7371
J. Phys. Chem. 1993,97, 7371-7374
Electron Spin Resonance Line-Shape Analysis of x-Doxylstearic Acid Spin Probes in Dibexadecyl Phosphate Vesicles and Effects of Cholesterol Addition Peter J. Bratt Pulsed EPR Laboratory, Department of Biology, University of London, Darwin Building, Gower Street, London WCl E 6BT, United Kingdom
Larry Kevan' Department of Chemistry, University of Houston, Houston, Texas 77204-5641 Received: November 13, 1992; In Final Form: March 17, 1993
Electron spin resonance spectra of a series of x-doxylstearic acids (x = 5,7,10,12, and 16), solubilized in anionic dihexadecyl phosphate vesicles containing 10,30,and 50 mol % cholesterol, are analyzed by employing spectral line-shape simulation. The spectra from the vesicle phase are well reproduced by using the microscopic ordermacroscopic disorder model of Freed and co-workers with Brownian rotational diffusion. The partial averaging of the magnetic interactions by local anisotropic motions in the vesicles is quantified mainly by the order parameter (S) and the rotational diffusion rate perpendicular to the alkyl chain (RI).The simulations show that the order parameter decreases with increasing x, suggesting a relatively extended conformation, unlike the U-shaped, bent conformation of the x-doxylstearic acid alkyl chain found previously in cationic dioctadecyldimethylammonium chloride vesicles. Furthermore, the headgroup region of the vesicle is less ordered in anionic dihexadecyl phosphate than in cationic dioctadecyldimethylammonium chloride vesicles. This result in liquid vesicle solutions corroborates the same x-doxylstearic conformations found infrozen vesicle solutions by electron spin echo modulation spectroscopy. The effect of cholesterol is found to decrease the observed order parameter for all doxy1 positions. This effect is attributed to more water penetration into the vesicle interface arising from the intercalation of the large rigid cholesterol molecule. The effect of cholesterol addition is supported by using 3-doxyl-5a-cholestane, a spin probe which resembles cholesterol. It is found that the addition of cholesterol up to 50 mol % has little effect on the observed electron spin resonance spectrum of cholestane.
Introduction Stable nitroxide radicals have found wide use as spin probes in studies of membrane mimetic systems such as vesicles and micelles.1-3 The x-doxylstearic acids (x-DSA) of the general formula shown below are especially useful, due to their low solubility in water and their tendency to be incorporated into heterogeneous aqueous systems containing amphiliphic molecules.4
correlation times by a full line-shape analysis is necessary. Recently, a suite of programs written by Freed and co-workers has been made available for the personal computer's suitable for simulating ESR spectra under these conditions. In a previous study16the dependence of the deuterium electron spin echo modulation (ESEM) depths was observed for x-doxylstearic acids, (where x = 5, 7, 10, 12, and 16) and 3-doxylSa-cholestane (see structure) in frozen solutions of anionic
r-doxylstearicacid Considerable insight into the motional dynamicsof amphiphilic systems can be obtained from appropriate magnetic resonance techniques. One such techniqueis the use of amphiliphicelectron spin resonance (ESR) active spin probes>-" which "mimic" the motional behavior of the surrounding environment. If the correlation time is calculated using a simple isotropic model12 then the value obtained is often unrealistically faste1' due to local averaging of the anisotropic interactions. A more sophisticated approach is to split the molecular motion into fast tumbling and slow tumbling components where the slow tumbling component is characterized by an order parameter which quantifies the local averaging of the anisotropic interactions.*J3J4 ESR spectra obtained from amphiphilicspin probes in micelles and lipid bilayers are typically in the intermediatemotional regime where the motions are on the time scale of the modulated interaction, with the spectral lineshape arising from both static and dynamic effect^.^ In such cases the evaluation of molecular 0022-3654/93/2097-737 1$04.00/0
3 - doxyl-bu- cholestane
dihexadecyl phosphate (DHP) vesicles and the conformation as a function of x was determined. In this work we have studied systematicallyby ESR line-shape analysis the average conformation of x-doxylstearic acid spin probes versus x in anionic DHP vesicles in the liquid state and fully corroborate the conformationsfound in the frozen state by ESEM spectroscopy. Effects of added cholesterol have also been studied. Experimental Section Dihexadecyl phosphate and the x-doxylstearicacid spin probes were purchased from Sigma Chemical and were used without further purification. They were stored as stock solutions in chloroform at -10 OC. 0 1993 American Chemical Society
Bratt and Kevan
1312 The Journal of Physical Chemistry, Vol. 97, No. 28, 1993
In order to form vesicle solutions, thin films were formed from 18 mM solutions of the surfactant in chloroform. The films were then sonicated in 20 mM tris(hydroxymethy1)aminomethane (TRIS) buffer solution adjusted to pH = 7.8 using a Fisher Model 300 sonic dismembrator operated at 35% output power with a 4-mm microtip a t 72 f 2 O C . The vesicle solutions were added to previously evaporated spin probe films and were allowed to stand for 24 h. This method solubilized the spin probe near the interface of the vesicle.17 The samples were purged with nitrogen and introduced into 1 mm i.d. by 2 mm 0.d. Suprasil quartz tubes and flame sealed. The ESR samples were equilibrated for 1 week before ESR spectroscopy. The amphiphile to probe ratio was 1000/1 in all samples. ESRspectrawererecordedwitha Bruker ESP 300spectrometer operating at X-band with 100-kHz magnetic field modulation at 77 K and 0.2-mW microwave power to avoid power saturation. The magnetic fields were measured with a Varian E-500 nuclear magnetic resonance gaussmeter, and the microwave frequencies in the 9 GHz range were directly measured with a Hewlett Packard 5350B microwave frequency counter. The resulting ESR spectra were transferred toan IBM compatible 33 MHz type 486 personal computer for off-line analysis. Computer simulations for the ESR line-shape analysis were performed on a type 486 personal computer using the program supplied by Freed and co-workers.15
Theory A collection of spin probes localized within a vesicle bilayer may be described as a random sample, in which collections of bilayer fragments are dispersed chaotically in space.’* However, within a given fragment, the liquid crystalline director is oriented uniformly in space implying substantial “microscopic order” in a morphology which may be described as “macroscopically disordered”. Freed and co-workers denoted this model as the MOMD (microscopic order-macroscopic disorder) m ~ d e l . l ~ - * ~ According to this model, rotational correlation times, ordering parameters, and the relative orientation of diffusion and magnetic axes are the independent molecular variables employed in the ESR line-shape analysis. Local ordering at the microscopic level means that the radical reorients in the presence of an orienting potential h imposed by the surrounding molecules in the vesicle bilayer which is characterized by a director d. Because there is no macroscopic order in the vesicle solution d will be distributed at random relative to the external magnetic field B. In oder to calculate the appearance of the ESR spectrum it is necessary to sum spectra weighted by sin 8 for all angles 8 between B and d. These simulations require four principal coordinate systems:23 (1) the laboratory axes determined by B, x , y , z; (2) the local coordinate system determined by d, x’!, y”, z”; (3) the molecular axes determined by the principal axes for ordering of the molecules relative to d as well as the principal axes for molecular diffusion; x’, y’, z’; and (4) the molecular axes determined by the principal axes of the magnetic hyperfine tensor A and g tensor g; x”’, y”‘, z’”. These coordinate systems have been illustrated and discussed more f ~ l l y . ~ ~ J ~ ~ ~ When the alkyl chains of the spin probe are fully aligned with the alkyl chains of surfactant molecules in the vesicle, the x’, y’, z’axes arecoincident with thex”, y”, ~“axes;that is, themolecular rotational diffusion axes are coincident with the local environmental axes determined by d. Since the spin probe molecules are approximately cylindrical, the molecular rotational diffusion tensor is axial so the orientation of thez components of thediffusion axes relative to the magnetic tensor axes (x’!’, y”’, z”3 is specified by a polar angle 9.In the simulation model 9 is a parameter that gives the tilt angle of the rotational diffusion axes relative to the magnetic tensor axes. To simulate an ESR spectrum the parameters involved are the principle values of the hyperfine and g tensors which are known,
t
100 G Figure 1. ESR spectra (solid lines) and simulations (dashed lines) of various x-doxylstearicacids in DHP vesicles at 298 K. The simulations are obtained using the parameters shown in Table I. the inhomogeneous linewidth (taken as 1.5 G23), the diffusion tilt angle 9,the components of the rotational diffusion coefficients RIIand RL which are respectively parallel and perpendicular to z’, and the orienting potential X which determines the order parameter S. The order parameter S is typically used as a physical parameter for interpretation since it can be determined experimentally from the ESR spectra of nitroxides in many cases.l* S is related to h by expressions 1 and 2 where P(8’) is an orientational distribution function and 8’ is the polar angle which p(ei) = e x p [ ( 1 / 2 ) ~ ( 3cos2et-l
- 111 x
Jexp[(1/2)X(3 c0s~19‘-~-1)] sin 8’dO’ (1)
s = ((3 COS* e
q )=
J P(8’)[1/2(3
cos2 8’-1)] sin 8’dY (2)
defines the orientation of z’to z”’. The calculational details have been given.15 For the simulations presented in this paper, calculations for 3 1 values of 8, the angle between d and B ranging evenly between Oo and 90°, were performed to obtain a set of eigenvalues and eigenvectors for each value of 8 which were integrated according to a sin 8 distribution to produce the composite spectra shown.
Results and Discussion Computer Simulations of the ESR Spectra. The ESR spectra at room temperature as a function of the various x-DSAIDHP systems are shown in Figures 1 and 2. The liquid crystalline phase transition of DHP vesicles is estimated to be above room temperature; this is supported by the asymmetric ESR spectra of x-DSA. These spectra have been simulated using the programs of Freed and co-workers.15 The program solves the stochastic quantum mechanical Liouville equation for a nitroxide spin probe within a magnetic field. The model of molecular motion used in these simulations is the isotropic and axially symmetric Brownian rotational diffusion model. It is possible to assume that the static g and A tensors of solid 5-doxylstearic acid are coincident, and are given as g,, = 2.0088, gyy= 2.0061, g,, = 2.0027, A,, = 6.26 G, A , = 5.85 G, and A , = 33.46 G.24 For the calculation of each subspectra 50 Lanczos steps were used with the following basis set truncationvalues: (lemx(6), lomx(4), kmx(2), mmx(2), and ipnmx(2). Using these parameters, it is possible to generate a simulated ESR line shape using the MOMD model and fit the experimental
ESR Spectra of x-Doxylstearic Acids DHP/x-DSA
The Journal of Physical Chemistry, Vol. 97, No. 28, 1993 7373 t 50 mole% Cholesterol
Oa41
12 DSA
\/
0 mole% Cholesterol
0.3 -
m 16 DSA
0.2
~
0.1 -
I
50 mole% Cholesterol
\ I
+
100 G Figure 2. ESR spectra (solid lines) and simulations (dashed lines) of various x-doxylstearic acids in DHP vesicles at 298 K with the addition of 50 mol 95 cholesterol. The simulationsareobtainedusing the parameters shown in Table 11.
TABLE I: Dynamical Parameters Obtained for Various x-Doxylstearic Acids in DHP Vesicles at 298 K X R1.s Ra, s S 5 0.19 x 108 0.30x 109 0.40 1 0.19 x 108 0.32x 109 0.31 10 0.28 X lo8 0.30 x 109 0.31 12 0.28 X lo8 0.30x 109 0.29 16 0.40X lo8 0.40 x 109 0.10
A dea 38 40 40 40 40
TABLE II: Dynamical Parameters Obtained for Various x-Doxylstearic Acids in DHP Vesicles at 298 K with the Addition of 50 mol % Cholesterol X
5 1
10 12 16
RL, s 0.28 X lo8 0.29 X lo8 0.30X lo8 0.30X lo8 0.50X lo8
Rn, s 0.30 x 109 0.30 x 109 0.30 x 109 0.28 x 109 0.50 x 109
S 0.29 0.29 0.20 0.20 0.01
deg 42 42 42 42 42
$5
ESR spectrum with a reasonable degree of accuracy. The correlation between the simulated and experimental spectra for 5-, 7-, lo-, 12-, and 16-doxylstearic acids at 298 K solubilized within DHP vesicles are shown in Figure 1. The dynamical motional parameters used to obtain these fits are summarized in Table I for 298 K. It is found that the appearance of the simulated spectra are most sensitiveto changes in Sand moderately sensitive to changes in RI. In Figure 2 the effect of cholesterol addition at 50 mol 3'% upon the various x-DSA spectra is shown together with simulated spectra. The dynamical motional parameters are summarized in Table 11. It can be seen that the addition of cholesterol at high concentrations has the effect of narrowing the line width. This may be quantified as a reduction in the order parameter S and an increasein RI.The effect of cholesterolon the order parameter S is shown in Figure 3. From Figure 3 it can be seen that the order parameter decreases with increasing distance of the doxyl nitroxide moiety from the acid headgroup located at the DHP vesicle interface. In order to explain these results, we firstly consider the likely solubilization sites and conformations of the spin probes. It has been shown that the carboxylic acid group of the doxyl spin probe tends to be solubilized near the vesicle interface with the hydrocarbon tail of x-DSA extending into the lipophilic core. The decrease in the order parameters S suggests that the probe is in an extended conformation with little chain bending in liquid DHP vesicle solutions. This result in liquid solutions is consistent with deuterium electron spin echo modulation analysis infrozen DHP
vesicle solutions which also indicate an extended conformation of x-DSA probes.16 This trend contrasts with similar ESR lineshape simulations of x-DSA probes in cationic dioctadecyldimethylammonium chloride (DODAC) vesicles in which the x-DSA probes are bent or U-shaped as a function of increasing x.25 Furthermore, the order parameter S for the system 5-DSA/ DODAC is much higher (S = 0.6)25than for the corresponding 5-DSA/DHPsystem (S= 0.4). This suggests that DHPvesicles are more hydrated at their interface than DODAC vesicles, which corresponds well with ESEM data in frozen solutions of these systems.16J7 General Effect of Cholesterol Addition. Upon the addition of cholesterol it can be seen from Figure 3 and Table I1 that the order parameter S decreases and RI increases. The gradual change with cholesterolmole fraction indicates an ordering effect with no sudden phase change. In general, the effect of cholesterol on a lipid bilayer increases the order parameter in the interfacial region.zGz9 This is due to the intercalation of the large planar cholesterol molecule between the alkyl chains, which also has the effect of stiffeningthe alkyl chain allowing an embedded molecule such as a doxyl spin probe to diffuse more easily between the chains. Furthermore, the intercalation of cholesterol hydrates the interfa~e.~O-3~ It can be seen from Figure 3 that the effect of cholesterol addition is to decrease the order parameter which implies that the x-doxylstearic acids are displaced from the hydrated interface, due to the lipophilic nature of the spin probes, deeper into the core of the vesicle. This movement is facilitated by the previously mentioned "chain stiffening" effect. In short, the effect of cholesterol is to locate the spin probe deeper into the lipophilic core of the vesicle which is a region of lower ordering. This is consistent with deuterium ESEM resultsl6 in frozen vesicle solutionswhich show increased deuterium modulation depths from deuterated water with increasing cholesterol addition for 5- and 7-doxylstearic acids suggesting increased hydration near the interface and reduced deuterium modulation depths for lo-, 12-, and 16-doxylstearic acids which suggest that chain stiffening near the interface prevents the doxyl moiety from sampling the interface. The ESR spectra obtained from 3-doxyl-5a-cholestane as a function of added cholesterol are shown in Figure 4. The general features of the spectra resemble those obtained for cholestane in various lipid bilayers35.36 in which the probe is constrained to rotate at an intermediate rate about the steroid long axis. Using the MOMD model with programs available for a personal computer, it has not been possible to simulatethis type of spectrum.
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7374 The Journal of Physical Chemistry, Vol. 97, No. 28, 1993
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implying that the interface is more hydrated resulting in the spin probes locating deeper into the vesicle core. Similar behavior is observed qualitatively for the 3-doxyl-Sa-cholestanespin probe.
1/11
7 30 mole % Cholesterol
References and Notes
10 mole% Cholesterol
0 mole% Cholesterol
I
Acknowledgment. This research was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, US. Department of Energy. P.J.B. is indebted to SERC for a Postdoctoral Fellowship.
I
)
100 Gauss
Figure 4. ESR spectra of 3-doxyl-Sa-cholestane in D H P vesicles at 298 K as a function of added cholesterol.
This is due to the constraints of the computer operating system which prevent convergence of the spectrum in the time scale of interest. It is possible, however, to observe qualitatively that the addition of cholesterol does not have a dramatic effect upon the spectra (Figure 4), but a broadening of the line widths is discernible,particularly when one compares the difference between 0 and 10 mol % cholesterol additions. In the deuterium ESEM studies in the frozen vesiclesystem, a large increase in modulation depth was observed when 10 mol %cholesterol was added. This was interpreted as being due to hydration of the interface. The broadening of the ESR spectrum observed in this study is consistent with an increase in ordering, due to the intercalation of cholesterol between the molecules of the bilayer which results in a decrease in the amplitude of motion resulting in a greater difference in the hyperfine splittings observed in complementary directionsand a decrease in the rate of motion resulting in broader resonances in the applied field parallel to the bilayer plane.3s However, the effect of cholesterol upon the cholestane spectrum is slight, suggesting that the effect of increased ordering at the interface is somewhatcounterbalancedby the lipophiliccholestane molecule locating deeper within the bilayer core in a similar manner to the x-DSA spin probes.
Conclusions This ESR line-shapesimulationstudy demonstratesthat anionic DHP vesicles are less ordered at their interface than cationic DODAC vesicles, and by implication are more hydrated. The simulated spectra are most sensitive to changes in the order parameter and the perpendicular component of the diffusionrate, RI. These results for liquid solutions correlate very well with deuterium ESEM data obtained from analogous frozen solutions. Upon addition of cholesterol the order parameter decreases
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