5170
J . Phys. Chem. 1992, 96, 5170-5174
Effect of Hydrophobic Molecules on N ,N-Dimethyldodecyiamine Oxide Micelles in Water Creger Oriidd,* Ciiran Lindblom, Lennart B.-A. Jobamson, and =ran Wikander Department of Physical Chemistry, University of Umel. S-901 87 Umel, Sweden (Received: November 5, 1991; In Final Form: January 13, 1992)
The micellar solution phase of the N,iV-dimethyldodecylamineoxide (DDAO)/2H20system was studied by pulsed field gradient FT-NMR and by a time-resolved fluorescence (TRF) quenching technique. Complementary ESR studies have also been performed on a micellar solution with high amphiphile concentration (27%w/w). The effect of solubilized hydrophobic molecules was investigated when increasing amounts of dodecane and tetramethylsilane(TMS) were added to the micellar solutions. The aggregation numbers obtained from TRF measuTcments were found to increase from about 75 to 95 by increasing the DDAO concentration from 2 to 20% w/w. The NMR diffusion data are compatible with a broad distribution in micellar size at all the amphiphile concentrations studied, provided that no dodecane or TMS was present in the system. On the other hand, if dodecane or TMS was added to the micellar system, spherical micelles were induced, having a narrow size distribution. The fluorescent quencher benzophenone is also found to induce a change in the micellar shape. It is concluded that at high amphiphile concentration (27%w/w) rodlike micellar aggregates are formed. NMR diffusion measurements show that the cubic phase in the DDA012H20system, occurring between the hexagonal and lamellar phases, is bicontinuous. Finally, it is also found that DDAO micelles cannot solubilize a hydrophobic peptide like gramicidin D, but liquid crystalline phases are formed.
Introduction Surfactants and most biological lipids self-assemble into various aggregate structures, depending on factors like temperature, concentration, and addition of solubilizate.’-s At low amphiphile concentration in water usually globular micelles are formed, while at higher concentrations they might change shape and become r~dlike.’-~One of the most important properties of surfactants and biological lipids is their ability to solubilize hydrocarbons, proteins, hydrophobic peptides, drugs, fat, and other substances the solubilizate normally not dissolvable in ~ a t e r . ~However, ?~ may affect the structure of the aggregate. It is well-known that solubilizationof nonpolar organic molecules in a micellar solution usually leads to an increase in the size of the micellar aggregate^.^ This is due to the fact that either the radius of the spherical micelles increases when the hydrophobic molecules are incorporated or the number of amphiphiles building up the micelles (Le., the aggregation number) increases. In the latter case the shape of the micelles may change, e.g., from a spherical to an oblate or prolate f ~ r m . ~Furthermore, ,~ for systems of amphiphila that do not form micelles but only lamellar liquid crystalline (La) phases at high water contents, like many membrane phospholipids, hydrophobic molecules such as alkanes or peptides of hydrophobic amino acids have been shown to induce reversed hexagonal phase
structure^."^ In the extraction process of membrane proteins, detergents play an important role,Is but a detailed understanding of the molecular mechanisms behind the process and why some detergents are more efficient than others is still lacking. So far, the choice of which detergent should be used for a particular membrane or protein is made by trial and error. This lack of knowledge is illustrated by the problem of finding the optimal detergent allowing for crystallizationof membrane proteins.I6 Obviously, further studies of the physicochemical properties of the amphiphiles utilized for this purpose and how these interact with various solubilizates are needed. It can be expected that such investigations also will give us information about the interaction between lipids and membrane proteins.17 It can also be mentioned that, in order to be able to incorporate membrane proteins into reconstituted phospholipid vesicles, the selection of the appropriate amphiphile to use is critical. Here, rhodopsin can be given as an example,lgand like in many other cases, NJV-dimethyldodecylamine oxide (DDAO) has been shown to be an efficient detergent for such a membrane reconstitution. In this communication we report studies of DDAO mainly in micellar solutions, but also in the cubic liquid crystalline phase
(Figure 1). The size and shape of the micelles have been determined in the presence and in the absence of hydrophobic solubilizates using time-resolved fluorescence (TRF) spectroscopy1*22and NMR diffusion method^?^-*^ Complementary ESR spin-label investigations have also been performed from which some structural information can be obtained.28
Experimental Section Materials. DDAO (research grade) was purchased from SERVA Feinbiochemica GmbH & Co., Heidelberg, Germany, and was used without any further purification. 2 H 2 0was manufactured by Dr. Glaser AG, Basel, Switzerland. 5-Doxylstearic acid was purchased from Molecular Probes Inc., Eugene, OR, and was used without further purification. Pyrene wm obtained from Fluka (melting point 429 K) and was recrystallized three times from diethyl ether. Benzophenone was obtained from Aldrich Chemical Co. (99+%, Gold Label). Tetramethylsilane (TMS) was obtained from Merck, Darmstadt, Germany. Dodecane and gramicidin were purchased from Sigma Chemical Co. and used without further purification. Methods and Results. 1 . TRF Measurements. Two identical solutions containing the appropriate amounts of DDAO in water were prepared. To both these solutions was added a weighed amount of pyrene. The molar ratio of the surfactant to pyrene was approximately lo5/ 1. To one of these solutions was added a weighed amount of benzophenone so that the molar ratio of DDAO/(pyrene + benzophenone)was approximately 100/1. The solutions were equilibrated during 2 days under gentle shaking at 25 OC. Timeresolved single photon counting fluorescence was monitored on a PRA System 3000 (Photochemical Research Assoc., Inc., London, Ontario, Canada). The excitation source was a thyratron gated flash lamp (Model 510 C) filled with deuterium gas and operating at 25 kHz. The excitation and emission wavelengths were selected by interference filters centered at 332.8 and 398.8 nm, respectively. The deconvolution software (DECAY V 3.0 a) used was developed by PRA. The samples were not degassed but sealed in quartz capillary tubes and thermostat4 to the measuring temperature to within 1 OC. The size of the DDAO micelles can be estimated from TRF experiments by using pyrene and its q u e n ~ h e r s . l ~ - The ~~ fluorescence decay of pyrene is monoexponential with a typical ) about 170 11s for all DDAO concentrations studied, lifetime ( T ~ of Le. 2-27 wt %. Upon addition of the quencher benzophenone, a rapid initial decay of the pyrene fluorescence is observed, while the fluorescence rate at longer times becomes equal to l / ~ fthe ,
0022-3654/92/2096-5 170$03.00/0 0 1992 American Chemical Society
The Journal of Physical Chemistry, Vol. 96, No. 12, 1992 5171
Effect of Hydrophobic Molecules on DDAO/H20
140 120
n
I
e
C
I 510
h
4:O
210
3:O
1.0
PPm
0
I
I
20
40
Figure 3. 'HNMR spectrum at 250 MHz of DDAO/2H20at 25 OC as obtained with the diffusion probe on the ACP-250 spectrometer. Spectral assignments: a, water; b, a-methylenes;c, methyls of the polar head group; d, &methylenes;e, bulk methylenes; f, terminal methyl. The assignments of the peaks were made with the aid of a double-quantum filtered COSY (DQF-COSY)43experiment of DDAO dissolved in deuterated chloroform on a Bruker AM-500 spectrometer.
I
60
100
80
DDAO/ Xw
Figure 1. Phase diagram of the system (DDAO)/H20,redrawn after Lutton.'2 The various phases are indicated in the diagram: L, = the micellar solution phase, HI = the hexagonal liquid crystalline phase, I2 = the cubic liquid crystalline phase, L, = the lamellar liquid crystalline phase.
z
120
I
TABLE I" DDAO DDAOTMS (1O:l) DDAOdodecane (16:l) DDA0:dodecane (1O:l)
nn
@ kapp i
10 11 11
69.4 78.2 75.9 70.5
3.5 3.4 3.1 2.9
k
Rn
1.9 1.7 1.6
25.7 22.7 23.4 25.2
O n H is the number of water molecules bound to each DDAO as obtained from the water diffusion data, DLi is the infinite dilution diffusion coefficient of the micelles obtained from the amphiphile diffusion data, k is the constant in eq 5, kaPqis the uncorrected value of k obtained from fits of the micelle diffusion data according to eq 6, and RH is the hydrodynamic radius of the micelles calculated from
equipped with an HR-50high-resolution VT diffusion probe for 5-mm samples (Cryomagnet Systems Inc., Indianapolis, IN). The same magnetic field gradient unit was used also on this spectrometer. All experiments were made at 25 "C except on the cubic m e 2. Aggregation numbers obtained by TRF measurements in the phase, where the temperature was varied between 20 and 50 "C. micellar phase of DDA0/2H20: open circles, DDAO + TMS/2H20; The temperature was kept constant within 1 "C by a heated air filled circles, DDAO/2H20. The uncertainty of N is within 10% at DDAO concentrationsof less than 10%w/w, while it is estimated to be stream around the sample, and it was measured by means of a within 30% at the higher concentrations studied. thermocouple placed close to the sample. Figure 3 shows a proton NMR spectrum at 250 MHz of a micellar solution of DDAO and rate of fluorescence decay in the absence of any quencher. From TMS in 2H20recorded with the diffusion probe on the ACP-250 the experimental data the aggregation number, N, can be calspectrometer. The amphiphile diffusion was measured through culated (cf. Analysis of Results). Figure 2 summarizes the values the peak height of the chain methylenes and the headgroup meof N found a t different DDAO concentrations. thyls. The self-diffusion coefficient of the amphiphile was mea2. NMR Measurements. The surfactant was dried under sured both for the isotropic cubic phase and for the micellar phase. vacuum, and the appropriate amounts of DDAO, hydrophobic The field gradient strength was calibrated at 25 "C using 2H20 molecules, and 2Hz0were added to 7- or 5-mm glass tubes. The with small amounts of H20with a self-diffusion coefficient of 1.9 samples containing cubic liquid crystalline phases were repeatedly X 10-9 m2/s.B The calibration was also checked against dodecane centrifuged back and forth to establish good mixing of the comwith a self-diffusion coefficient of 8.6 X m2/s at 25 0C.30 ponents. The samples were left for some days for equilibration The high-field gradient strength used in the measurements of the before the experiments were done. cubic phase was calibrated against a diffusion coefficient of 2.0 The self-diffusion coefficient was measured with the FourierX mz/s of water-free glycerol at 25 0C.31 This calibration transform pulsed magnetic field gradient spin-echo t e c h n i q ~ d ~ # ~ ~was J ~ also in agreement with an earlier calibration made on water as described in previous papers from this laborat~ry.~'The pulse and a cubic phase consisting of 35.1% w/w potassium octanoate sequence (R&/~--T?~~)Ns was utilized in the diffusion exin 2H20. periment, where RD is the waiting time between repetitive scans The Micellar Solution Phase. The self-diffusion coefficients and NS is the number of scans collected in each experiment. The of DDAO and water were determined as a function of amphiphile magnetic field gradient pulses of width 6 and strength g are concentration in micellar solutions in ZH20.Similar experiments separated by a time interval A and placed at each side of the 7 were made with small amounts of TMS and dodecane solubilized pulse. Two dummy scans were rejected at the beginning of each in the micelles. The molar ratios of DDAO and the added experiment. The attenuation of the signal measured through the molecules are displayed in Table I. peak height is described by eq 1, and the fitting of the experimental Several experiments were made with different (constant) values data to this equation was made on a personal computer as deof RD, 7 , A, and g while 6 was varied. Typical settings on these scribed previou~ly.~' parameters for the investigation of the micellar diffusion coefficients were as follows: RD, 1-2 s; T , 50-200 ms; A, 60-210 ms; A = A0 exp("y2gWD(A - 6/3)] exp(-2r/Tz) (1) g, 0.04-0.12 T/m. The corresponding settings for the water diffusion coefficients were the following: RD, 10-60 s; T , 30-100 The diffusion experiments were performed on a Bruker MSLms; A, 40-1 10 ms; g, 0.01-0.02 T/m. The observed diffusion 100 spectrometer equipped with a proton diffusion probe manucoefficients did not depend on a variation of these experimental factured by Bruker Analytische Mestechnik, Karlsruhe, Germany. parameters. The magnetic field gradient pulses were generated with a slightly The self-diffusion coefficients obtained are shown for water in modified Bruker B-Z 18B unit as described earlier.27 Some exFigure 4 and for DDAO in Figure 5 . periments were also made with a Bruker ACP-250 spectrometer
a
I
5
I
I
10 15 DDAO % w t
I
20
25
Oradd et al.
5172 The Journal of Physical Chemistry, Vol. 96, No. 12, 199’2 1.0
!
7
k
0.8 0.9 .
0
85 ‘C
86 *c
75 ‘C 86 ‘C
1 \
:::I, 0.8
\
,
0.5
0
t
10
,
20
30 0
10
,A 20
55
-c
45
*c
35 ‘C 25 ‘C
30
15 ‘C
DDAO X w / w
Figure 4. Water self-diffusion constants at various concentrations.
Shown are the quotient of the observed water diffusion and the diffusion of pure water, Ow/@, vs the concentrationof DDAO. (a) DDA0/’H20. The three lines correspond to different numbers of bound water molecules/DDAO, nH, according to eq 3. The values of nH are 5 (upper), 10 (middle), and 20 (lower). (b) DDAO + TMS/2Hz0. The line corresponds to nH = 10. (c) DDAOdodecane = 16:l. The line corresponds to nH = 11. (d) DDA0:dodecane = 1O:l. The line corresponds to n~ = 11.
V
1 mT
Figure 6. Experimental ESR spectra of 5-doxylstearic acid solubilized in 27% w/w DDAO solutions at various temperatures. The different temperatures are given to the right in the figure. The total scan range is 8 mT.
trometer equipped with an E-238-type (TMllo mode) cavity. Temperature regulation was achieved by means of a Model V-6040 variabletemperature regulator. The ESR spectra of 5doxylstearic acid solubilized in a micellar solution of DDAO a t 27% w/w DDAO a t different temperatures are displayed in Figure 6. (u
’ 2. .
e
\
0 .
100
d.
C .
*
m
0
50
”
n
10
20
30
10
20
30
DDAO % w / w
Figure 5. DDAO self-diffusionconstants at various concentrations. (a) DDAO/’H20. The line fit correspondsto L& = 69.4 X lo-’’ m2/s, k,, = 3.5. (b) DDAO + TMS/ZH20.The line fit corresponds to Pd= 78.2 X m2/s, k = 3.4. (c) DDA0:dodecane = 16:l. The line fit corresponds toDb,qP= 75.9 X 10-l’ m2/s, /cam = 3.1. (d) DDA0:dodecane = 101. The line fit correspondsto Pi = 70.5 X m2/s, kapp= 2.9.
The Cubic Phase. The diffusion coefficient of DDAO for the cubic phase was measured a t different temperatures in the range 20-50 O C . The settings in this experiment were 7 = 30 ms, A = 40 ms, and RD = 2 s. The field gradient strength, g, was constant a t each temperature, and it was varied between 1.368 and 0.836 T/m a t different temperatures. The gradient pulse width, 6, was varied from 0.5 to 9.5 ms. The diffusion coefficient of DDAO in the cubic phase is measured to be equal to 1.0 X mz/s a t 25 OC, and a plot of In (0)vs 1/ T gives a straight line with an apparent energy of activation for the diffusion process of 35 kJ/mol. 3. ESR Measurements. The spin-label was dissolved in chloroform-methanol (2:l v/v). A suitable amount of the label was transferred to glass vials. The solvent was removed under an Nz atmosphere and finally pumped off under vacuum. Appropriate amounts of the amphiphile and 2Hz0 were added to the thoroughly dried label. The glass vials were flame-sealed, and the content was thoroughly mixed by repeated centrifuging at 40 O C for several hours. They were stored for equilibration during some days. Small amounts of the samples were transferred to glass capillary tubes which were subsequently sealed and thereafter run on the ESR spectrometer. The label/amphiphile ratio was always kept at 1/103 on a molar basis. All ESR spectra were recorded using a Varian Model E-109 X-band (9 GHz) spec-
Analysis of Results -TRF. After a rapid initial decay the long-time fluorescence rate of pyrene becomes equal to the rate of fluorescence in the absence of the quencher. This means192othat the rate of migration of pyrene and benzophenone among the aggregates is negligible. For such a system one can show that the mean aggregation number can be calculated from the characteristics of the fluorescence de.cayI9 F(t) = F(0) exp{-t/rr - ([QI/[DDAOl)N[l - exp(-k,OlI (2) provided that the concentration of DDAO is much larger than the critical micelle concentration (cmc). In eq 2, kq denotes the rate of quenching and [Q] is the concentration of the quencher benzophenone. Thd aggregation number, N, was evaluated graphically from F(t) determined in the presence and in the absence of quenchen. Equation 2 is based on the assumption that the micelles are monodisperse. However, for a polydisperse system it is still possible to analyze the decay with eq 2, provided that the quenching is sufficiently rapid and the migration between the aggregates is negligible. An average aggregation number is then obtained. NMR. WaferDiffusion. The analysis of the water diffusion data is based on a two-site model involving free water, having a diffusion coefficient Df,and water bound to the micellar aggregates, having the diffusion coefficient Db. Fast exchange yields an observed diffusion coefficient, Dw, given by Dw = Dhfb 4- Ddl - fb) (3) wherefb is the fraction of water in the bound site. The contribution to Dw from water bound to monomeric DDAO is very small and will be omitted in the following analysis. 4 is equal to the micellar diffusion coefficient, Dmirwhich is taken from the diffusion data of the amphiphile (see below). Dfis obtained from an expression describing the diffusion of small molecules obstructed by spherical particles3’ Dr = @/(I + @/2) (4) where DY is the self-diffusion coefficient of pure water, taken to mz/s at 25 0C,29and 4 is the volume fraction of be 1.9 X obstructing particles, i.e., the DDAO micelles and the water associated to these.
The Journal of Physical Chemistry, Vol. 96, No. 12, 1992 5173
Effect of Hydrophobic Molecules on DDAO/H20 With this model, the expected value of the ratio Dw/Dpcan be calculated for different numbers of bound water molecules per DDAO, nH, which are then compared with the experimental results. Figure 4a shows the experimentally found Dw/@ for DDAO in 2 H z 0and the calculated result for three different values of n H . (The discrepancies in this fit are discussed below.) Figure 4 W shows experimental results obtained with solubilized molecules together with the best fit of nH. Amphiphile Diffusion. If the micelles are considered as hard spheres diffusing in a medium with hydrodynamic interactions, the concentration dependence of the diffusion coefficient can be written as33-35
Dmi = Pmi(l- k#)
(5)
where D L is the diffusion coefficient at infinite concentration, # is the volume fraction of DDAO and bound water, and k is a constant. In the analysis, however, only the volume fraction of DDAO will be accounted for, giving an apparent value of k, kaW By using ItH calculated from the water diffusion experiment, the volume of bound water can be included, and this gives a value of k between 1.6 and 1.9, comparable with theoretical calculation~.~~-~~ When the amphiphile concentration is low (
