J. CHEM. SOC. FARADAY TRANS., 1994, 90(18), 2717-2724 Effects of Surfactant Charge and Structure on Excited-state Protolytic Dissociation of 1=Naphthol in Vesicles Yurii V. Il'ichev, Kyrill M. Solntsev and Michael G. Kuzmin" Department of Chemistry, Moscow State University, 117234 Moscow, Russian Federation Helge Lemmetyinen Institute of Materials Chemistry, Tampere University of Technology, P.O.Box 589,FIN33101 Tampere, Finland ~ ~ ~ ~~~~~~~~~ The kinetics of protolytic photodissociation of 1-naphthol in the bilayer membrane of cationic vesicles of di- dodecyldimethylammonium bromide (DDAB) and dioctadecyldimethylammonium bromide (DOAB) have been studied in comparison with the same reaction in vesicles of a zwitterionic lipid dipalmitoylphosphatidylcholine (DPPC).Evidence for the existence of two fractions (two types of site) of 1-naphthol molecules in vesicles of cationic surfactants which differ strongly in their rate constants for excited-state proton transfer was found, similar to the case for zwitterionic vesicles. The rate constants of the excited-state proton transfer for both fractions are much higher in bilayer membranes of cationic surfactants than for zwitterionic lipids (DPPC and egg lecithin). The activation enthalpy of excited-state proton transfer (ESPT) for both fractions of ArOH in the membrane of DDAB is ca. 40 kJ mol-', which is much higher than in homogeneous solutions and zwitterionic surfactants. Fluorescence kinetic data for DOAB vesicles allow no reliable conclusions to be drawn as to the temperature dependence of excited-state protolytic dissociation rate constants in these vesicles because the reaction rate is too fast. No significant decrease in the excited-state proton-transfer rate constants at the mem- brane phase-transition temperature of vesicles of cationic surfactants is observed, in contrast to the zwitterionic lipids.All these features characterize distinctions between the properties of the membranes of the vesicles of cationic and zwitterionic surfactants in proton-transfer reactions. Proton transfer, being a fundamental chemical reaction, con- stitutes an important step in many processes in cellular biology.' The elucidation of the dynamics and mechanism of this reaction is the key to understanding how proton-controlled processes are realized in biological membranes.Protolytic photodissociation (proton transfer between an acid in the singlet excited state and water molecules) provides a simple model reaction for proton-transport processes in membranes and could provide a deeper insight into the mechanisms of very complex systems which function in bio- membranes. Steady-state and time-resolved fluorescence measurements have been used extensively to study the kinetics of excited- state protolytic dissociation in proteinsg." and other microheterogeneous systems which can be considered as models of biomembranes [reversed micelles and water: in-oil (w/o) microemulsions,' '-" micelles,' 7-33 and o/w micro emulsion^^^]. The aim of this work was to study the kinetics of protolytic photodissociation of 1-naphthol in a bilayer membrane of cationic vesicles and compare them with the same reaction in zwitterionic lipid vesicles.Our prefious studiedp8 demon- strated some peculiarities in naphthol photodissociation in bilayer membranes of some phospholipids [egg lecithin (EL) and dipalmitoylphosphatidylcholine (DPPC)] when com-pared with aqueous solutions and other microheterogeneous systems. Time-resolved fluorescence data suggested the exis- tence of two fractions (two types of localization site) of naph- thol molecules (ArOH) in lipid membranes. These fractions differ strongly in their rate constants for excited-state proton transfer. Furthermore, temperature-dependent measurements pointed to a strong effect of phase transitions in DPPC bilayer membranes on ArOH photodissociation.8 The physi- cochemical nature of these two localization sites was not identified.Two possibilities, transmembrane and lateral inho- mogeneity of the lipid bilayer, were discussed.8 To obtain additional information on these phenomena we studied the reaction in bilayer membranes with a different structure and charge. We used artificial cationic surfactants with different chain lengths and, hence, different main phase- transition temperatures (T,: didodecyldimethylammonium bromide (DDAB, T', = 17"C) and dioctadecyldimethylam- monium bromide (DOAB, T', = 35°C).35 Data obtained in these systems were compared with kinetic results in vesicles of zwitterionic phospholipids (DPPC and EL).Experimental 1-Naphthol was purified by vacuum sublimation (ca. 10 Pa for 5 days). Agreement of the absorption and fluorescence spectra with literature data and single-exponential fluores- cence decay in several solvents served as criteria of purity. DPPC (99%)was purchased from Sigma. DDAB and DOAB both from Eastman-Kodak, were kindly provided by Prof. F. Menger (Atlanta University, Georgia, USA). All surfactants were proved to be highly free from fluorescent impurities and were used as received. Highly purified deionized water (Millipore MilliQ) was used to prepare solutions for fluores- cence measurements. The overall concentration of the naph- thol in solutions did not exceed mol 1-' in any of the experiments. To prepare vesicles we use the injection method proposed and tested for zwitterionic phospholipid^^^*^' and cationic surf act ant^:^^ an ethanol solution (80 mmol 1-', 0.15 ml) of a surfactant was injected into an aqueous solution (2.85 ml) of 1-naphthol.Aqueous solutions were heated to ca. 50°C before ethanol was injected into the solutions. The pH was controlled by an ionometer with a glass electrode and was ca. 6 in all cases. It was shown previ~usly~~-~~that this method gives monodisperse stable unilamellar vesicles. The size of vesicles is strongly dependent on the initial concentration of the sur- factant in the alcohol, e.g. the diameter of the DPPC vesicles varies from 30 to 120 nm for the concentration range 3-40 mmol 1-'.36 Although we did not specially determine the diameter of the DPPC vesicles, we used a higher concentra- tion of surfactants (ca. 80 mmol 1-') in an attempt to achieve the complete solubilization, thus the size of our DPCC vesi- cles is similar to the largest value reported.36 The diameter of EL vesicles obtained by this method in our previous work' was ca. 150 nm. The diameter of the vesicles of DDAB was shown36 to vary from 220 to 320 nm for an initial concentration of surfactant in ethanol in the range 0.29-0.87 mol 1-'. The diameter of the vesicles obtained in the present work was assumed to be <200 nm since we used a much lower concentration of DDAB (0.08 mol 1-'). The hydrodynamic diameters of the cationic vesicles were determined by quasielastic light scat- tering (QELS) using a 1096 Correlometer (Langley-Ford).? The autocorrelation function of fluctuations in the scattering intensity was analysed by the method of cumulants.Hydro- dynamic diameters were calculated from the directly deter- mined diffusion coefficients of the vesicles. Vesicular solutions were freed from dust by filtration through a 1.2 ,um Millipore filter. No significant loss of naphthol or change in vesicular size occurred after filtration. At 20°C the diffusion coeff- cients and the hydrodynamic diameters of the vesicles were 1.0 x lo-' cm2 s-' and 420 nm for DDAB and 1.9 x lo-' cm2 s-' and 220 nm for DOAB. Unexpectedly, our method of preparation of vesicles produces small unilamellar DOAB vesicles and large DDAB vesicles (we have no direct evidence that they are unilamellar). Ultrasonication of opalescent DDAB solutions using a Branson 1200 sonicator for 30 min at 50°C did not change the opalescence of the solution and fluorescence spectra of 1-naphthol in this solution.To evaluate the possible effects of the size of the vesicles on the temperature dependence of the ESPT rate constants we measured the temperature dependence of the hydrodynamic diameter of the vesicles. The diameter of DPPC vesicles is Inknown not to be very sensitive to temperat~re.~~ our experiments the diameter of DOAB vesicles decreased by only 20-25 nm when the temperature was increased from 20 to 55"C, but for DDAB vesicles a significant decrease of the diameter occurred from 420 to 190 nm when the temperature was increased from 20 to 55°C. This effect was accompanied by a small long-wavelength shift (8 nm) of the ArO- fluores- cence maximum and the disappearance of opalescence.Absorption spectra were recorded on a Shimadzu MPS-2000 spectrophotometer. Fluorescence and excitation spectra were measured with a Shimadzu RF-5000 spectrofluorimeter (both slits being 1.5 nm). Fluorescence decay curves were recorded with a time-correlated single-photon-counting instrument (Edinburgh Instruments 199). A synchronously pumped, cavity-dumped dye laser (Spectra-Physics Model 375; an Nd :YAG laser as a pump source) frequency-doubled with a KDP crystal (model 390; A= 310 nm) was used as an excitation source (pulse width <100 ps).In most experiments the typical time resolution was 24.2 ps per channel. An apparatus function was registered at the excitation wave-length. Decay curves were analysed by the non-linear least- squares method using the Edinburgh program for the IBM PC. The accuracy of fits was estimated by the x2 parameter and by inspecting the weighted residuals. Decay curves were deconvoluted as multiexponential functions with a variable time shift between an exciting pulse and fluorescence curve. Results and Discussion The fluorescence spectra of 1-naphthol in water and vesicle suspensions of various surfactants at the room temperature, 7 The authors are grateful to A.A. Yaroslavov and M. F. Zanso-hova (Dept. of Polymer Sciences, Moscow University) for these mea- surements. J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 at the same surfactant concentration (4 mmol 1-and pH x 6 are shown in Fig. 1. An excitation wavelength of 315 nm was used. Fluorescence kinetics in ArOH and ArO -emission bands in DOAB, DDAB and DPPC vesicular solutions at the same conditions are shown in Fig. 2. 1-Naphthol in the S, state is well known4' to be a very strong acid (pK,* = 0.41) and to dissociate in aqueous solu- tion with a very high rate con~tant,4~*~~ k, = (2-3) x loio s-'. Therefore the fluorescence obtained from ArOH excita- tion in water consists almost entirely of the broad emission band of *ArO- formed due to excited-state protolytic disso- ciation.In contrast to water, a distinct short-wavelength band is present in the fluorescence spectra of 1-naphthol in vesicles. In addition, a blue shift of the naphtholate anion emission band is observed in these aggregates. No noticeable variation in fluorescence excitation spectra was observed for different emission wavelengths (360, 440 and 480 nm) in the systems investigated. The excitation spectra (Fig. 1) confirm that naphthol in the ground state, exists only in the proto- nated form and *ArO- formation is a result of ArOH photo- dissociation. At room temperature the ArO- to ArOH fluorescence intensity ratio (Z'/Z) was found to increase in a series of sur- factants: DPPC < DDAB < DOAB.This change in Z'/Z indi-cates an increase in the proton-transfer rate constant in this series. To discover the effect of the nature of the bilayer on excited-state proton-transfer reactions we studied the fluores- cence kinetics of *ArOH and *ArO- in suspensions of these vesicles at various temperatures. Note that because of the very fast (< 0.05 ns40-42) photodissociation of ArOH in the aqueous phase, the fraction existing in the volume phase will not contribute to the ArOH fluorescence kinetics observed in the solution of vesicles. It was previously shown' that the fluorescence spectra of 1-naphthol in a suspension of DPPC vesicles depend on the DPPC concentration because of the incomplete binding of ArOH to bilayer membranes at the low lipid concentrations. With increasing DPPC concentration Z'/Z decreased along with ArO-fluorescence, the maximum being shifted to shorter wavelengths.This indicates more complete solu- bilization of naphthol and retardation of the ESPT efficiency. The fluorescence kinetics of *ArO- at [DPPC] < 3 mmol 1-' was described by a triexponential function with two decay times, the fast decay time being equal to the anion life- time in aqueous solutions and the slow decay time being close to the anion lifetime in non-ionic micelles. In this work in cationic vesicles of DDAB and DOAB we also found that /'/I decreased with increasing surfactant con- h 4-.-C 3 300 400 400 500 L/nm Fig. 1 Fluorescence excitation (A) and emission (B) spectra of 1-naphthol in water (a)and vesicles of various surfactants: DPPC (b), DOAB (c) and DDAB (4; A,, = 480 nm for excitation spectra; Ae, = 3 15 nm for emission spectra; T = 20 "C J.CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 v)c v) C cC 8 8 channels channels channels !:,),I1 ,,,,(, I .....................I. , , , , , 0.00 100.0 200.0 300.0 400.0 500.0 Fig. 2 Fluoroescence decay curves of 1-naphthol(Aern = 370 nm, ArOH) and its anion (Aern = 480 nm, ArO-) at 20°C and pH z 6 in vesicle suspensions of various surfactants ([surfactant] = 4 mmol 1-l): (a)DDAB, (b)DOAB, (c)DPPC. 24.2 ps per channel. centration, but in contrast to zwitterionic vesicles no varia- tion in the ArO-fluorescence maximum was detected. Moreover, at all the surfactant concentrations studied, the fluorescence kinetics of *ArO- can be fitted by a biexponen- tial function with one rise time and one decay time (Table 1).These effects, together with a noticeable long-wavelength shift and a distinct change in the vibration structure in the excita- tion spectra measured in vesicles (Fig. 1) imply that most of 1-naphthol and its anion is located in bilayer membranes of vesicles (a less polar medium than water). Thus we suggest ki *ArO,-+ H+ -*ArOH, factant concentration can be explained mainly by changing the structure of the vesicles but not by the greater binding of naphthol. Previously6-’ we found that the fluorescence kinetics of naphthols in the membranes of DPPC and EL vesicles show the existence of two fractions of ArOH possessing different reactivities [probably localized in different parts of ‘heterogeneous’ (with respect to proton transfer) membranes] and discussed the kinetics of ESPT reactions in vesicles in terms of the extended pseudophase model: a vesicle suspen- sion with ArOH was considered to consist of a bulk water phase and two different microphases of the lipid membrane. Excited-state protolytic dissociation of these two fractions of ArOH solubilized in the membrane at neutral pH is described by the following scheme: ki 1 *ArOH,, -ArO,,- + H+ Ar0,-+ H+ -ArOH, IArOH,,-ArO,,-+ H’ Scheme 1 J.CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 Table 1 Parameters of the fluorescence decay curves of 1-naphthol and its anion in vesicles of various surfactants ArOH ArO -T/"C cfmmol I-' zl/ns z2/ns 7Jns AJCA, A2/XAi x2 7;fn.s z;/ns AJA, x2 zo/nsa DDAB, phase-transition temperature 17 "C 11 4.0 1.10 2.24 0.69 0.31 1.22 1.02 21.21 -0.86 1.59 5.25 15 4.0 1.11 2.2 1 0.74 0.26 1.86 -' ---5.10 24 4.0 0.60 1.25 0.52 0.48 1.05 0.75 15.56 -0.80 1.34 4.72 35 4.0 0.24 0.73 1.49 0.36 0.59 1.11 0.50 13.23 -0.75 1.30 4.26 45 4.0 0.20 0.53 3.10 0.74 0.26 1.03 0.26 11.89 -0.65 1.31 3.86 55 4.0 0.14 0.32 3.33 0.67 0.33 1.15 0.16 9.90 -1.17 1.24 3.59 25 2.5 0.62 1.30 0.67 0.33 1.29 0.60 14.34 -0.79 1.76 (0.76 18.31 -0.67 1.72)* 25 4.0 0.67 1.36 0.60 0.40 1.14 0.67 20.00 -0.87 1.92 (0.80 18.79 -0.84 1.64) 25 5.5 0.78 1.49 0.69 0.3 1 1.22 0.73 --0.7 1 1.43 (0.95 19.05 -0.77 1.59) DOAB, phase-transition temperature 35 "C 15 4.0 0.15 0.67 1.64 0.29 0.67 1.18 0.49 17.22 -0.69 1.22 5.10 25 4.0 0.14 0.52 1.88 0.32 0.67 0.98 0.43 15.77 -0.64 1.27 4.68 35 4.0 0.17 0.48 2.02 0.33 0.66 1.08 0.36 14.34 -0.70 1.17 4.26 ---4.0041 4.0 0.14 0.46 1.46 0.39 0.58 0.98 -55 4.0 0.2 1 0.61 2.87 0.69 0.28 1.10 0.33 12.1 1 -0.64 1.35 3.59 25 2.5 0.14 0.50 2.63 0.35 0.64 1.02 0.40 14.71 -0.62 1.33 25 5.5 0.19 0.67 2.65 0.30 0.6 1 1.17 0.55 17.70 -0.69 1.05 DPPC, phase-transition temperature 41 "C 22 4.0 1.26 4.78 0.37 0.63 1.51 0.51 11.26 -0.25 1.25 4.8 1 28 4.0 0.87 4.00 0.32 0.68 1.21 0.44 1 1.03 -0.30 1.40 4.55 31 4.0 1.07 4.32 0.36 0.64 1.14 0.48 11.60 -0.30 1.45 4.42 40 4.0 1.29 3.97 0.35 0.65 1.29 1.08 10.87 -0.50 1.32 4.03 55 4.0 1.00 3.18 0.43 0.57 1.17 0.84 11.34 -0.41 1.39 3.59 (I Lifetimes of 1-naphthol in pentanol.' Large values of x2 are due to modulation of decay curve by sine-shaped noise. All missing data are due to unsatisfactory results of fitting.Data in parentheses were obtained at lower time resolution (190 ps per channel) in order to record the longer-lived component more accurately. Here ArOH, and ArOH,, are two fractions of the ground- room temperature we found a biexponential decay. A third state naphthol molecules (solubilized in two different types of exponent with a very small amplitude appeared only at high site); k, and k,, are the excited-state protolytic dissociation temperatures (Table 1, Fig. 3). In DOAB vesicles a much rate constants for these two fractions; zp, zg and z;', zip are better fit was found for the triexponential approximation at the lifetimes of *ArOH and *ArO-, respectively, in the all temperatures, although the amplitude of the exponent absence of protolytic reactions in these sites. This scheme with the longest decay time (1.5-3 ns, depending on the assumes that exchange between these two types of site is too temperature) was <6% (Fig.4, Table 1). The amplitude of slow (compared with the rates of the excited-state reactions) this exponent increased markedly with the surfactant concen- to influence the kinetics of these reactions. At pH x 6 the tration (Table 1). A third exponent can probably be attrib- reverse reaction of *ArO- protonation can be neglected.For uted to the fluorescence of surfactant impurities. This each fraction of ArOH molecules standard expression^^^ for component was neglected in the discussions of excited-state the fluorescence quantum yields of ArOH (6)and ArO (6') proton transfer. should be valid: The fluorescence kinetics of the ArO- anions was fitted with reasonable accuracy by a biexponential function and can be described by a rise time (z;) and a decay time (zi). The and rise time of the ArO- fluorescence is close to the faster decay times of the ArOH fluorescence; however, in all cases the rise l/z = 1/ro+ k (2) time is slightly longer than the decay time. At the same time, the anion decay time is much longer than any of the *ArOH where +o and 4; are the fluorescence quantum yields of decay times.This provides evidence for the irreversibility of ArOH and ArO-, respectively, in the absence of the protoly- *ArOH dissociation at pH x 6. In principle, the fluorescence tic reaction; z is the observed lifetime of *ArOH. kinetics of *ArO- formed via the protolytic dissociation of According to Scheme 1 the fluorescence decay of ArOH (8') ArOH in both the microphases and in the bulk phase should is described by a biexponential function : be described by three rise times and three decay times. 2 However, we can observe only the fastest rise time in one of F(t) = 1 Aiexp(-t/zi) (3) the microphases (the rise time in the aqueous phase, as was i= 1 already mentioned, is too short to be recorded) and one where cli = Ai/C Ai are the relative concentrations of each decay time.The decay times of *ArO- in both microphases fraction, which are equal to the relative initial amplitudes of are probably similar. A substantially shorter decay time of each exponent of the ArOH fluorescence. We fitted the ArOH *ArO- in the aqueous phase (ca. 10 ns) can be hidden owing fluorescence decay kinetics as biexponential and tri-to the relatively small fraction of ArOH present in the esponential functions. Table 1 lists the parameters obtained volume phase (it can be observed as a second decaying com- for the ArOH and ArO- decay curves. For DDAB vesicles at ponent at low surfactant concentrations). Because of these J. CHEM. SOC. FARADAY TRANS., 1994, VOL.90 3 n channels Fig. 3 Fluorescence decay curves of 1-naphthol (ArOH) in DDAB vesicles at various temperaturesrc (top): (a) 11, (b) 15, (c) 24, (6)35, (e)45, (f)55. Results of fitting (weighted residuals and values of xz) are presented for each temperature (bottom). xz: (a) 1.18, (b) 1.86, (c) 1.05, (d)1.11, (e) 1.03, (f) 1.15; (a)-(c) biexponential fit, (d)-(f) tri-exponential fit. complications the fitted amplitude of the rising exponent was smaller than the amplitude of the decaying exponent. The excited-state protolytic dissociation rate constants for both fractions of ArOH, k, and k,,, were calculated from the decay times in the following way k, = l/z, -1/zp (4) k,, = l/z, -1/z; (5) where the decay times in the absence of protolytic reactions, zp and zg, for both fractions were assumed to be identical and equal to the lifetime of *ArOH in pentanol, which has a vis- cosity and relative permittivity similar to those in the interior of the membranes (this lifetime is known to be slightly sensi- tive to the nature of the solvent, if not protolytic photoreac- tion occurs, and sensitive to the temperature).Table 2 lists the rate constants obtained for ESPT in DPPC, DDAB and DOAB vesicles at various temperatures. All these calculations of ki assume that radiationless decay processes for all fractions of ArOH* in the bilayer vesicles are the same as those in a viscous solvent such as pentanol. To I - I I 1 0 100 200 300 400 channels 3 0 -4 c I 0 100 200 300 400 channels 13L3 .I 1 0 -2 Fig. 4 Fluorescence decay curve of 1-naphthol (ArOH) in DOAB vesicles at 25°C fitted by biexponential (top, x2 = 1.25) and tri- exponential (bottom, x2 = 0.98) functions. Weighted residuals are also shown. check this assumption and to exclude the possibilities of other sources of the acceleration of decay of ArOH* in the microphase we compared (Fig. 5) the temperature depen- dences of the relative fluorescence quantum yields of ArOH and ArO- obtained from the fluorescence spectra and those calculated from kinetic data obtained by single-photon counting : For each surfactant we used as To the lowest temperature of the experiment: 11 “C for DDAB and 15 “C for DOAB.The coincidence between $J(T)/+(To) measured from the fluorescence spectra and calculated from the kinetic data (zi and a,) at various temperatures reflects only a quantitative consistence between spectral and kinetic data, but the coin- cidence between the measured and calculated values of g5’(T)/g5’(To)confirms that the decrease in zi at higher tem- peratures is really caused by an increase in the proton- transfer rate constants k’; rather than by an increase in any 2722 Table 2 Rate constants of ESPT of 1-naphthol in vesicles of various surfact ants' T/T kJ1OP8 s-l k,,/lO-Bs-l k'/10 -8s-1 DDAB (4 mmol l-'), phase-transition temperature 17"C 11 7.2 2.6 8.0 C15 7.0 2.6 -24 14.6 5.9 11.2 35 39.2 11.4 17.6 45 47.4 16.3 35.9 55 68.7 28.5 59.7 DOAB (4 mmol I-'), phase-transition temperature 35 "C 15 64.7 12.9 18.5 25 69.4 17.1 -35 56.2 8.5 25.5 41 68.7 19.3 -55 44.7 13.6 27.6 DPPC (4 mmol 1-I), phase-transition temperature 41 "C 22 5.9 <0.1 17.5 28 9.3 0.3 20.5 31 7.1 <0.1 18.5 40 5.3 <0.1 6.7 55 7.2 0.4 9.1 ~~ (I All values of rate constants were calculated by using eqn.(4) and (5). k' values were calculated using the rise time of ArO- in In. (5). 1.20 I B 1 1.00 [ I s kY E .. 0.60' s v 0.40 E a 0.20 E 0.000.00 '10.0 20.00 30.00 40.00 50.00 60.00 TI"C Fig. 5 Temperature dependences of relative fluorescence quantum yields of ArOH[(a), (b)] and ArO- [(c), (43 obtained from fluores- cence spectra [(a), (c)] and calculated by using eqn.(6) and (7) from time-resolved measurements [(b),(41in DDAB (A) and DOAB (B). Dashed lines show the phase-transition temperatures (1 7 "C for DDAB and 35 "C for DOAB). J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 radiationless decay processes [induced internal conversion or intersystem crossing, quenching by counterion of surfactant (Br-), or by impurities etc.]. The temperature dependence of the measured and calculated +'( T)/+(T) presents an apparent activation enthalpy averaged over all fractions of ArOH. A difference between cationic vesicles and zwitterionic lipid vesicles is evident from the comparison of the data listed in Table 2 and in Fig. 6-8. This is especially true for DDAB and DPPC since the data for DOAB are less precise owing to the very short lifetime of the first fraction of *ArOH.The first observation is that the protolytic dissociation rate constants are much higher in bilayer membranes of cationic vesicles than those in phosphatidylcholine and EL mem-branes. This effect is more pronounced for the fraction of naphthol molecules characterized by the lower proton-transfer rate constant kI,. This is why both ArOH fractions dissociate in DDAB and DOAB bilayers with comparable rate constants even at room temperature. The higher values of the protolytic dissociation rate constant in the cationic vesicles than those in zwitterionic vesicles are in good agree- ment with the data in micelles: in cationic micelles the disso- ciation rate constants are an order of magnitude greater and pK,* values are one unit lower than in non-ionic mi~elles~~ 23.00 A 22.00121 .oo -20.00 1 .oo B 0.80 0.60 E 0.40 I0.20 3.00 3.10 3.20 3.30 3.40 3.50 3.60 103 KIT A, Arrhenius plots of the protolytic photodissociation rate constants of 1-naphthol in DDAB vesicles calculated by using eqn.(4) and (5) from the decay times of *ArOH [(a) and (b)] and from the rise time of *ArO- (c)and of the relative fluorescence quantum yields (4, [In (#'/#), # and #' are in relative units, the ordinate scale is shifted arbitrarily]. B, Dependence of the relative contribution of each ArOH fraction us. inverse of the temperature: (a) APA,, (b) A,/ZA,. The dashed line shows phase transition temperature (17"C).J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 23.0 I 22.01 1 .ooE R I U I I I Io.*oj I I 0.60; 0.40: 0.20 0.00 3.00 3.10 3.20 3.30 3.40 3.50 103 KIT Fig. 7 A, Arrhenius plots of the protolytic photodissociation rate constants of 1-naphthol in DOAB vesicles calculated by using eqn. (4) and (5)from decay times of *ArOH [(a)and (b)] and from the rise time of *ArO- (c) and of relative fluorescence quantum yields (d) [In(+'/+), + and 9' are in relative units, the ordinate scale is shifted arbitrarily]. B, Dependence of the relative contribution of each ArOH fraction us. the inverse of the temperature: (a) A,/ZA,, (b) A,/ZA,. The dashed line shows the phase-transition temperature (35 "C).because of the electrostatic interaction contribution to the Gibbs energy of the reaction. In DDAB and DOAB vesicles no essential change of the rate constants of excited-state protolytic dissociation was found at the main phase-transition temperature in contrast to DPPC vesicles (Fig. 6-8). The amount of the fraction of naphthol molecules that have a lower protolytic dissociation rate constant in cationic vesicles in the liquid-crystalline state (at temperatures higher than the phase transition) decreases rapidly with temperature in contrast to the situation with zwitterionic lipid vesicles, where it is almost constant. Some increase in the fraction with lower rate constants is observed at a phase-transition temperature only for DDAB vesicles (Fig.6). Moreover, a substantial difference was found between DDAB and DOAB vesicles. A very strong increase of both the rate constants with increasing temperature was observed in the liquid-crystalline state of DDAB vesicles, which indi- cates a relatively high activation enthalpy of the proton transfer. Below the phase-transition temperature of DDAB vesicles (17"C) the rate constants were measured only for two temperatures (1 1 and 15 "C) and they are close to each other. 23.00 A C- 21.00 20*oolc 19.00~~""""~""""'~""""3.00 3.10 3.20 !"""3.30 3.40 lo3 KIT 1.oo t , DIDI 0.80 0.201, aI,, ,,,,,,I, ,,,,,( ,';,;,, , ,,,()(,,, 0.003.00 3.10 3.20 3.30 3.40 103 K/T Fig.8 A, Arrhenius plots of the protolytic photodissociation rate constants of 1-naphthol in DPPC vesicles calculated by using eqn. (4) and (5) from the decay time of *ArOH (a) and from the rise time of *ArO-(b). B, The dependence of the relative contribution of each ArOH fraction us. the inverse of the temperature: (a) A,/ZA,, (b) A,/XA,. The dashed line shows the phase-transition temperature (41"C). Fig. 6 shows the temperature dependence of the excited- state protolytic dissociation rate constants in an Arrhenius plot. In the liquid-crystalline state of DDAB vesicles a linear dependence with activation enthalpies of AH' 39 and 40 kJ mol-' was observed for the faster and slower dissociating fractions, respectively. These values are much higher than those in the zwitterionic vesicles of DPPC and EL, where the activation enthalpies for k, were 9 and 21 kJ mol-' for the gel and liquid-crystalline states of the DPPC bilayer, respec- tively.' In homogeneous solutions and in micelles the activa- tion enthalpies of protolytic dissociation are of the same order of magnitude, 10-25 kJ mol-l.'The abnormally high activation enthalpy for DDAB vesicles can be attributed par- tially to the decrease in the size of the vesicles with increasing temperature, as was mentioned in the Experimental section. The temperature dependence of 4'/4 (Fig. 6) yields an apparent activation enthalpy of AH* = 17 kJ mol-'. This is smaller than the activation enthalpies obtained from the kinetic data because of the substantial decrease of the frac- tion of slowly dissociating ArOH molecules with increasing temperature.In DOAB vesicles some modest irregular variations of both k, and k,, with temperature are observed at tem- 2724 peratures below and above the phase-transition temperature. As was mentioned earlier, the deconvolution of the kinetic curves is much less accurate for DOAB vesicles than for DDAB and DPPC vesicles due to the substantially faster decay. The evaluated activation enthalpy is <10 kJ mol-'. The ratio of the total fluorescence quantum yields 4'/4 (Fig. 7) is almost constant over the whole temperature range from 15 to 55"C, which confirms the low value of the activation enthalpy of the protolytic dissociation in DOAB vesicles.All these features characterize differences in the properties of membranes of the vesicles of cationic and zwitterionic sur- factants and demonstrate the possibility of using ESPT lumi- nescent probes for investigations of the structure and properties of membranes. Nevertheless, for a more com-prehensive discussion of the solubilization effects in vesicles on the kinetics of the protolytic reactions, more detailed information on the localization of the probes in the bilayer is necessary. Conclusion Two types of site for 1-naphthol molecules which differ strongly in their ESPT rate constants were found to exist in the membranes of cationic vesicles of DDAB and DOAB. This situation is similar to that seen for vesicles of zwitter- ionic lipids (EL and DPPC). The ESPT rate constants for both sites are much higher in bilayer membranes of cationic surfactants than those in zwitterionic surfactants because of the electrostatic interaction contribution to the Gibbs energy of the reaction. The fraction of the slower-dissociating 1-naphthol molecules in cationic vesicles is significantly smaller than in EL and DPPC vesicles.The temperature dependence of ESPT rate constants for both fractions of ArOH and DDAB vesicles follows the Arr- henius equation above the phase-transition temperature. The activation enthalpy of ESPT for both fractions of ArOH in membranes of DDAB vesicles is significantly greater than in membranes of zwitterionic lipids. In DOAB vesicles these activation enthalpies are, however, much smaller.This can be attributed partially to the effect of the temperature on the size of DDAB vesicles. 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