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Effect of Vesicles of Dimethyldioctadecylammonium Chloride and Phospholipids on the Rate of Decarboxylation of 6-Nitrobenzisoxazole-3-carboxylate† Maria Virginia Scarpa,*,‡,§ Pedro S. Araujo,§ Shirley Schreier,§ Antonio Sesso,| Anselmo G. Oliveira,‡ Hernan Chaimovich,§ and Iolanda M. Cuccovia§ Depto. de Fa´ rmacos e Medicamentos, Fac. Cieˆ ncias Farmaceˆ uticas, UNESP, Araraquara, Depto. Bioquı´mica, Instituto de Quı´mica, USP, Sa˜ o Paulo, Depto. Patologia, Fac. Medicina, USP, Sa˜ o Paulo, Brazil Received June 10, 1999. In Final Form: September 30, 1999 Sonicated mixtures of dimethyldioctadecylammonium chloride (DODAC), egg phosphatidylcholine (PC), dimyristoyl phosphatidylcholine (DMPC), and dipalmitoyl phosphatidylcholine (DPPC) were used to analyze vesicle effects on the rate of decarboxylation of 6-nitrobenzisoxazol-3-carboxylic acid (Nboc). Electron microscopic images of the vesicles were obtained with trehalose, a know cryoprotector. Phase diagrams and phase transitions temperatures of the vesicle bilayers were determined. Nboc decarboxylation rates increased in the presence of vesicles prepared with both phospholipids and DODAC/phospholipid mixtures. Quantitative analysis of vesicular effects was done using pseudophase models. Phospholipids catalyzed up to 140-fold while the maximum catalysis by DODAC/lipid vesicles reached 800-fold. Acceleration depends on alkyl chain length, fatty acid insaturation of the lipids, and the DODAC/phospholipid molar ratio. Catalysis is not related to the liquid crystalline-gel state of the bilayer and may be related to the relative position of Nboc with respect to the interface.
Introduction The rate of spontaneous decarboxylation of 6-nitrobenzisoxazole-3-carboxylate ion (Nboc) can vary by 8 orders of magnitude depending on the nature of the solvent.1,2 This extraordinarily large effect results from the influence of the solvent on the formation of the free Nboc, anion from the ion pair, hydrogen bonding from the solvent to Nboc, and solvent effects on the relative stabilities of the initial state and the charge-delocalyzed transition state.3 The decomposition of Nboc is generally faster in organic solvents as compared with water because of hydrogen bonding stabilization of the initial state by water.3 The rate of Nboc decomposition has been used to probe the effect of supramolecular aggregates and polymers on chemical reactivity and to relate the properties of the reaction site with reaction rates. Nboc decomposition, catalyzed by cationic micelles, can be affected by salt addition.4-6 Other supramolecular systems such as didodecyldimethylammonium chloride small assemblies,7a reverse micelles,7b crown ethers,8 and microemulsions9 have been used as a medium for Nboc decomposition. † Part of the Special Issue “Clifford A. Bunton: From Reaction Mechanisms to Association Colloids; Crucial Contributions to Physical Organic Chemistry”. * To whom correspondence should be addressed. Current address: Instituto de Quı´mica da Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes 748, Sa˜o Paulo, SP, Brazil, E-mail: imcuccov@ quim.iq.usp.br. Fax: 55 11 815 5579. ‡ UNESP. § Depto. Bioquı´mica, Instituto de Quı´mica, USP. | Depto. Patologia, Fac. Medicina, USP.
(1) Kemp, D. S.; Paul, K. G. J. Am. Chem. Soc. 1975, 97, 7305. (2) Kemp, D. S.; Cox, D. D.; Paul, K. G. J. Am. Chem. Soc. 1975, 97, 7312. (3) (a) Grate, J. W.; McGill, R. A.; Hilvert, D. J. Am. Chem. Soc. 1993, 115, 8577. (b) Ferris, D. C.; Drago, R. S. J. Am. Chem. Soc. 1994, 116, 7509. (4) Bunton, C. A.; Minch, J. Tetrahedron Lett. 1970, 3881. (5) Bunton, C. A.; Minch, M.; Sepulveda, L. J. Phys. Chem. 1971, 75 (5), 2708. (6) Bunton, C. A.; Minch, M. J.; Hidalgo, J.; Sepulveda, L. J. Am. Chem. Soc. 1973, 95, 3262.
Catalytic antibodies developed against 2-bromoacetamido1,5-naphthalenedisulfonate catalyze the decomposition of a Nboc analogue by 104 fold.10 The effect of vesicles on the rate of decomposition of Nboc has been described using, among other amphiphiles, phospholipid and dimethyldioctadecylammonium chloride (DODAC) vesicles.11 The maximum rate acceleration produced by sonicated DODAC aggregates is approximately 400-fold, calculated by comparing the maximum observed rate constant kψmax with the rate constant in water kw at 30 °C.11a DODAC catalysis was not analyzed quantitatively and was attributed to local hydrophobicity. Phosphatydilcholine sonicated vesicles reportedly increase the rate of decomposition of DODAC by less than 3-fold.12 The effect of zwitterionic micelles on the rate of decarboxylation of Nboc depends critically on the position of the dipole with respect to the hydrocarbon chain.12 Thus, lysolecithin micelles increase the reaction rate by 30% while sulfobetaine micelles catalyze by 2 orders of magnitude.12 Here we focus our attention on the investigation of the effect of vesicles of phospholipids with varying chain length and (positive) charge density at the vesicle interface on the rate of decarboxylation of Nboc. The variation in charge density was obtained by changing the mole ratio of lipid/ DODAC used for vesicle preparation. Kinetics were analyzed quantitatively using pseudophase models, and rate constants in the vesicle pseudophase kv were calculated.13,14 The value of kv was 30-140-fold larger that kw (7) (a) Germani, R.; Ponti, P. P.; Savelli, G.; Spreti, N.; Cipiciani, A.; Cerichelli, G.; Bunton, C. A. J. Chem. Soc., Perkin Trans 2 1989, 1767. (b) Germani, R.; Ponti, P. P.; Spreti, N.; Savelli, G.; Cipiani, A.; Cerichelli, G.; Bunton, C. A.; Si, V. J. Colloid Interface Sci. 1990, 138, 443. (8) Shah, S. C.; Smid, J. J. Am. Chem. Soc. 1978, 100, 1426. (9) Bunton, C. A.; Buzzaccarini, F. J. Phys. Chem. 1981, 85, 3139. (10) Lewis, C.; Kramer, T.; Robinson, S.; Hilvert, D. Science 1991, 253, 1019. (11) (a) Kunitake, T.; Okahata, Y.; Ando, R.; Shinkai, S.; Hirakawa, S. J. Am. Chem. Soc. 1980, 102, 7877. (b) Patel, M. S.; Bijma, K.; Engberts, J. B. F. N. Langmuir 1994, 10, 2491. (12) Bunton, C. A.; Kamego, A. A.; Minch, M. J.; Wright, J. L. J. Org. Chem. 1975, 40, 1321.
10.1021/la9907477 CCC: $19.00 © 2000 American Chemical Society Published on Web 01/12/2000
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in lipid vesicles and increased with the mole percent of DODAC, reaching a kv/kw ratio of approximately 800-fold. Our results suggest that these effects can be attributed to local medium effects and changes in Nboc location at the bilayer with changing amphiphile ratio. Experimental Section Materials. Egg phosphatidylcholine (PC) was purified from egg yolk as described;15 dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) (Avanti Polar Lipids Inc.) were used as received. Dimethyldioctadecylammonium chloride (DODAC) was prepared from dimethyldioctadecylammonium bromide (DODAB) (Eastman Kodak Co.) by ion exchange.16 6-Nitrobenzisoxazol-3-carboxylic acid (Nboc) was kindly furnished by Dr. Gianfranco Savelli (Perugia University, Italy) and used as received. All salts were analytical grade, and all solutions were made with bidistilled and deionized water. Methods. The decarboxylation of Nboc was followed spectrophotometrically, measuring the appearance of the salicylonitrile product at 410 nm.4 All reactions were performed in Tris/ HCl 0.01 M, pH 8.1 buffer. The reactions were initiated by adding 0.04 mL of an aqueous solution of a 0.001 M Nboc solution to 2.0 mL of the reaction mixtures in temperature-equilibrated (30 °C, unless stated) spectrophotometric cells. The Nboc stock solution was maintained at -18 °C. With pure phospholipids the scattering-derived absorbance is high and the reaction slow at this temperature; therefore, aliquots were obtained at convenient times and added to n-propanol (1:1, v/v). At this n-propanol volume ratio vesicles dissolve and the absorbance at 400 nm can be related to product concentration without scattering artifacts. Vesicle Preparation. Films of phospholipids and/or DODAC were prepared using chloroform solutions of phospholipids and DODAC and evaporating the solvent under a N2 flux. Residual solvent was removed, maintaining the films under vacuum, in a desiccator, for at least 2 h. The films were maintained at -18 °C. Vesicles were prepared by hydrating the films with the adequate volume of buffer (usually 0.01 M total concentration of lipids and 15 mL of buffer or water), letting the film swell for 30 min and vortexing it five times during the hydration period. Samples were subsequently irradiated using a probe-type sonicator (Braunsonic 1510, B. Braun Melsungen AG) operated at a nominal output of 90 W, for 12-30 min, depending on the lecithin concentration, until constant values of scattering-derived absorbance (400 nm) were obtained. The temperature of the sample was maintained in all cases above the phase transition temperature Tc of the mixture. Samples were centrifuged at 23000g (Hitachi, Himac CR 20 B2, 30 min, 10 °C) in order to remove the titanium particles formed during the sonication. Phase-Transition Temperature. A solution (1.5 mL) containing vesicles (0.002 M total lipid) was added to a spectrophotometric cell, and the light-scattering-derived absorbance was measured at 250 nm.17 The temperature of the sample was increased (0.5 °C/min) in a Beckman DU-7 temperaturecontrolled spectrophotometer. The temperature in the cell, at the time of absorbance measurement, was checked with a digital thermometer coupled with a J-thermopar. Electron Microscopy (EM). Vesicles were prepared in Tris/ HCl buffer 0.01 M, pH 8.1 with and without 0.3 M sucrose or trehalose. A vesicle suspension (0.005 mL) was placed onto an Au-Ir specimen support disk. Plunging the disks in a N2 slush (13) Menger, F. M.; Portnoy, C. E. J. Am. Chem. Soc. 1967, 89, 4698. (14) (a) Quina, F. H.; Chaimovich, H. J. Phys. Chem. 1979, 83, 1844. (b) Chaimovich, H.; Bonilha, J. B. S.; Zanette, D.; Cuccovia, I. M. In Surfactants in Solution; Mittal, K. L., Lindmann, B., Eds.; Plenum Press: New York, 1984; Vol. 2, p 1121. (c) Bunton, C. A.; Nome, F.; Quina, F. H.; Romsted, L. S. Acc. Chem. Res. 1991, 24, 357. (d) Chaimovich, H.; Cuccovia, I. M. Prog. Colloid Polym. Sci. 1997, 103, 667. (15) Singleton, W. S.; Gray, M. S.; Brow, M. L.; White, J. L. Am. Oil Chem. Soc. 1965, 29, 353. (16) Cuccovia, I. M.; Sesso, A.; Abuin, E. B.; Okino, P. F.; Tavares, P. G.; Campos, J. F. S.; Florenzano, F. H.; Chaimovich, H. J. Mol. Liq. 1997, 72, 323. (17) Carmona-Ribeiro, A. M.; Chaimovich, H. Biochim. Biophys. Acta 1983, 733, 172.
Figure 1. Effect of lipid vesicles on the rate of decarboxylation of Nboc; [Nboc] ) 6.7 × 10-4 M: (1) DMPC; (O) DPPC; (b) PC. at -210 °C rapidly froze the vesicles, free of fixative. The frozen samples were fractured at -150 °C (4 × 10-6 Torr) in a Baltzers BAF 301 freeze-fracture apparatus. Shadowing was carried out with 95% C and 5% Pt at 45 °C. Replicas were reinforced with C at an angle of 90°, transferred to a slab with water, and carefully washed with water. The replicas were very fragile and were destroyed by more drastic washing procedures. The replicas were examined on a Formvar-coated grid (400-mesh) at 80 kV in a 1010 JEOL Electron Microscope.16
Results Sonicated phospholipid vesicles prepared with PC, DMPC, and DPPC catalyzed the decarboxylation of Nboc (Figure 1). The [lipid] dependence of kψ varied with the nature of the phospholipid, and a significant rate increase was observed only at relatively high lipid concentrations (Figure 1). Previous data showing only a 3-fold PC catalysis,12 obtained at 0.002 M lipid, are in good agreement with the present data (Figure 1). A pseudophase model (Scheme 1) can be used to Scheme 1
quantitatively analyze vesicle rate effects when the substrate Nboc equilibrates faster than it reacts, and reaction can take place in the aqueous phase (w) or the vesicular pseudophase (v), respectively.13 The first-order rate constant kψ, is given by eq 1, where kw and kv are the first-order rate constants in water and in the vesicle, respectively, KNboc is the binding constant of Nboc to vesicles, and Cv is the lipid concentration.
kψ )
kw + KNbockvCv (1 + KNbocCv)
(1)
A linearized form of eq 1 (eq 2) was used (not shown) to calculate the values of kv and KNboc.13
1/(kw - kψ) ) 1/(kw - kv) + 1/(kw - kv)(1/KNbovCv) (2) The lipid concentration Cv used corresponded to 70% of the total lipid concentration. This correction is necessary because sonicated phospholipid vesicles have 70% of the lipids in the external surface,18 and from data obtained (18) Hauser, H.; Oldani, D.; Phillips, M. C. Biochemistry 1973, 12, 4507.
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Table 1. Values of Nboc Binding Constants KNboc, First-Order Rate Constants in the Vesicle kv Calculated with a Pseudophase Model, and kv/kw Ratios for Lipid Vesicles KNboc (M-1) kv (s-1) kv/kw
PC
DMPC
DPPC
50 2.5 × 10-4 34
21 1.0 × 10-3 136
21 5 × 10-4 68
Figure 4. Effect of DODAC/DPPC vesicles on the rate of decarboxylation of Nboc; [Nboc] ) 2.6 × 10-5 M. DODAC mole %: (.) 0; (]) 10; ([) 20; (3) 30; (1) 40; (2) 50; (9) 60; (4) 70; (0) 80; (O) 90; (b) 100. Table 2. Catalysis of Nboc Decarboxylation by Vesicles as a Function of % DODAC kψmax/kw
Figure 2. Effect of DODAC/PC vesicles on the rate of decarboxylation of Nboc; [Nboc] ) 2.6 × 10-5 M. DODAC mole %: (.) 0; (]) 10; ([) 20; (3) 30; (1) 40; (2) 50; (9) 60; (4) 70; (0) 80; (O) 90; (b) 100.
Figure 3. Effect of DODAC/DMPC vesicles on the rate of decarboxylation of Nboc; [Nboc] ) 2.6 × 10-5 M. DODAC mole %: (.) 0; (]) 10; ([) 20; (3) 30; (1) 40; (2) 50; (9) 60; (4) 70; (0) 80; (O) 90; (b) 100.
in similar systems, added Nboc can be expected to permeate slowly through the bilayer.19 The calculated values of kv and KNboc are shown in Table 1. Using a value of kw of 7.35 × 10-6 s-1 (refs 1 and 2) and taking the kv/kw ratio as an indication of catalytic efficiency, vesicles of PC and DPPC catalyze the reaction 34-fold and 68-fold, respectively, and DMPC catalyzes it by a factor of 136.
% DODAC
PC
DMPC
DPPC
100 90 80 70 60 50 40 30 20 10 0
554 518 563 574 637 508 365 236 208 199 34
554 585 758 782 763 673 600 630 635 283 136
578 472 497 414 313 201 165 126 110 52 68
The KNboc is of the same order of magnitude for the three lipids (Table 1). The charge effect on the rate of decarboxylation of Nboc was investigated by increasing the molar fraction of DODAC in vesicles of PC, DMPC, and DPPC (Figures 2-4). The values of kψ increase with total amphiphile concentration, ([DODAC] + [phospholipid]), at any DODAC/amphiphile ratio, reaching a plateau at high amphiphile concentration. The values of kψ in this plateau kψmax depend on the DODAC/amphiphile ratio and on the phospholipid nature (Figures 2-4). The kψmax/kw ratios can be taken as a phenomenological description of the maximum observed catalytic effects when the mole % DODAC is varied in different lipid mixtures (Table 2). These data show that the apparent catalysis increases with mole % DODAC with all phospholipids. The data in Figures 2-4 were analyzed quantitatively using a pseudophase model with explicit consideration of ion exchange (PPIE), which adequately describes the effect of a charged supramolecular aggregate on the unimolecular decomposition of an oppositely charged substrate (Scheme 2).14 The PPIE model, as reviewed recently, has been previously applied to a series of chemical reactions in
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Scheme 2
vesicles.20 The rate of Nboc decomposition in charged vesicles can be analyzed with eq 3.14,20
kψ )
kw + kvKNboc/Cl•(Clb/Clf) (1 + KNboc/Cl•(Clb/Clf))
(3)
The subscripts f and b refer to the ion concentrations free in the bulk solution and bound to vesicles, respectively. KNboc/Cl is the ion-exchange constant between Nboc and Cl- and is given by14,20
Figure 5. Effect of the mole % DODAC of DODAC/PC vesicles on the rate of decarboxylation of Nboc: (b) kψmax; (4) kv.
KNboc/Cl ) Nbocb × Clf/Nbocf × Clb Clb and Clf are given by
Clb ) (1 - R)Cv - Nbocb
(4)
Clf ) RCv + Nbocb + Yad
(5)
where Clf and Clb are the analytical chloride concentration free and bound to the vesicles, respectively, and Yad is the chloride concentration added with the Tris/Cl buffer. In all experiments, Yad is 0.005 M. R, the degree of ion dissociation of the vesicle, is given by
R ) (1 - β) and β ) Clb/[DODAC]
(6) (7)
At each DODAC/lipid mole ratio we supposed that the number of sites in the vesicles is constant and that the major sites binding the Nboc are the positive amphiphiles. From the data obtained with pure phospholipids (see above), we conclude that the binding of Nboc to the phospholipid fraction is negligible at the lipid concentration used in experiments with mixtures. Note that in the experiments shown in Figure 1 the lipid concentrations are higher and the rates lower than those in Figures 2-4. Therefore, it can be assumed that in mixed DODAC/lipid vesicles the contribution of the reaction in the pure lipid phase can be neglected and the system can be analyzed using the PPIE model. Equations 3-6 were used to fit the data, yielding the lines in Figures 2-4. We took the value of R to be 0.078 (ref 21) and used as adjustable parameters kv and KNboc. Assuming that the DODAC concentration (Cv) accessible to Nboc is the total added DODAC, all kinetics can be fitted with a single value of 50 for KNboc/Cl. The kv best fit values, however, are strongly dependent on the %DODAC (Figures 5-7). Considering the variety of systems, the fits shown in Figures 2-4 are remarkably good. If we assume that only 60% of the DODAC is accessible to Nboc and use the value 0.13 for R, as obtained previously,21 a value of KNboc/Cl of 70 is obtained upon fitting all data in Figures 2-4. The values of kv, calculated for 100% DODAC accessibility, are very similar to those of kψmax, and both are plotted against the mole % DODAC for PC, DMPC, and (19) Cuccovia, I. M.; Kawamuro, M. K.; Krutman, M. A. K.; Chaimovich, H. J. Am. Chem. Soc. 1989, 111, 365. (20) Chaimovich, H.; Cuccovia, I. M. Prog. Colloid Polym. Sci. 1997, 103, 67. (21) Cuccovia, I. M.; Feitosa, E.; Chaimovich, H.; Sepulveda, L.; Reed, W. J. Phys. Chem. 1990, 94, 3722.
Figure 6. Effect of the mole % DODAC of DODAC/DMPC vesicles on the rate of decarboxylation of Nboc: (b) kψmax; (4) kv.
Figure 7. Effect of the mole % DODAC of DODAC/DPPC vesicles on the rate of decarboxylation of Nboc: (b) kψmax; (4) kv.
DPPC in Figures 5-7. With both PC and DMPC, kψmax and kv increase with % DODAC to values that are slightly higher than those obtained in pure DODAC vesicles. In contrast, the values of kψmax and kv with mixed DODAC/ DPPC vesicles increase with % DODAC, reaching the values obtained with pure DODAC vesicles. With all DODAC/amphiphile vesicles the increase of kψmax and kv is not linear with % DODAC, increasing sharply above 20% DODAC. The values of the kψmax/kw ratios for DODAC/ amphiphile mixtures are presented in Table 2. The rates of Nboc decomposition with vesicles presented so far were obtained at a single temperature, 30 °C. The physical state of the vesicle’s bilayer could be responsible for the dependence of kψmax and kv on amphiphile
Effect of Vesicles on Decarboxylation
Figure 8. Phase diagram for DODAC/PC vesicles: (2, 4) Tris/ HCl buffer; (b, O) water; (b, 2) beginning of transition; (4, O) end of transition.
Figure 9. Phase diagram for DODAC/DMPC vesicles: (2, 4) Tris/HCl buffer; (b, O) water; (b, 2) beginning of transition; (4, O) end of transition.
composition, since Tc changes with bilayer composition. The Tc’s of DODAC, DMPC, DPPC, and egg PC are 44,22 23,23 41.5,23 and -15 °C,24 respectively. The Tc’s of vesicles prepared in water and in Tris/HCl buffer with mixtures of DODAC with PC, DMPC, and DPPC were determined by turbidity measurements, as described in Methods. The Tc’s obtained for DODAC (Figures 8-10, 100% DODAC), DMPC (Figure 9, 0% DODAC), and DPPC (Figure 10, 0% DODAC) were comparable to literature values.22-24 The temperatures at the beginning and at the end of the transition at different DODAC/amphiphile ratios are presented in Figures 8-10. These plots are phase diagrams defining regions where the bilayers are in the gel state Lβ and the fluid state (liquid crystal) LR and regions where both the gel state and the liquid crystalline state coexist, LR + Lβ.25 The phase diagrams for DMPC and DPPC are not affected by the presence of buffer while for PC the phase transitions in buffer are sharper than in those water (Figures 8-10). For mixtures of DODAC and PC, we obtained Tc’s only above 40% DODAC. For PC, DMPC, and DPPC/DODAC mixtures, there is a large region where the gel and the liquid crystalline phases coexist, suggesting (22) Conroy, J. P.; Fox, K. K. Chem. Phys. Lipids 1995, 78, 129. (23) Marsh, D. CRC Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1990. (24) Jain, M. K.; Wagner, R. C. Introduction to Biological Membranes; John Wiley & Sons: New York, 1980. (25) Lee, A. G. Biophys. Biochim. Acta 1977, 472, 285.
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Figure 10. Phase diagram for DODAC/DPPC vesicles: (2, 4) Tris/HCl buffer; (b, O) water. (b, 2) beginning of transition; (4, O) end of transition.
phase separation with phospholipid-rich and DODACrich regions. As can be see in Figures 9 and 10, for both DODAC/DMPC and DPPC mixtures, the vesicle membrane is in the gel state at 30 °C, except for 10% DODAC/ DMPC. For DODAC/PC ratios above 40 mol % DODAC, vesicles exhibit LR + Lβ phases at 30 °C. To check the effect of Tc on the rate of decarboxylation of Nboc, we performed a limited amount of kinetics with DODAC/PC to verify if the gel-liquid crystalline transition of the bilayer determines the extent of vesicle catalysis (Figure 11). Rate constants of Nboc decarboxylation, as a function of temperature, were obtained at different DODAC % using a single total amphiphile concentration (0.01 M). No change in the slope of the Arrhenius plot was obtained, indicating that, under these conditions, vesicle catalysis does not depend critically on the detailed physical state of the bilayer. Amphiphile sonication does not necessarily produce vesicles.26 In fact, there is current controversy on the nature of the aggregates produced by pure sonicated DODAC, although vesicles have been clearly demonstrated using other preparation methods.16,26 In freeze-fracture experiments using several DODAC/lipid ratios, we could not demonstrate the presence of vesicles. Vesicle images were only obtained when the samples were prepared in the presence of 0.3 M trehalose (Figure 12). The replicas without trehalose were fragile, and only upon trehalose addition did we obtain manipulation-resistant replicas. The diameter of sonicated DODAC vesicles in the presence of trehalose, estimated from the EM images, was 50 nm, which is in good agreement with the diameter previously determined by light scattering for vesicles prepared without trehalose.21 Discussion Rate comparisons in micelles and vesicles have been made easy by the use of models that permit the evaluation of rate constants of the reaction in the aggregate.20,27 When a substrate such as Nboc distributes between an uncharged vesicle and a bulk aqueous solution, a simple distribution model permits the calculation of the association constant and the value of the rate constant at the interfacial reaction site.13 In pure lipids the association constant of Nboc was larger for PC vesicles, probably reflecting the larger fluidity of (26) Pansu, R. B.; Arrio, B.; Roncin, J.; Faure, J. J. Phys. Chem. 1990, 94, 796. (27) Fendler, J. H. Membrane Mimetic Chemistry; Wiley-Interscience: New York, 1982.
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Figure 11. Arrhenius plots for the decarboxylation of Nboc in DODAC/PC vesicles. Mole % DODAC: (a) 100%; (b) 70%; (c) 50%; (d) 40%; (e) 30%; (f) 20%. The total amphiphile concentration was 0.01 M.
PC at 30 °C, when compared with DMPC, despite both bilayers being in the liquid crystalline state at that temperature.23,24 Although the bilayer of DPPC is in the gel state at 30 °C,23 the value of the association constant of Nboc was equal to that obtained in DMPC vesicles (Table 1). These data, taken together, suggest that Nboc does not penetrate deeply into the hydrophobic segment of the bilayer. The three lipids catalyzed Nboc decarboxylation, and the largest catalytic factor was obtained with DMPC (Table 1). A comparison of the data in Figure 1 with that in Figures 2-4 shows that Nboc associates more to mixed vesicles containing DODAC than to vesicles prepared with pure lipid, since maximum rate constants are obtained at much lower amphiphile concentrations. Added DODAC, therefore, contributes with an added electrostatic factor to Nboc binding. The values of rate constants in the vesicle for substrates oppositely charged to the interface were obtained using pseudophase models with explicit consideration of ion exchange.14 Using PPIE for the analysis of Nboc decomposition in vesicles, we made a number of assumptions regarding the value of the extent of ion dissociation from the interface (R) as well as the relative composition of the external and internal faces of the bilayer. Notwithstanding, we obtained excellent fits of the equations describing the PPIE model and the experimental data (Figures 2-4). Given the assumptions made in the quantitative analysis, the values of the calculated
kv’s cannot be taken as absolute but can be confidently used as a tool to compare the effect of the vesicles on the Nboc decarboxylation rates. As expected for a substrate that tightly binds to the interface, the best-fit values of kv are similar to the values of kψmax.14 Another feature, resulting from the quantitative analysis of the effect of vesicles on the rate, is that the best-fit values of KNboc/Cl are the same with the three lipids, suggesting that the preferential adsorption site of Nboc is ion-pair formation with DODAC at the interface. Catalysis by mixed DODAC/lipid vesicles, as estimated by the kψmax/kw ratios, reached approximately 800-fold with 70% DODAC and DMPC, suggesting that in that mixture Nboc is localized in a particularly hydrophobic solubilization site, not present in other DODAC phospholipid mixtures. It is well established that the Tc of mixed membranes changes with composition.24,25 With mixed lipids the variation of Tc with composition does not lead to ideal behavior unless the chain lengths and head groups are very similar.24,25 In the present case both the chain lengths and the head group structures of the mixtures are different. Hence, the changes in the vesicle effect on decarboxylation rates could reflect simply a change in the state of the bilayer. Two independent evidences, presented here, strongly suggest that the effect of increasing the % DODAC on the decarboxylation rate is not related directly to thermotropic changes of the vesicle bilayer. First, the
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Langmuir, Vol. 16, No. 3, 2000 999 Chart 1
Figure 12. Freeze-fracture electron micrographs of DODAC vesicles prepared in 0.3 M trehalose. A, B, and C are different regions of the same preparation.
best-fit values of both association constants with the pure lipid vesicles and the best-fit values for the selectivity constants indicate that the substrate resides preferentially at the interface. The phase diagrams indicate that at 30 °C essentially all DODAC/lipid mixtures are composed of phase-separated bilayers containing both phospholipidand DODAC-rich regions. Moreover, the effect of temperature on the rate did not show discontinuities at the expected Tc for the DODAC/PC vesicles. Previous work, using the same reaction, has shown discontinuities, at the Tc, in Arrhenius plots for aggregates formed by bromide salts of derivatives of (Cn)2N+(CH3)2, particularly for n ) 12, 14, and 16.11a The discontinuity was less pronounced for (C18)2N+(CH3)2Br-(DODABr).11a This discrepancy may be attributed to experimental differences related to (a) the nature of the counterion (chloride versus bromide), (b) the salt concentration (0.016 M KCl and 0.02 M borate buffer versus 0.005 M total chloride), and, most importantly, (c) the total amphiphile concentration (1 mM versus 10 mM), which affects mainly the degree of Nboc binding to the aggregate.11a The medium sensitivity of the rate of decarboxylation of Nboc led to a widespread use of this reaction as a probe of medium effects in micelles, bilayers, macrocyclic hosts, polymers, and catalytic antibodies. A variety of factors ranging from solvent effects on the dissociation of the initial ion pair between the Nboc anion and the counterion to the relative stabilization of the initial and transition states determine the overall medium effect on the reaction rate. The influence of solvent donor-acceptor properties and polarity on the rate of decarboxylation of Nboc has been analyzed with several models such as the unified solvation model.3 The observation of rate changes upon transferring the reaction site from bulk solution to the interface of a supramolecular assembly cannot be interpreted simply in mechanistic terms and directly related to a single property of the interfacial reaction site. In water, the relevant Nboc species is a separated ion pair, and for such species hydrogen bonding to the initial state sharply decreases the rate.3 Transferring the reaction site to the interface of a vesicle can lead to several
consequences which will depend not only on the medium but also on the relative charge on the reaction site as well as the detailed substrate position at a site where properties vary sharply over molecular distances.27 The change in the kv values with the increase in the DODAC/amphiphile ratio can be ascribed to a change in the position of the phospholipid monomer in the vesicle with the increase of the DODAC concentration. At low DODAC/amphiphile ratio the Nboc can bind at the ammonium group of the phospholipid that is located near the aqueous phase. As the relative proportion of DODAC increases, there will be an increasing tendency for Nboc to bind at a DODAC head group. Nboc can then occupy a more internal site, nearer to the hydrophobic core of the membrane. The change in microenvironment going from a more hydrated to a more hydrophobic environment can lead to the change in kv. In Chart 1 we represent the possible positions of Nboc under conditions of low DODAC and high DODAC/phospholipid ratios. Previous analysis of this reaction, contrasting micellar and vesicle-forming ammonium amphiphile effects on Nboc decarboxylation, has suggested that the interaction mechanisms between the aggregate and the initial state and the transition state, respectively, are different.11b It is tempting to speculate that the differences of the initial and transition state associations with the vesicle are most important for DODAC, explaining therefore the increase in catalysis with the DODAC %. Acknowledgment. Granting Agencies: FAPESP, CNPq, and Pro-Reitoria de Pesquisa da USP. M.V.S. was a recipient of PICDT- CAPES and CNPq fellowships. We thank He´lio Correˆa, from Laboratory of Molecular Pathology, Department of Pathology of Faculty of Medicine, USP, for his invaluable assistance in the preparation of freeze-fracture replicas. LA9907477
