J . Phys. Chem. 1985,89, 1547-1550
1547
Electron Spin-Echo Studies of Doxylstearic Acid Spin Probes in Ionic Micelles. Nitroxide Interactions with Deuterium in the Stern Layer E. Szajdzinska-Pietek,+Ren6 Maldonado, Larry Kevan,* Department of Chemistry, University of Houston, Houston, Texas 77004
Stuart S. Berr, and Richard R. M. Jones* Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109 (Received: August 22, 1984; I n Final Form: December 18, 1984) Two-pulse electron spin-echo modulation studies have been carried out for a series of x-doxylstearic acids (x = 5, 7, 10, 12, and 16) in frozen aqueous solutions of tetramethyl-d12-ammonium dodecyl sulfate, dodecyltrimethyl-d9-ammoniumbromide, and hexadecyltrimethyl-d9-ammoniumbromide. Weak hyperfine effects due to interactions of the nitroxide group with counterion or headgroup deuterium result in spin-echo modulation. The modulation depths have been measured as a function of x and the results are discussed in terms of possible nitroxide locations and resultant chain conformations of the spin probe in the micelles.
Introduction Stable nitroxide radicals are widely used as spin probes in studies of biological membranes and membrane mimetic systems.' Among others, x-doxylstearic acids of the formula
+
HOCO(CH,),-,-C-(CH,),,-~CH,
are particularly useful as they are sparingly soluble in water but can be readily incorporated into heterogeneous aqueous systems containing amphiphilic molecules. These detergent-like molecules have generally been found to be oriented approximately parallel to the amphiphilic components in bilayers, membrane preparations, and cells with their polar headgroups at the bilayer/membrane surface.2" In micelles the carboxyl group of rhe spin probe also appears to be at the polar micelle surface leaving the hydrophobic tail of the probe molecule containing the doxy1 group to seek a location in the micelle in response to the hydrophobic, hydrophilic, and steric forces acting.'~~ This carboxyl location is further supported in the present work. Electron spin-echo ( B E ) techniques recently developed in this and other laboratories9 enable one to obtain more detailed information about the location of the spin probe in specifically deuterated frozen micellar systems by monitoring modulation effects due to interactions of the nitroxide group with deuterium on surfactant molecules. In the present work we have used this method to study micellar solutions of dodecyltrimethylammonium bromide (DTAB-d9) and hexadecyltrimethylammonium bromide (HTAB-d9) with deuterated headgroups and tetramethylammonium dodecyl sulfate (TMADS-d12) with deuterated counterions. The location of the deuterium in micelles of these surfactants must necessarily be in the Stern layer.lb Thus by measuring deuterium modulation of several x-doxylstearic acids as a function of x we are able to obtain the proximity of the spin probe nitroxide group with respect to the polar headgroup region or Stern layer of the micelles.
Experimental Section The spin labels 5-, 7-, lo-, and 12-doxylstearic acids from Molecular Probes,Inc. as well as 16-doxylstearic acid from Aldrich Chemical Co. were stored under nitrogen at -10 OC upon arrival and were not purified further. These materials were aliquoted in chloroform solutions under nitrogen and the chloroform was evaporated under a stream of nitrogen at room temperature. The 'On leave from the Institute of Applied Radiation Chemistry, Technical University of Lodz, Poland.
0022-3654/85/2089-1547$01.50/0
resulting films of doxylstearic acid were stored at -10 O C under nitrogen and were used within 1 week. Tetramethyl-d12-ammonium dodecyl sulfate (TMADS-d12)was prepared as follows. Trimethyl-+amine (MSD Isotopes) was vacuum transferred into 5 mL of absolute ethanol (US. Industrial Chemical Co., U.S.P.,freshly opened sealed pint) to give a 55.8 wt.% solution. Weights were determined by difference; 2.63 g of this solution containing 0.0215 mol of the amine was syringed at ice temperature with a cold, dry syringe previously wet with cold absolute ethanol into 30 mL of absolute ethanol contained in a two-neck 50-mL round-bottom flask equipped with a magnetic stir bar, spirit thermometer, and addition funnel with Teflon stopcock all sealed with Teflon to avoid grease, under dry nitrogen, and cooled to -15 OC with a dry ice/2-propanol bath. Through the addition funnel methyl-d, iodide (Aldrich 99+ atom % D, Gold Label), 1.4 m, 3.3 g, 0.023 mol, dissolved in 5 mL of absolute ethanol was added dropwise with stirring while maintaining -1 5 OC. The reaction was stirred for 2 h at this temperature, then at ice temperature for 12 h sealed off from the atmosphere. The white tetramethyl-d9 ammonium iodide salt was isolated by vacuum removal of the solvent, washed twice with small amounts of dry acetone, removed by pipet, and dried under vacuum to a constant weight of 4.81 g, 0.225 mol, 99% yield. The iodide salt was dissolved in 10 mL of distilled, deionized water and 2.62 g, (0.0113 mol of silver(1) oxide (Aldrich, 99+%) added. The Ag20 was crushed with a spatula and the mixture heated to 70 O C for 1 h with magnetic stirring. The solids which changed from black to yellow during the course of the reaction were filtered off and washed with a small quantity of water, and the hydroxide salt was isolated by lyphilization of the water yielding 2.35 g or 101% trimethyl-d12-ammoniumhydroxide. The material was taken up in another 10 mL of distilled, deionized water and filtered off through a 0.45-pm cellulose nitrate syringe filter to remove traces of AgI. The colorless filtrate was brought to pH 7 by addition of a 1-butanol solution of dodecyl bisulfate prepared as described previously8 from isomerically pure dodecanol and chlorosulfonic (1) See for example: ( a ) 'Spin Labeling Theory and Application", Berliner, Lawrence J., Ed.; Academic Press: New York, 1976. (b) Fendler, J. H., Fendler, E. J. "Catalysis in Micelles and Macromolecular Systems"; Academic Press: New York, 1975. (2) Seeling, J.; Limacher, H.; Bader, P. J . Chem. SOC.1972, 94, 6364. (3) Hubbel, W. L.; McConnell, H. H. Proc. Natl. Acad. Sei. U.S.A. 1969, 64, 20. (4) Libertini, L. J.; Wagoner, A. S.; Jost, P. C.; Griffth, 0. H.Proc. Narl. Acad. Sei. U.S.A. 1969, 64, 13. ( 5 ) Bales, B. L.; Leon, V. Biochim. Biophys. Acta 1978, 509, 90. (6) Jost, P.; Libertini, J. L.; Herbert, V. C. J . Mol. Biol. 1971, 59, 77. (7) Bales, B. L.; Kevan, L. J. Phys. Chem. 1982, 86, 3836. (8) Szajdinska-Pietek, E.; Maldonado, R.; Kevan, L.; Jones, R. R. M. J . Am. Chem. SOC.1984, 106,4675. (9) Kevan, L. In 'Time Domain Electron Spin Resonance", Kevan, L., Schwartz, R. N., Eds.; Wiley-Interscience: New York, 1979; Chapter 8.
0 1985 American Chemical Society
Szajdzinska-Pietek et al.
1548 The Journal of Physical Chemistry, Vol. 89, No. 8, 1985
12r
M
>2
TMADS
0.5
1.o
TIME ,
- hI2
2.0
1.5
--
HTAB
ps
dg
Figure 1. Two-pulse electron spin-echo decay envelopes at 4.2 K of
5-doxylstearic acid probe in TMADS micellar solutions. The base lines have been offset vertically to avoid overlap. The normalized modulation depth is alb. I
acid. The aqueous butanol solution of tetramethyl-d12-ammonium dodecyl sulfate was worked up also as described previouslys for the perhydrogenated compound to yield 3.56 g (46% based on the starting amine) of the surfactant after four recrystallizations from ethanol. Anal. (Galbraith Laboratories, Inc., Knoxville, TN). Found C, 54.61; H D, 14.43; N , 3.73; S, 9.40. Calcd: C, 54.66; H + D, 14.05; N , 3.98; S,9.12. Dodecyltrimethyl-d9-ammonium bromide (DTAB-dg) and hexadecyltrimethyl-d9-ammoniumbromide (HTAB-d9) were prepared as follows. To 4.5 g (0.018 mol) of 1-bromododecane (Aldrich, 98%) and to 5.5 g (0.018 mol) of I-bromohexadecane (Aldrich, 99%) each dissolved in 30 mL of absolute ethanol contained in two-neck round bottom 50-mL flasks equipped with magnetic stirrer, spirit thermometer, and addition funnel with Teflon stopcock all sealed with Teflon, under dry nitrogen, and cooled to -1 5 O C with a dry ice/2-propanol bath was added 2.69 g of a 55.8 wt % ethanol solution of trimethyl-dg-amine (0.22 mol, prepared above and transferred as above) dropwise through the addition funnel with stirring. The amine was washed into the flask with an additional 5 mL of absolute ethanol and the flask closed to the atmosphere. The contents were allowed to stir for 12 h at 3 ‘C (ice bath) and for 72 h at room temperature. The addition funnel was replaced with a reflux condenser topped with a CaC12 drying tube and the ethanol refluxed for 3 h to complete the reaction. The ethanol and any remaining trimethylamine were removed by vacuum evaporation and the resulting solid was recrystallid four times from acetone/methanol (1O:l v / ~ and ) dried to constant weight under vacuum. The yield of HTAB-d9 was 3.21 g or 48% based on the starting bromide. Anal. (Galbraith Laboratories, Inc.). Found: C, 61.25; H D, 13.43; N, 3.69. Calcd: C, 61.09; H D, 13.76; N , 3.75. The yield of DTAB-d9 was 2.92 g or 51% based on the starting bromide. Anal. Found: C, 56.84; H + D, 13.56;N, 4.39. Calcd: C, 56.76; H + D, 13.66; N , 4.41. Surfactant solutions of 0.1 M were prepared with triply distilled water deoxygenated by bubbling with dry nitrogen. Doxylstearic acid probes were dissolved in these solutions to concentrations of about 0.4 mM by the procedure described previously.’ Samples were prepared in a nitrogen atmosphere and sealed in 3-mm-0.d. Suprasil quartz tubes. These were frozen to 77 K by rapid immersion into liquid nitrogen. The two-pulse ESE spectra were recorded at 4.2 K on a homebuilt spectrometerlo a t a frequency of about 9.15 GHz using exciting pulse widths of 50 ns.
+
+
+
0.5
1.o TIME,
1.5
2.0
ps
Figure 2. Two-pulse electron spin-echo decay envelopes at 4.2 K of 5-doxylstearic acid probe in DTAB and HTAB micellar solutions. The base lines have been offset vertically to avoid overlap.
Results Two-pulse electron spin-echo envelopes were recorded from the MI = 0 14N hyperfine transition of the nitroxide electron spin resonance spectrum. The results for 5-doxylstearic acid in anionic micelles of TMADS-d12and in cationic micelles of DTAB-d9 and HTAB-d9 are shown in Figures 1 and 2, respectively. They are compared with the spectra of reference solutions containing perprotiated surfactants. All the echo decay curves exhibit prominent modulation with the period of 0.08 being due to nitroxide interactions with water as well as with surfactant protons. In TMADS-dI2, DTAB-d9, and HTAB-d9 solutions an additional modulation period of about 0.5 ps is clearly seen which corresponds to nitroxide interactions with surfactant deuterium. Similar ESE patterns were obtained with other x-doxylstearic acid probes in the same micelles. The echo decay curves are scaled to the highest echo intensity observed, and all are seen to extrapolate to the noise level at times longer than 2.5 ps. To approximately remove the unmodulated echo decay, smooth curves are drawn through the modulation maxima and minima. The difference between these curves at the first modulation minima divided by the amplitude of the upper curve is defined as the normalized modulation depth as shown in Figure 1. Similar values are obtained at the second modulation minima. The results obtained in this way are plotted vs. the doxy1 group position, x , on the stearic acid chain in Figures 3 and 4 for anionic and cationic micelles, respectively. A smooth decrease of the normalized modulation depth is seen in going from x = 5 to x = 10 in TMADS-dI2 and from x = 5 to x = 12 for DTAB-d, and HTAB-d9. After reaching the minimum the normalized modulation depth increases with further increase of x . In cationic micelles the strongest normalized deuterium modulation is observed with 16-doxylstearic acid. Discussion
The spin probes distribute between micelles according to Poisson statistics.l’ From recent small-angle neutron scattering results12 (1 1) (a) Kalyanasundaram, K.; Gratzel, M.; Thomas, J. K. J. Am. Chem.
(10) Ichikawa, T.; Kevan, L.;Narayana, P. A. J . Phys. Chem. 1979,83. 3378.
SOC.1975,97, 3915. (b) Infelta, P. P.;Gratzcl, M,J. Chem. Phys. 1979, 70, 179.
Doxylstearic Acid Spin Probes in Ionic Micelles
The Journal of Physical Chemistry, Vol. 89, No. 8, 1985 1549
aggregation numbers of 80 for TMADS-d12,65 for DTAB-d9, and 145 for HTAB-dg can be estimated at 0.1 M. These numbers seem reasonable by comparison with the best available data at lower concentrations of s~rfactant.'~The micellar concentrations of these solutions are then about 12.5 mM for TMADS-I,,, 1.5 mM for DTAB-d9, and 0.7 mM for HTAB-d9. From a Poisson distributionllaof 0.4 mM doxylstearic acid in 0.1 M TMADS-d12, approximately 19% of the micelles are occupied by one spin probe and another 5% have two or more spin probes per micelle. In DTAB-d, micelles the corresponding occupancies are 14% for single occupancy and -3% for multiple occupancy and in HTAB-dg the respective occupancies are -20% and -7.5%. So in all the systems singly occupied micelles dominate strongly. It is noteworthy that no ESR line broadening due to spin-spin interactions has been observed in these systems. So any contribution to the ESR or ESEM data from multiply occupied micelles is expected to be minimal. At 0.1 M surfactant concentration, the micelles are thought to be spherical or nearly so. With DTAB, Anacker, Rush, and Johnson13bsaw no further increase in the intensity of scattered light from concentrations just above the critical micelle concentration (cmc) up to above 0.1 M surfactant. Also, Ozeki and Ikeda14find that at all surfactant concentrations up to the solubility limit, but below 1.8 M added NaBr, no second cmc indicative of rods can be seen. No comparable data exists for TMADS, but by analogy to the sodium salt, one would expect small, nearly spherical micelles up to perhaps 0.6 M surfa~tant.'~For HTAB micelles, Reiss-Husson and Luzzati,I6 from radii of gyration of micelles calculated from small-angle X-ray scattering data, find evidence for a sphere-to-rod transition at 0.14 M surfactant at 27 OC. This transition is temperature dependent. Raising the temperature to 50 OC moves the sphere-to-rod transition to 0.47 M HTAB in the absence of salt. Thus at room temperature we expect predominantly spherical micelles for this surfactant at 0.1 M. Neutron scattering data also show no evidence for rods in any of these systems at 0.10 M surfactant.12 The deuterium modulation depth depends on the number of interacting deuteriums and their average distance from the spin probe. Calculation of the average number of surfactant headgroups or counterions a spin probe interacts with allows extraction of information concerning the relative distances of the spin probe from the Stern layer as the position of substitution on the stearic acid chain is varied. Deuterium modulation is generally only detectable for interaction distances less than 0.6 nm.? The volume of intersection of a sphere of this radius with the spin at its center and at the center of a Stern layer of outer dimension dictated by the micelle radius and minimum thickness dictated by the largest ion or headgroup determines the maximum number of interacting deuterated species. We have employed a space-filling spherical model to calculate the micelle radius based on the aggregation numbers above and known partial molar volume^.^' The interaction volume calculated in this way is only large enough for one counterion or headgroup. Consideration of the probable occupancy of this volume by a counterion or headgroup results in 9-10 deuteriums interacting with the spin probe for all three systems studied. We conclude that the nitroxide groups at different
-
~
~~
(12) Berr, S.S.;Jones, R. R. M.; Johnson, J. S.,Jr.; Magid, L.; Triolo, R. Work reported to the National Center for Small-Angle Scattering Research, Oak Ridge National Laboratory, Oak Ridge, TN, manuscript in preparation. (13) (a) For TMADS see Mysels, K. J.; Princen, L. H. J. Phys. Chem. 1959,83,3378. (b) For DTAB see Anacker, E. W.; Rush, R. M.;Johnson, J. S. J . Phys. Chem. 1964,68,81. (c) For CTAB and DTAB see Guveli, D. E.; Kayes, J. B.; Davis, S.S. J. Colloid Interface Sci. 1979, 72, 130. (d) For a view which denies micelle growth with concentration see Liana, P.; Zana, R. J . Colloid Interface Sci. 1981, 84, 100. (14) Ozeki, S.;Ikeda, S.J . Colloid Interface Sci. 1982, 87, 424. (15) Missel, P. J.; Mazer, N. A.; Benedek, G. B.; Carey, M. C. J . Phys. Chem. 1983,87, 1264. (16) Reiss-Husson, R.; Luzzati, V. J . Phys. Chem. 1964, 68, 3504. (17) (a) Krumgalz, B. S.J . Chem. Soc., Faraday Trans.1 1980,76, 1887. (b) Perron, G.; Desrosiers, N.; Desnoyers, J. E. Can. J. Chem. 1976,54,2163. (c) DiPaola, G.; Belleau, B. Can. J . Chem. 1975, 53, 3452. (d) Immirzi, A.; Perini, B. Acta Crystallogr. 1977, 1733, 216.
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Figure 3. The dependence of normalized deuterium modulation depth on the doxyl group position in stearic acid spin probes for a TMADS-d12 micellar solution. ~
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