received October 13, 1986; accepted December 16, 1986 Ca2+ induces fusion of didodecyl phosphate (DDP) vesicles and the mechanism of this process has been investigated. The kinetics of aggregation and fusion were monitored by turbidity measurements and by a resonance energy transfer fusion assay, respectively. It appears that fusion represents the ratelimiting step of the overall reaction, since aggregation reveals a Ca2÷ threshold concentration of 1.4 mM while fusion occurs only above 1.75 mM. Interestingly, Mg2+ does not induce fusion although massive aggregation was observed. Furthermore, it is shown that Mg2+ inhibits the Ca2+-induced fusion of DDP vesicles. The occurrence of fusion is further confirmed by electron microscopy and takes place only above the phase transition temperature (28°C) of the DDP bilayer, i.e., an overall fluid membrane is required for the initiation of fusion. To probe Ca2+-triggered changes in the bilayer-water interface 31p NMR measurements were performed. As a function of the Ca2÷ concentration a splitting of the NMR signal was observed. An assignment was made for phosphate head groups on the inner and outer leaflets of the DDP vesicle bilayer. At a Ca2÷ threshold concentration of 0.13 mM, there is a shift of the signal arising from the inner phosphate head groups, which indicates that Ca 2÷ leaks across the bilayer. This process presumably occurs via an unstable inverted structure that may closely resemble the fusion intermediate. The upfield shifts and the substantial line broadening of the 3lp NMR signal suggest that the binding of Ca2÷ causes a dehydration of the head groups and a concomitant reduction of the head group mobility. Since these alterations occur on separate vesicles, i.e., prior to aggregation, the involvement of a specific trans (interbilayer) Ca2+-phosphate complex, triggering the actual fusion process, is discussed. In a process secondary to these Ca2+-induced physical alterations of the bilayer-water interface, an isothermal phase transition occurs, resulting in an upward shift of the phase transition temperature from 28°C to a temperature higher than 65°C. The results are discussed in the light of comparable results previously obtained on the fusion of didodecyldimethylammonium bromide vesicles. The significant differences between both systems are related to differences in head group hydration thus emphasizing the crucial role of membrane (de)hydration in the mechanism of membrane fusion. © 1987 Academic Press, Inc.

INTRODUCTION S t u d i e s o f t h e d y n a m i c a l t e r a t i o n s in aggregate morphology and membrane structure of s i m p l e s u r f a c t a n t vesicles ( 1 - 8 ) are n o v e l a n d r a p i d l y d e v e l o p i n g a r e a s in m e m b r a n e m i m e t i c c h e m i s t r y ( 9 - 1 1). T h e fact t h a t t h e structure of these simple amphiphiles can be readily and systematically modified provides v e r s a t i l e possibilities for studies o f t h e r e l a t i o n b e t w e e n t h e s u r f a c t a n t s t r u c t u r e a n d t h e s e dy-

To whom correspondence should be addressed.

n a m i c a l t e r a t i o n s . O b v i o u s l y , w i t h t h e struct u r a l l y m o r e strictly d e f i n e d p h o s p h o l i p i d s s u c h possibilities are l i m i t e d . T w o i m p o r t a n t d y n a m i c a l t e r a t i o n s are fusion and the morphological transformation f r o m a l a m e l l a r to a h e x a g o n a l H u p a c k i n g o f t h e a m p h i p h i l e s . B o t h are essential p h e n o m e n a in n u m e r o u s p h y s i o l o g i c a l l y r e l e v a n t p r o cesses ( 1 2 - 1 4 ) a n d h a v e also b e e n d e m o n strated t o o c c u r b e t w e e n a n d in b i l a y e r s c o m p o s e d o f s y n t h e t i c a m p h i p h i l e s (3, 15). A s b o t h a l t e r a t i o n s r e s u l t f r o m (i) a (partial) d e h y d r a t i o n o f t h e h e a d g r o u p s a n d (ii) t h e f o r m a t i o n 125 0021-9797/87 $3.00

Journal of Colloid and Interface Science, Vol. 120, No. 1, November 1987

Copyright © 1987 by Academic Press, Inc. All fights of reproduction in any form reserved.

126

RUPERT,

HOEKSTRA,

of a "trans complex" between the head groups of two apposed bilayers and the fusogenic agent, it has been suggested that they most likely proceed via the same unstable inverted intermediate (3). Membrane dehydration, to lower the strong repulsive forces (16-18), and the formation of a "trans complex" have also been suggested to represent key events in the fusion of phospholipid vesicles (19-21). In the present work we report a detailed kinetic and membrane structural analysis of the CaZ+-induced fusion of didodecyl phosphate (DDP) vesicles. The kinetic analysis of the fusion process was performed using the previously employed resonance energy transfer (RET) fusion assay (1, 2, 22) in combination with turbidity measurements. Compelling evidence for fusion was also obtained using electron microscopy (EM). The fluorescence polarization of the apolar probe diphenylhexatriene (DPH) (23) was measured to determine the influence of the bilayer fluidity on the fusion process and the effect of the binding of the fusogenic agent Ca z+ on the membrane fluidity. 31p NMR measurements were carded out to clarify the phase behavior of the DDP vesicles and the influence of the binding of Ca2+ on the phosphate head groups in more detail. The results indicate that the fusion step is rate limiting in the overall fusion process and that fusion only occurs when the vesicle bilayer is in the liquid crystalline state.

AND ENGBERTS

Vesicle preparation. Sodium didodecyl phosphate (DDP) vesicles were prepared by the ethanol injection method (25). Hereto 10 mg of didodecyl phosphoric acid was dissolved in 100 #1 of ethanol containing 0.30 M N a O H (slightly higher than equimolar with respect to didodecyl phosphate). With a preheated Hamilton microsyringe, 50 ~tl of this solution was injected into 2 ml 5 m M Hepes/5 m M sodium acetate buffer (pH 7.4) thermostated at 55°C. DPH-labeled vesicles were prepared by adding small aliquots ofa DPH stock solution (in tetrahydrofuran) to the vesicle solutions, and were subsequently equilibrated for 1 h at 50°C. The DPH-to-DDP ratios were ca. 1:11,000 and 1:1000 in the temperature- and Ca2+ concentration-dependent fluorescence polarization measurements, respectively. The effect oftetrahydrofuran on the vesicles (0.05% for both measurements) can be neglected (15). Vesicle aggregation. Aggregation of DDP vesicles, induced by Ca2+ or Mg2+ at 40°C, was measured by monitoring continuously the turbidity change at 350 nm using a Perkin-Elmer Lambda 5 spectrophotometer, equipped with a thermostated cell holder and a stirring device. The initial rate of aggregation is defined as the first derivative (at t = 0) of the curve describing the change in turbidity as a function of time. Fusion measurements. Vesicle fusion was monitored continuously with the resonance energy transfer (RET) fusion assay, as previEXPERIMENTAL SECTION ously described (1, 2, 22). Vesicles containing Materials. Didodecylphosphoric acid was 0.8 mole% each of N-NBD-PE and N-Rh-PE obtained from Alpha Chemicals, mp 59.1- were prepared by solubilizing appropriate 60.2°C (literature, 58-59°C (24)) and used amounts of didodecyl phosphate and fluorowithout further purification. N-(7-Nitrobenz- phores in ethanol/NaOH. Fusion measure2 - oxa - 1,3 - diazol - 4 - yl)phosphatidyleth- ments were carried out in Hepes/sodium acanolamine (N-NBD-PE) and N-(lissamine- etate buffer (pH 7.4) with equimolar amounts rhodamine B-sulfonyl)phosphatidylethanol- of labeled and nonlabeled DDP vesicles. The amine (N-Rh-PE) were purchased from Avanti total amphiphile concentration was 59 #M. Polar Lipids, Inc. Diphenylhexatriene (DPH) After equilibration at the desired temperature was obtained from Aldrich, 4-(2-hydroxy- (see legends) fusion was initiated by injecting ethyl)- 1-piperazineethanesulfonic acid (Hepes) the vesicle solution into the buffer solution from Sigma Chemicals, and calcium and containing the appropriate cation. NBD flumagnesium chloride from Merck. orescence (Xex -- 475 nm, Xer~= 530 nm) was Journal of Colloid and Interface Science~ Vol. 120, No. 1, November 1987

FUSION OF DIDODECYL PHOSPHATE VESICLES

127

monitored continuously by using a PerkinElmer MPF43 spectrophotometer 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 after the addition of cetyltrimethylammonium bromide (final concentration 1%, w/v), corrected for the sample dilution, as 100% 0nfinite dilution).

scope operating at 80 kV. The cryo EM experinaents were performed as previously described (15). The samples on carbon-coated grids were examined in a Philips EM400 electron microscope equipped with a Philips cooling holder (PW 6591) operating at 80 kV and at a low electron dose.

Fluorescence polarization measurements.

Cation-induced aggregation of D D P vesicles was observed in the presence of both Mg 2+ and Ca 2+. The cation threshold concentrations were 1.0 and 1.4 raM, respectively (Fig. 1), while the extent of aggregation in terms of the absolute turbidity at t = oo (not shown) was higher for Mg 2+ than for Ca ~+. These results are indicative of a higher binding constant for Mg z+ in this system. The maximal initial rates of aggregation at cation concentrations above 5 m M were, however, higher for Ca z+. The absence of a direct correlation between the extent and the initial rate of aggregation is not surprising in view of the complex dependence of the aggregation process on electrostatic repulsions, van der Waals interactions, and vesicle size (29). We note that phosphatidylserine (PS) vesicles display a higher binding constant for Ca 2+ than for Mg 2+ (19), in contrast to DDP vesicles. This difference may be attributed to the possibility that Ca 2+ is chelated

Measurements of the fluorescence polarization were carried out in the spectrofluorometer equipped with a polarization accessory. D P H was excited at 360 nm; the emission wavelength was 428 nm. The degree of fluorescence polarization (P) was calculated from the equation (27) P =

11)/(1 H+

where Ijt and I± are the fluorescence intensities detected with the polarizers oriented parallel and perpendicular, respectively, to the direction of polarization of the excitation light. The value of I i was corrected for the intrinsic polarization of the instrument (28). 31p NMR measurements. 1H_decoupled 31p N M R measurements were performed using a 10-mm tube at 80.988 MHz on a Nicolet NT200 instrument equipped with a temperature controller and a deuterium lock. Chemical shifts (ppm) were determined relative to the external reference of hexachlorocyclotriphosphazene in CDC13 (+ 19.9 ppm downfield from 85% H3PO4). Accumulated free induction decays were obtained from 400 or 17,000 transients (see legends) with an interpulse time of 3.2 and a pulse angle of 19 #s (ca. 90°). Tetrahydrofuran-d4 in a concentric 2-mm tube was used as an internal deuterium lock. Electron microscopy. Aliquots of DDP vesicles, before or after the addition of CaC12, were stained with a 1% (w/v) solution ofuranyl acetate as previously described (2). The carbon-coated Formvar grids were pretreated by glow discharge in air. The samples were examined in a Philips EM 300 electron micro-

RESULTS AND DISCUSSION

Cation-induced aggregation and fusion.

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FIG. 1. Effectof the Ca2+(C)) and Mg2+(A) concentration on the initial rate of aggregation (in arbitrary units) of DDP vesicles.[DDP] = 58 × 10-6 M, pH 7.4; incubation temperature 40.0°C. Journal of Colloid and Interface Science, Vol. 120, No. 1, N o v e m b e r 1987

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RUPERT, HOEKSTRA, AND ENGBERTS

between the phosphate and the carboxylic unit of a PS head group, while Mg 2+ only binds to the phosphate moiety (30). With DDP vesicles, however, either cation can bind only to the phosphate head group which would lead to a lower binding constant for Ca 2÷ than for Mg 2÷. Besides causing the vesicles to aggregate, Ca 2+ also induces fusion as revealed by the resonance energy transfer assay. The Ca 2+ threshold concentration is 1.75 m M (Fig. 2) while the initial fusion rate levels offabove 5.7 mM. The maximal extent of fusion reaches a value of ca. 45% implying that about half of the vesicle population resists fusion, presumably as a result of a different bilayer packing and/or head group hydration (2). Clearly, the overall fusion process involves two consecutive steps: aggregation followed by merging of the bilayers, i.e., the actual fusion process. The above results indicate that fusion is the ratedetermining step in the overall process. A similar conclusion has been reached for fusion of PS and DDAB vesicles (1, 21), induced by Ca z+ and the dianion of dipicolinic acid (DPA2-), respectively. The threshold concentrations for the DDP/Ca 2+ system are 70 times higher than those for the DDAB vesicles, in-

dependent of the chemical nature of the fusogenic agent (2). It is likely that this difference originates mainly from differences in the hydration of the head group region. Ab initio calculations reveal that a phosphate head group is more strongly hydrated than a dimethylammonium head group as reflected both by the number of water molecules, 4-5 and 2, respectively in the first hydration shell, and by the different hydration energies per water molecule (31-33). We conclude therefore that dehydration, which has been proposed to play an essential role in the induction of fusion (16-18), occurs more readily for DDAB head groups than for DDP head groups. This is further supported by recent observations showing that the hydration forces for bilayer systems of longer-chain analogs of DDAB are small or negligible compared with the electrostatic double-layer forces (34, 35). Although Mg 2+ is capable of inducing aggregation (vide supra), no fusion was observed (Fig. 2) even at concentrations as high as 100 mM. A similar result has been reported for large unilamellar PS vesicles (36, 37). In this case it was inferred that Mg 2+ cannot form a dehydrated "'trans MgZ+/PS complex" between two adjacent vesicles, which represents a prerequisite for fusion to occur (19). As shown in Fig. 3, incubation of DDP vesicles with Mg 2+ 50 5.¢ inhibits CaZ+-induced fusion. When both cations are present at equimolar concentrations 4O 4.0 the fusion process is almost completely suppressed, which is consistent with a higher 30 "~ 3.O binding constant of Mg 2+. Thus Mg 2+ comu_ petes effectively with Ca 2+ for the same binding ._g 1 "8 sites at the phosphate head groups of the DDP u_ 2.0, 2o ,~ .,~ vesicle, thereby inhibiting the formation of the #J Ca2+/DDP "trans complex" necessary for fu1. 10 sion. Apparently, partial dehydration of the bilayer-water interface and reduction of the 10 0 surface charge by binding of Mg z+ do not in[Cation] (raM) duce the synergism of Mg 2+ on CaZ+-induced FIG. 2. Effectof the Ca2+ concentration on the initial fusion as seen with small unilamellar PS vesfusionrate (0) and the extentof fusion(n) of DDP vesicles. icles (36). The ratio of labeled to nonlabeled vesicleswas 1:1. Total Electron microscopic evidence for fusion. [DDP] = 58 × 10 -6 M ; pH 7.4; incubation temperature 40.0°C. Alsoshown is the effectof Mg2+(~) on the initial Electron microscopy provided additional and fusion rate. compelling evidence for Ca2+-induced fusion

/

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FUSION OF DIDODECYL PHOSPHATE VESICLES

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Influence of the membrane Jluidity on fusion susceptibility. The ability of a vesicle to fuse

18 and 33°C (Fig. 5).2 This change consists of a main transition (22-33°C), with a phase transition temperature (To) of 28°C and a pretransition (18-22°C) with the midpoint at 20°C. The fluorescence polarization values above Tc fall in the same range as those for DDAB vesicles (2). Below T~, however, a remarkably higher value is found for DDP (0.39) than for DDAB (0.15) (2). This polarization value is even higher than the value found for the C18 analog of DDAB, dioctadecyldimethylammonium bromide (P = 0.30) (38). Therefore it is likely that the packing density of the hydrocarbon chains below the phase transition is considerably higher for DDP than for DDAB vesicles. This may be explained in terms of the larger head group area of the dimethylammonium bromide moiety as compared to that of the sodium phosphate unit. 3 This not only would allow a much higher packing density but also would lead to a different orientation of these amphiphiles in the bilayer below the phase transition (40). Because DDAB vesicles show a significantly enhanced fusion activity at the pretransition (2), an accurate determination of such a pretransition for DDP vesicles was, therefore, deemed worthwhile. Although the plot of the fluorescence polarization vs temperature suggests a pretransition around 20°C (Fig. 5) additional evidence was required. From measurements on phospholipid vesicles it is known that the linewidth of the 3~p NMR signal increases at the pretransition (41). A typical 31p NMR spectrum of DDP vesicles is shown in Fig. 6. The linewidth of this signal and the fact that there is no splitting of the signal as a result of large bilayer curvature (42) are consistent with a vesicle size of ca. 1000 A, in agreement with the electron microscopic results (Fig. 4a). The

depends strongly on the physical state (gel or liquid crystalline) of the vesicle bilayer as vesicles are more prone to fusion in the fluid state (2, 2 l). To characterize the phase behavior of DDP vesicles, the fluorescence polarization (P) of diphenylhexatriene (DPH)-labeled DDP vesicles was measured as a function of temperature. The phase change occurs between

2 The phase transition temperature of DDP vesicles is pH dependent (Rupert, L. A. M., Hoekstra, D., Engberts, J. B. F. N., to be published). 3 This is expressed in the shape factor v i a l with v = volume per molecule, a = head group area, and l = length of the fully extended hydrocarbon chain (39). The shape factors for DDP and DDAB are 1.5 and 0.95, respectively.

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of DDP vesicles. [DDP] = 58 × 10-6 M; [CaC12] = 4.76 × 10 -3 M; pH 7.4; 40.0°C. Ratio of labeled to nonlabeled vesicles was 1:1.

of DDP vesicles. Figure 4a shows the original vesicles (mean diameter ca. 900 A) aggregated under the influence of the positively charged staining agent uranyl acetate. Examination of negatively stained samples of vesicles that had been preincubated with Ca 2+ for 1 min revealed the presence of large (4 ca. 7000 A), fused vesicles (Fig. 4b). In contrast to the DDAB/DPA 2- system where the large fused vesicles represent the stable end products of the fusion reaction (1), the DDP/Ca 2+ system subsequently undergoes an additional transformation from vesicles to large hexagonal Hn tubes (Fig. 4c). The dark striation on the tube in Fig. 4d shows the hexagonal packing of the tube (15). A detailed analysis of this intriguing morphological transformation will be reported elsewhere (3, 15).

Journal of Colloidand InterfaceScience, Vol. 120,No. l, November1987

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RUPERT, HOEKSTRA, AND ENGBERTS

FIG. 4. Electron micrographs of negativelystained DDP vesiclesaggregatedunder the influence of the staining agent uranyl acetate(a), 1 min (b), and 1 h after the addition of Ca2+(c). Cryo electronmicrograph (d) shows the hexagonalcharacter of the tube. The marker line represents3500 A, except for (d) (280 A). intensity of the 31p N M R signal as a function In Fig. 7 the initial fusion rate and the extent of temperature showed an abrupt change at of fusion are shown as a function of temper20°C, where it vanishes in the noise (Fig. 6). ature. It turns out that the fusion capacity of The linewidth increases even more strongly DDP vesicles decreases with decreasing bilayer than in the case of phospholipid vesicles (41, fluidity. Fusion is completely suppressed below 42). These results are indicative for a pretran- 30°C, i.e., below the phase transition tempersition at 20°C since, as shown for phospholipid ature. This behavior differs from that of vesicles, a suppresion of the axial rotation of DDAB vesicles where fusion does occur below the phospholipid molecule in the bilayer will To, although strongly attenuated. Presumably result in an enhanced influence of the chem- the packing of the hydrocarbon chains of DDP ical-shift anisotropy on the linewidth (42, 43). below Tc is too tight to permit the necessary The phosphate moiety of a phospholipid pos- reorientations of these chains in the actual fusesses, in spite of the suppressed rotation of sion step. Even at the pretransition where a the acyl chain-glycerol backbone moiety, the change in the hydrocarbon chain conformapossibility of a fast rotation around the glyc- tion perturbs the bilayer packing, no fusion of erol-phosphate bond. The phosphate head DDP vesicles is observed. group of DDP, however, lacks this fast rotation Ca2+-induced alterations of DDP bilayer mode relative to the magnetic field, and con- properties'. Studies of the DDAB/DPA 2- syssequently, the contribution of the chemical- tem revealed that alterations in the head group shift anisotropy to the linewidth will be much regior[ are essential for fusion to occur (2). For larger. DDP vesicles, the phosphorus nucleus can act Journal of Colloid and InterfaceScience, Vol. 120, No. 1, November 1987

131

FUSION OF DIDODECYL PHOSPHATE VESICLES

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as an intrinsic probe in studies aimed at characterizing the bilayer-water interface by 3]p NMR. 4 The effect of Ca 2+ on the chemical shift (A6o) and the intensity of the 31p N M R signal of DDP vesicles are shown in Fig. 8. Figure 9 portrays the splitting of the 31p N M R signal when the vesicles are incubated with increasing concentrations of Ca 2+. The approximate ratio of the integrated signals of the lowfield and upfield signal is 1:1. A similar splitring has also been found for the PS/Ca 2+ system. Following the explanation of Kurland et al. (30) for their results on the PS/Ca 2+ system, we suggest that the upfield line corresponds to the weight-averaged signals of free and Ca2+-complexed phosphate head groups on the outer surface of the DDP vesicles. When EDTA, which strongly complexes Ca 2+, is added to a DDP vesicle solution incubated with Ca 2+ (at Ca 2+ concentrations below the aggregation threshold concentration) the original 31p N M R signal reappears. This observation reveals that the Ca2+-induced alterations are completely reversible. It is known that the addition both of Na + to diethyl phos4 Our efforts to probe changes in the bilayer-water interface of DDP vesicles as a function of the Ca2+ concentration were unsuccessful because binding of Ca2+ repelled the positively charged probe molecules (c.q. acriflavine and ethidium bromide) from the vesicle surface into the bulk water.

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FIG. 6. Effect of lowering the temperature on the intensity (I) of the 3~p NMR signal of DDP vesicles. Inset: a typical signal at 40°C. [DDP] = 5.8 × 10-3 M; pH 7.4; 400 transients.

phate (44) and dipalmitoylphosphatidylcholine vesicles (43) and of Mg 2+, Ca 2+, and Zn 2+ to adenosine triphosphate (45) leads to small downfield shifts. Thus it seems unlikely that the observed upfield shift for DDP is caused only by charge neutralization. For phosphates in a rather rigid bilayer, stereoelectronic effects (46) are not expected to be important. This also applies for an effect due to an increase in the O - P - O bond angle (46) because Ca 2+ has only minor effects on the 31p N M R signal of

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FIG. 7. Effect of the temperature on the initial fusion rate (O) and the extent of fusion ([~) of DDP vesicles. The ratio of vesicles labeled with N-NBD-PE and N-Rh-PE to nonlabeled was 1:1. Total [DDP] = 58 × 10-6M; [CaC12] = 3.85 × 10-3 M; pH 7.4. Journal of Colloid and Interface Science,

Vol. 120, No. 1, November 1987

132

RUPERT, HOEKSTRA, AND ENGBERTS

[ 3

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FIG. 8. Split 31p N M R signal as a result of the addition of CaCI2. [DDP] = 5.8 × 10-3 M; [CaC12] = 1.5 X 10-4 M; pH 7.4; 40.0°C; 17,000 transients.

dimethyl phosphate (47). However, lowering of the polarity or proton-donating ability of the medium surrounding the phosphate head group could induce such an upfield shift (48). Therefore the upfield shift can be reconciled with a dehydration of the head group. 5 A dehydration of the head group below the aggregation threshold concentration (1.0 m M at these vesicle concentrations) has also been found for the DDAB/DPA 2- system (2). At concentrations above 0.13 m M Ca2+ the signal of the head groups in the inner leaflet of the bilayer shifts, indicating that the bilayer becomes permeable to Ca 2+. It has been reported that lipids, which are able to form a hexagonal Hu phase can facilitate Ca 2+ transport across bilayers (14, 49-51). Since DDP can also form such a hexagonal phase (3, 15), we assume that the transport of Ca 2+ across the DDP bilayer occurs via the formation of an inverted structure similarly reported for phosphatidic acid and cardiolipin (49). Support is found in the fact that in the PS/Ca2+ s A comparison with the protonation of the dianion of O-phosphateserine (30) is misleading, because this, contrary to the protonation of the monoanion of a diester, gives an increase in the O - P - O band angle and therefore an upfield shift (46). Journal of Colloid and Interface Science, Vol. 120, No. I, November 1987

system the low-field line does not shift (30), while PS favors a lamellar packing. The leakage of Cd 2+ over a dihexadecyl phosphate bilayer (52) presumably proceeds by the same mechanism. If the signal derived from the head groups in the inner and outer leaflets would be completely separated, a relative intensity of 0.5 would be expected. However, the signal intensity decreases to maximally 25% suggesting a substantial line broadening 6 presumably because of a reduced mobility of the head groups. This is supported by relaxation time measurements on the PS/Ca2+ system, as reported by Kurland et al. (30). Thus the binding of Ca2+ to DDP vesicles results in a dehydration and a concomitant reduction of the mobility of the head groups well below the aggregation threshold concentration, i.e., on separate vesicles. Conceivably, under these conditions a "cis Ca2+/DDP complex" is formed, i.e., complexation of adjacent DDP molecules within the lateral plane of the bilayer. Because fusion occurs only between vesicles in the aggregated state, well above the aggregation threshold concentration (see above), it is clear that both processes are not sufficient to cause immediate fusion upon aggregation. The same conclusion was drawn for the DDAB/DPA 2- system (2). Thus the fusion event requires an additional process, most likely the formation of a "trans complex" between the fusogenic agent and the head groups of two apposed bilayers, which triggers the fusion reaction. If the area ofinterbilayer contact is large enough and the amphiphile is coneshaped, such a "'trans complex" can eventually also lead to a morphological transformation from a lamellar to a hexagonal HII packing of the amphiphilic molecules (3). Because both processes are triggered by a "trans complex," they are essentially different for the Ca2+ transport over the DDP bilayer which, apparently, is triggered by a "cis complex." This, 6 Direct measurements of the linewidth were hampered by overlap of both lines at low Ca 2+ concentrations and by an unfavorable signal-to-noise ratio at the highest Ca 2÷ concentrations.

133

FUSION OF DIDODECYL PHOSPHATE VESICLES

however, does not exclude the possibility that the transiently stable inverted structures of the intermediates in these processes resemble each other. It has been shown that the binding of DPA 2- to DDAB vesicles influences only the head group packing (2). This contrasts with the DDP/Ca 2÷ system where the influence of Ca2÷ binding propagates into the hydrocarbon region of the bilayer. As is shown in Fig, 10, the fluorescence polarization of DPH, as a function of the Ca 2+ concentration, changes after an initial gradual increase rather abruptly and steeply at 1.25 m M CaC12. The high polarization value (P = 0.35) found for the gel state indicates again the large influence of the phosphate head groups, as compared with the dimethylammonium head groups, on the bilayer packing. The gel to liquid crystalline phase transition temperature of the CaZ+-DDP complex is shifted to a temperature above 65°C. A similar isothermal phase transition has also been observed for the PS/Ca 2÷ system

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FIG. 10. Ca2+-dependent membrane fluidity of DDP vesicles as determined with DPH fluorescence polarization. [DDP] = 58 X 10-6 M; [DPH] = 5.0 X 10-8 M; pH 7.4; 40.2°C.

and it has been postulated that this phase change is a prerequisite for fusion (53). However, the observations that (i) this phase transition is a slow process on the time scale of fusion (20) and that (ii) such an isothermal phase transition is absent in the DDAB/DPA 2system (2) are not consistent with this hypothesis. Instead we contend, as discussed above, that a local perturbation of the waterbilayer interface is crucial to fusion (2). ACKNOWLEDGMENTS

0

The investigations were supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO). J. F. L. van Breemen (Laboratory of Biochemistry, University of Groningen, The Netherlands) is gratefully acknowledged for taking the micrographs. We thank Rinske Kuperus for expert secretarial assistance.

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REFERENCES 0.20

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0.80

[Ca2"]tmM) PIG. 9. Effect of Ca 2+ on the relative intensity (Iro0 and the change in chemical shift (A6p) of the 31p NMR signal of the phosphate head groups on the inside (O) and the outside (I-q) of the DDP vesicles. [DDP] = 5.8 X 10-s M; pH 7.4; 40.0°C; 400 transients.

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