Chemistry and Physics of Lipids, 53 (1990) 231--241 Elsevier Scientific Publishers Ireland Ltd.

231

Interaction of surfactants with model and biological membranes. II. Effect of N-alkyl-N,N,N-trimethylammonium ions on phosphatidylcholine bilayers as studied by spin probe ESR

J. Gallovfi, F. D e v f n s k y a n d P. Balgav~ °Faculty of Pharmacy and Institute of Physics and Biophysics, J.A. Comenius University, 832 32 Bratislava (Czechoslovakia) (Received October 7th, 1987; revision received September 17, 1989; accepted October 13th, 1989) The interaction of N-alkyl-N,N,N-trimethylammonium (C,TMA, n =6-18) salts (iodides and/or bromides) with model membranes prepared by hydration of egg yolk phosphatidylcholine (EYPC) over aqueous salt solutions has been studied by m-doxyl stearic acid (m-DSA, m = 12 and 16) spin probe method. In disoriented EYPC bilayers the C, TMA salts decrease the orientational order parameter $33 of m-DSA evaluated from the powder pattern ESR spectra. This effect is maximal for C6TMA. In oriented EYPC bilayers prepared by the parallel-beam sputtering method and hydrated over saturated NaCl solution the order parameter $33 calculated from the angular dependence of the nitrogen hyperfine splitting is decreased in the presence of C6TMA. The order parameter $11 obtained from the angular dependence of line positions indicates deviation of m-DSA motion from axial symmetry. CoTMA increases the probability of gauche conformations of the lipid chains by about 13-14%, and decreases the effective energy difference between the trans and gauche conformations by about 420--480J/mol, at molar ratio of EYPC/ C6TMA = 2:1. The angular dependence of linewidths is analysed by employing a theory of spin relaxation based on the strong collision model for molecular reorientations. The correlation time 70 of the reorientation of an axis orthogonal to the doxyl ring of 16-DSA is decreased in the presence of C6TMA, while that of 12-DSA is not influenced by it. The ratio of ~2/%is increased in the presence of C6TMA for the both spin probes. The results are explained using the free-volume model of the CnTMA-EYPC membrane interaction.

Keywords: phosphatidylcholine bilayers; N-alkyl-N,N,N-trimethylammonium ions; free volume; trans-gauche isomerization; spin probe ESR.

Introduction

Surface active quaternary ammonium ions exhibit a wide range of biological activities - they are powerful bactericides and fungicides, plant growth regulators, spermicides, myorelaxants, etc. Studies with model phospholipid membranes indicate that the prime mechanism of their action could be an irreversible perturbation of the cytoplasmic membrane. It has been found that Nalkyl-N,N,N-trimethylammonium (C,TMA) ions increase the ionic conductivity of black lipid Correspondence to: E Balgav~.

membranes [1,2] and the efflux of ions and organic compunds from lipid vesicles [2,3], influence the gel-liquid crystal phase transition temperature of multilamellar dispersions of synthetic phospholipids in water [4,5], change the polar head group conformation of phospholipids in the liquid crystalline state [6,7], and destabilize the bilayer structure of black lipid membranes [1,2] and multilamellar lipid dispersions [7-9]. It has been also reported that CnTMA ions decrease the order parameter of fluorescent probe DPH incorporated in the bilayer hydrophobic region [10]. In the present paper, we study effects of C TMA ions on the spin probe

0009-3084/90/$03.50 (~) 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

232 (m-doxyl stearic acid) order parameters and rotational correlation times in egg yolk phosphatidylcholine (EYPC) bilayers. Material and methods

N-alkyl-N,N,N-trimethylammonium iodides were prepared as follows: 0.1mol of N-alkylN,N-dimethylamine was dissolved in 30 cm 3 of dry methylcyanide and while mixing, 0.11 mol of iodomethane was dropwise added at laboratory temperature. After the exothermic reaction ceased the reaction mixture was boiled under reflux in an inert atmosphere of nitrogen for an additional 14 h, the volatile materials were removed by distillation in vacuum and the quaternary ammonium salt was crystallized AT 6 times from dry acetone (or acetone/methanol mixture) to TLC purity. The yields were generally very high, in some cases quantitative. N-alkyl-N,N,N-trimethylammonium bromides were prepared by two methods. Method A: to a mixture of approx. 0.2 mol trimethylamine in dry methylcyanide (the mixture was prepared by dissolving of the required amount of undercooled (to aP3Prox. - 2 0 to -25°C) trimethylamine in 30 cm of cool (approx. -18°C) methylcyanide) 0.1 mol of the appropriate 1-bromoalkane was added and the reaction mixture left for overnight at laboratory temperature. Then the mixture was boiled under reflux in an inert atmosphere of nitrogen for 12 h and worked up as mentioned above. The yields were around 90% of the theory. Method B: to a well stirred mixture of 0.1 N-alkyl-N,N-dimethylamine in 30 cm 3 of dry methylcyanide a solution of 0.2 mol of bromomethane in methanol was added (the solution was prepared by absorbing of the required amount of anhydrous bromomethane, b.p. 4°C, in 30 cm 3 of dry methanol at laboratory temperature). After reacting for 12 h at 25°C the reaction mixture was heated under reflux in an inert atmosphere of nitrogen for an additional 12 h and worked up as above. The yields were around 90% of the theory. EYPC was isolated from fresh hen eggs and purified according to Ref. 11. Spin probes 12doxyl stearic acid (12-DSA) and 16-doxyl stearic

acid (16-DSA) was purchased from Syva (Palo Alto, U.S.A.). Oriented EYPC bilayers deposited on glass surfaces were prepared by the parallel-beam sputtering method (introduced in Ref. 12) as described in detail in [13]. Briefly, the EYPC + CnTMA + m - DSA mixture dissolved in absolute ethanol was atomized with a stream of nitrogen gas. The beam of atomized solution then passed through an orifice before deposition on the glass plate. Thereafter, the glass plate in a sample holder was inserted into a glass tube (o.d. 5 mm), and the traces of solvent were removed by a diffusion pump evacuation. Disoriented EYPC bilayers were prepared by deposition of the EYPC + CnTMA + m-DSA mixture dissolved in ethanol on a pulverized glass. After insertion of the powder into a glass tube the traces of solvent were removed by evacuation as described above. Finally, the lipid bilayers on the glass plate or on glass powder were hydrated over saturated salt solution (or pure H 2 0 ) and sealed in the tube. To some glass powder samples an excess of water (~12 tzl HEO/mg EYPC) was added using a microsyringe. Electron spin resonance spectra were recorded by an ERS 230 X-band ESR spectrometer (ZWG AdW Berlin, G.D.R.) using the 100-kHz modulation technique and a GX goniometer (Radiopan, Poland). Microwave frequency was measured using a q3-54 frequency meter equipped with a $I3q-43 frequency converter (Moscow, U.S.S.R.). The precision of temperature setting was -+0.5 K. For the m-DSA spin probes, the principal z-axes of the hyperfine splitting tensor A and g-factor ~ are almost parallel to the long molecular axis, and their motion in the lipid bilayers is supposed to be axially symmetric about the director (see Refs. 14 16 for references). Because the parallel-beam sputtering method produces well oriented bilayers with the director perpendicular to the glass surface [12,13], the time averaged components of the A and ~ tensors parallel (A II, gll) and perpendicular (A ±, g±) to the director can be obtained from the ESR spectra recorded at different director orientations y with respect to the magnetic field:

233 A(3,) = [A 2 sin2 3, + A~ cos2 3,1t/2

g(3') = Ig~ sin2 3" + g~ c°s2

(1)

3'1t'2

(2)

The All, A l , gll and gx values were calculated from the experimental angular dependence of A(3") and g(3") according to Eqns. (1,2) using a least-squares method. Assuming a cylindrical symmetry about the long molecular axis, the order parameters Sii defined as the ensemble average over all probe molecules

Sii = (3 cos 2 0 i - 1 ) : 2

(3)

(with angles between the/-axis of A tensor and the director denoted by O/) were calculated from the experimental gll, g-, All, and A± values according to equations SH = [fs(gll - g±) + S33(gyy - g,z)]:(gxx - gyy) (4) $33 =

522

=

IrA( A II -- A ±)]: [ A z , -

-- all

--

where gii and A , are the cartesian principal values of diagonalized ~ and A tensors, respectively, and fg and fA are polarity correction factors (see Refs. 14--16 for references). The principal values of the ~ and A tensor of rn-DSA spin probes used in the present paper were taken from literature [17]:

Azz = 3.35 mT

(11)

The order parameter 533 in disoriented samples ! I was calculated from experimental A±, All values using Eqns. (5,8,10,11) and A , values (9). In oriented samples the linewidths are dependent on sample orientations 3,. From this angular dependence, the m-DSA spin probe correlation times were calculated using a motional-narrowing theoretical model of Luckhurst et al. [19-21]: Provided the correlation time z for m-DSA spin probe reorientation is small, the ESR line shape is lorentzian with a width

where m is the nitrogen nuclear spin quantum number associated with a line. When the correlation time is greater than the inverse of the resonance frequency % and nuclear spin is assumed to be quantized parallel to the magnetic field for all orientations % the model predicts the angular dependence of the linewidth coefficients in Eqn. (12) to be of the form

(8)

gzz = 2.0027

t

All ~ All

(6)

fA=(A,,, + Ayy + A~z):(2A± + AII)

Ayy = 0.58 mT

p

All(m, 3,) = A(3,) + B(3,)m + C(3')m 2

(7)

gyy = 2.0061

!

× [ 1 - (All - A ± ) : { A , , - (A~x + Ayy):2}] (10)

(5)

fg = (g,,x + gyy + gzz):(2g± + gll)

Axx = 0.65 mT

¢

A±'~ A± +0.14

+ A y y ) : 2]

S33

gxx = 2.0088

values of the effective outer and inner hyperfine t I splittings A ±, All could be deduced. According to Gaffney [18], the effective splittings are related to the true splittings A ±, A I1"

(9)

The m-DSA spin probes in disoriented bilayers display axially symmetric powder pattern spectra. From these spectra, experimental

B(3') = Bo + B2P2(cos 3') + B4P4(cos 3')

(12)

(13)

where PL(COSy) are the Legendre polynomials and BL(AL, CL) are the angular linewidth coefficients. In our experiments, the widths of the three hyperfine lines were determined from that of the central line (m = 0) and the heights of the outer two lines (m =---1) relative to the central peak. These linewidths were then used to determine the linewidth coefficients B(3' ) and C(3') at different orientations 3'. The angular linewidth coefficients B L (and CL) were obtained by a non-linear least squares fit of the theoretical

234 expression in Eqn. (13) to the experimental B(7) and C(7) values. The angular linewidth coefficients depend on both the correlation time(s) of spin probe molecular reorientation and its ordering. The strong collision model for molecular reorientation (assuming that the reorientations before and after collision are uncorrelated) predicts for angular linewidth coefficients of rigid cylindrically symmetric spin probes in uniaxial samples

0.~0

Saa 0.15

°° I 0.05

o.o

!

q.2

0.4

o.o

CsTMA:EYPC B 0 = 4w0[g(2,°)A(E,°)r0(1 _ $323) Jr 2g(2'2)A(E'2)T2] : 15~

(14)

B E = 8to0S33[g(2'°)A(2'°)%(1 - $33) - 2g(2'2)A(2'2)~'2] : 21 ~ CO= [A(2'°)2%(1 - $23) + 2A(2'E)2z2] : 12 C2

=

Fig. 1. The dependence of the orientational order parameter Sa3 for the 16-doxyl stearic acid spin probe in the disoriented phosphatidylcholine bilayers on the C6TMA/EYPC molar ratio. The samples were hydrated over saturated NaCI solution, t = 23°C.

(15) (16)

13S33[A(2'°)2,ro(1 - $33 ) - 2A(2'2)2'r2] : 84

(17) where ~ is t h e partially averaged g-factor, and g(i,i) and A(~'i) are the irreducible components of the ~ and A tensor, respectively. The %, 7"2 values in Eqns. (14---17) were calculated from the experimental Bt. , C~. values, using g(i,i) and A (i'i) values calculated from their cartesian counterparts (9) according to following equations: g(2,0) = (2/3)0.5[gzz _ (gxx + gyy):2] : fg

(18)

g(2,2) = [gx~ _ gyy]:2fg

(19)

A (2'°) = (2/3)°5[Azz - ( A ~ + Ayy):2]:fA

(20)

A (2"2) = [A~x - Ayy] :2f, t

(21)

where fs, fa are the polarity correction factors (7,8).

Results and discussion

The presence of N-alkyl-N,N,N-trimethylammonium ions in the EYPC bilayers affect the

ESR spectra of m-DSA spin probes in both the oriented and disoriented samples. This indicates a perturbation of the bilayer structure. Figure 1 demonstrates this perturbation effect as a change in $33 order parameter of the 16-DSA spin probe in dependence on the C6TMA concentration in the disoriented EYPC samples at low lipid hydration. It is seen that the bilayer perturbation increases with the surfactant concentration. Qualitatively similar effects have been observed in spin probe ESR studies of the interaction of amphiphilic amines (local anesthetics and beta blockers) and amine oxides (bactericides) with various model and biological membranes [22--

27]. The order parameter Sa3 in control sample (without any surfactant added) decreases with the increased bilayer hydration. For example, it was equal to 0.182-+ 0.005, 0.126_+ 0.007 and 0.070 _+0.006, for 16-DSA in samples hydrated over saturated NaCl solution, over pure water and prepared in excess water, respectively. This is in accord with the hydration-induced increase in lipid area [28-30] which facilitates lipid chain motion owing to decreased inter- and intramolecular friction. Since the values of $33 in samples prepared in excess water were outside the region of validity of Eqns. (5,7,8,10,11) used for the evaluation of spectra [18], all the experiments were performed with samples hydrated

235 0.22

Saa 0.18

0.14

0.10

0.06

0.02 4

I 8

i 12

I 16

20

I1 Fig. 2. The dependence of the odentational order parameter $33 for the 16-doxyl steadc acid spin probe in the disoriented pbosphatidylcholine bilayers on the C . T M A alkyl chain length n at the molar ratio of C . T M A / E Y P C = 1:4. Open symbols, hydration over saturated NaCI solution; closed symbols, hydration over pure H20; circles, C . T M A iodides;

squares, C,TMA bromides, t=23°C. Horizontal dashed lines, samples without C.TMA.

over saturated salt solutions. Independent of the salt solution (i.e. of the hydration degree), the perturbation effect of C,TMA ions on the bilayer structure has been observed to increase with the decrease of surfactant chain length. This effect is demonstrated in Fig. 2 for two limiting values of hydrations used. It is clearly seen that the S3a order parameter of the 16-DSA spin probe increases with the increase of alkyl chain length n and approaches that of control samples for n ~ 4--18. As described above, the values of the Sa3 order parameter are dependent on the bilayer hydration. To ascertain that the C,TMA chain dependence of S3a is not caused by different hydration degrees for different E Y P C + C,TMA bilayers, their hydration has been studied by gravimetry. Because the effects of C,TMA ions on the order parameter in disoriented bilayers were maximal at highest surfactant concentrations and at lowest hydrations this study has been performed at a molar ratio of C,TMA/EYPC = 1:2 and hydration over saturated NaCl aqueous solution (76% relative humidity). It has been found that after a prolonged (up to 13 days) hydration of disoriented samples over NaCI the final (maximum) molar fractions of H20 were 0.87 -+ 0.02, 0.87 -+ 0.04 and 0.85-+ 0.04 for the pure EYPC, E Y P C + C6TMA, and EYPC + C14TMA bilayers, respec-

tively. It is seen that the hydration level of bilayers is independent of the alkyl chain length of CnTMA and within an experimental error is the same as in the control sample without C,TMA. It has been suggested that the chain lengthdependent decrease of $33 is caused by the formation of free volume between the lipid chains in the bilayer centre due to the insertion of surfactant molecules between the lipid molecules [7,22--27]. The insertion of C~TMA molecules into the lipid part of the membrane is anisotropic. C, TMAs bear a polar group with the positive charge on nitrogen. This group can interact with negatively charged phosphate of lipids in the polar region in the membrane. The alkyl chain of CnTMA will orient parallel to the hydrocarbon chains of lipid molecules. At this location the packing density of lipids must be influenced due to lateral expansion of the membrane and formation of free volume below the C,TMA alkyl chain end. If the alkyl chain is short, the free volume created below its end will be large. As alkyl chain of CnTMAs becomes comparable to lipid hydrocarbon chains the free volume will decrease to zero. The formation of free volume would be expected to lessen intermolecular interaction energy between the lipid chains at their ends and to provide greater degree of freedom for their trans-gauche isomerization. However, the decrease of $33 could be also caused by the changes in the motion of the long axis of the lipid molecule as a whole. To study these effects in detail, further experiments have been performed with oriented bilayers. Because the effects of C,TMA ions on the order parameter found in disoriented bilayers were maximal at shortest alkyl chain length and lowest hydration degree, more detailed studies with oriented bilayers were performed in the presence of C6TMA at a molar ratio of C6TMA/ EYPC-- 1:2 and hydration over saturated NaCl aqueous solution. At this molar ratio, the only phase forming in our "surface" samples deposhed on the glass material was the multilamellar bilayer phase, as proved by 31p-NMR experiments (data not shown). This is in accord with our previous study [7] which has shown that in

236

the "bulk" samples the multilamellar bilayer phase is stable up to molar ratios as high as C6TMA/EYPC=0.8 at the weight ratio of H20/EYPC ~ 1 : 1. However, the lamellar phase may transform into unilamellar vesicles and/or mixed micelles with further increase of water content. Formation of bilayer "fragments" in the presence of C,TMA ions has been observed by De Smedt et al. [8] and that of unilamellar vesicles by Hauser [31]. Uiailamellar vesicles in excess water are transformed into multilamellar phase with the increase of ionic strength [31]. The oriented multibilayer samples studied in the present work are thus stabilized by the low degree of hydration and, consequently, high ionic strength. Figure 3 demonstrates typical angular dependence of A ( y ) and Fig. 4 that !

!

I

I

I

I

!

4.030

gZ(r) 4.028 ~1:+4. • °,, "dj...

4.026 "" ..%1.

4.024 4.022 4.020 0

l

I

0.2

0,4

I

I

0.6 0.8 1.0 cos2), Fig. 4. The angular dependence of the line position g(y) for the 16-doxyl stearic acid spin probe in the phosphatidylcholine bilayers, t = 23°(2; dotted line, least-squares fit to Eqn. (2)•

!

A2(r ) s

of g ( y ) as obtained with these oriented samples. The values of order parameters calculated from such data are shown in Table I and Fig. 5. As clearly seen from these results, $33 order parameter decreases in the presence of C6TMA and this decrease is more pronounced in the case of 16-DSA spin probe. This is in accord with results obtained with disoriented samples. Furthermore, the disordering effect of C6TMA ions is more pronounced at lower temperature. The information about probability of gauche conformations Pg and about effective energy difference Eg between trans and gauche conformations can be extracted from the $33 values. According to Seelig [32,33] the order parameter for rotation about a single C---C bond S~ can be obtained from $33 values of m-DSA spin probes:

.+

.,4°.~"

I 0

I

I

I

I

I

0.2

0.4

0.6

0.8

1.0

cos2),

Fig. 3• The angular dependence of the hyperfine splitting A(y) for the 12-doxyl stearic acid spin probe in the phosphatidylcholine bilayers, t = 23°C, C6TMA/EYPC = 1:2 (mol:mol); dotted line, least squares fit to Eqn. (1). TABLEI

Order parameters S, and asymmetry parameter ~/of order matrix in oriented EYPC bilayers at 23°C. The accuracy of $1], Sag is -+0.008, the accuracy of $22 is -+0.016, and that of ~ is -+0.16. Spin probe

Sample

$33

16-DSA

EYPC + H20 EYPC + C6TMA + H20

0.251 0.219

0.051 0.060

-0.302 -0.279

1.41 1.55

12-DSA

EYPC + H20 EYPC + C6TMA + H20

0.480 0.465

-0.186 -0.220

-0.294 -0.245

0.22 0.05

S2 2

Sl 1

237 !

I

I

0.32

s~

formations is given by

,~ ,,,,,"

S33

Pg=

0.30

{1 - (1 - 0.): [(1 + 0.)2 + 40.]1/2} : 2

2S 2 + 1 = 3:[(1 + o') z + 4o'] 1/2

j*~

(23) (24)

0.28

,,/~"

0.26

, S

Marsh [16,34] extended this analysis excluding which are disfavoured by intermolecular packing, and found

g+g+ conformations pg =

[1 - (1 + 80") -'/2] :2

(25)

1 - S 2 = 90.: [1 + 80" + (1 + 80")1'2] 0.20 t-

[i

3A

i 3.5

i 3.6

i

3.7

,O00/r IX"] Fig. 5. The temperature dependence of the orientational order parameter $33 for the 16-doxyl stearic acid spin probe in the phosphatidylcholine bilayers. Open symbols, without C6TMA iodide; dosed symbols, with C6TMA iodide at C6TMA/EYPC = 1 : 2 molar ratio. S33 =

SnSo

(22)

where n is the number of C - - C single bonds between the doxyl and carboxyl group of m-DSA spin probe, and SO is the order parameter for motion of the m-DSA molecule as a whole. Supposing that S~ is approximately constant between C12 and C16 carbons, S~ can be calculated from $33 values obtained with 16-DSA and 12DSA spin probes. Provided the rotations about adjacent C---C bonds are interdependent and g±g~ combinations are disfavoured, Seelig [32,33] found that the probability of gauche con-

(26)

The effective energy difference between gauche and trans conformations at the temperature T is then given by 0. = exp ( -

Eg/RT)

(27)

where R is the molar gas constant [17,32--34]. The values for Eg and Pg Obtained in our experiments are given in Table II and compared with the Eg value found by Marsh [16] for dipalmitoylphosphatidylcholine (DPPC) bilayers in the liquid crystalline state. The values of Eg are all considerably larger than the 2 - - 3 k J / m o l value found for the rotational potential in liquid hydrocarbons and diluted normal paraffins [35]. As noted by Marsh [16], this indicates that Eg must be considered a pseudopotential containing contributions from intermolecular effects as well as the intrinsic intramolecular rotational potential. From Table II it can be seen that C6TMA increases the probability of formation of gauche

TABLE II Effective energy, Eg (kJ. mol-~), and probability, Pg, of gauche conformations of the lipid chains in oriented EYPC bilayers at 24 -+ 1"C. The value of Eg for DPPC was taken from [17]. Model A: Eqns. (23---24), model B: Eqns. (25--26). Model

Sample DPPC + H20

A

Eg Ps

B

E~ Pg

7.54

EYPC + H20

EYPC + C6TMA + H20

6.15 0.126

5.73 0.143

5.82 0.123

5.34 0.139

238

conformations and decreases the energy difference Eg. This is what was expected from the free volume model of C6TMA-bilayer interaction. Both theoretical models [Eqns. (23--24), (25-26)] yield comparable values for the effects of C6TMA ion--an increase of Pg by about 13--14% and a decrease of Eg by about 420---480 J/ mol at C6TMA/EYPC molar ratio of 1:2. The values of $1~ indicate deviation of ordering from axially symmetric case and the effect of C6TMA iodide upon it. Because the order parameters Sii are not independent (see Eqn. (6)), it is convenient to introduce asymmetry parameter rl of the order matrix S, which is defined by n : (522 -- a l l ) : 533

(28)

In the case of axial symmetry a l l - 522 = - 0 . 5 5 3 3

(29)

and 77= 0. Hemminga [36] suggested that the asymmetry parameter for the cholestane spin probe could be a measure for the degree of lateral ordering in lipid bilayers. Since we have found in Raman spectroscopy experiments that C,TMA ions increase the lateral order in DPPC bilayers in the liquid crystalline state [37], it was interesting to compare this result with the values of 7/parameter. Unfortunately, the experimental accuracy of ~/values was rather poor to see any effect due to presence of C6TMA in the bilayer (see Table I and Fig. 6). It seems to be surprising that the asymmetry parameter found for the 16-DSA spin probe is higher than that for 12DSA. Notable is also an increase of ~ with the temperature as demonstrated for the case of 16-DSA spin probe in Fig. 6. A similar temperature effect was observed by Hemminga [37] in the case of cholestane spin probe, and was ascribed to long-range interactions resulting in cooperativity of the liberational motion of the lipid chains. However, it would be premature to ascribe the effects observed in our study to this type of interactions. We suppose that only a comprehensive line-shape model, which includes the effects of both long axis motion and tramgauche isomerization and which is valid in all

I

I

I

2.0 1.8

t

1.6 1.4 1.2 t.0 0.8I

I

I

I

-20

-10

0

10

Fig. 6. The temperature dependence of rameter ~ of the 16-doxyl stearic acid phosphatidylcholine bilayers. Open C6TMA iodide; closed symbols, with C6TMA/EYPC = 1:2 molar ratio.

I

20

the asymmetry paspin probe in the symbols, without C6TMA iodide at

motional regimes, would allow the extraction of information about the asymmetry from the ESR spectra of flexible m-DSA spin probes in lipid bilayers [17,38]. However, such a model needs rather extensive computer calculations. Since the asymmetry parameter indicates deviation from the axial symmetry, the only conclusion at the present stage of spectra evaluation is that this deviation is higher at higher temperature and for 16-DSA spin probe. As noted above, not only splittings and line positions, but also linewidths are dependent on sample orientations 7- Several groups of authors (see Refs. 14,17 for references) developed various theoretical models describing the angular lineshape dependence. In our present study we decided to follow a motional-narrowing theoretical model of Luckhurst et al. [19 20] (see Eqns. (12--21)) which was successfully used in the analysis of ESR spectra of m-DSA spin probes in oriented lamellar phase of the sodium decanoate/n-decanol/HzO system [21]. The typical angular dependence of the linewidth coefficients B and C as observed in our experiments is shown in Fig. 7. The angular linewidth coefficients B L

239

,

C(~,)~,~T]0.00

, .... +~.....

E

~" '"

0.06

,~

"" "'..

~" '

0.02 I 0

I

I o.

-B(),) O.Oe

,0

...

,,,: '"

'"+,..

~nT] 0.06 • """

+,

..... ¢ ..... +....

o.0: [-1T' II

0

I

0.2

i

0.4

I

I

0.6 cos 20i8

1.0

Fig. 7. The angular dependence of the B and C linewidth coefficiens for 16-doxyl stearic acid spin probe in phosphatidylcboline bilayers, t=23°; open symbols, without C6TMA iodide; closed symbols, with C6TMA iodide at C6TMA/EYPC = 1:2 molar ratio; dotted lines, least-squares fits to Eqn. (13).

(and CL) obtained by a least-squares fit of the experimental B(T) (and C( y )) values to the theoretical expression in Eqn. (13) are listed in Table III. The theoretical angular dependence, calculated with these values, is plotted as the dotted lines in Fig. 7. The agreement between

theory and experiment is seen to be rather good for both the B and C coefficients. The ~'0, ~'2 values in Eqns. (14--17) can be extracted from the experimental data in Tables I and III, using g(i,i) and A u'O values calculated according to Eqns. (18--21). The results are presented in Table IV. The values of ~'0 obtained from the C L coefficients differ slightly from those found from the B L coefficients. The differences are similar to those found in analogous studies of liquid crystals [19-21]. However, the ~'2 values differ significantly, and in some cases we obtained even physically impossible (negative) values from the C L coefficients (missing data in Table IV). This failure can be caused by both the experimental data and the theoretical model: Our experimental B and C data were not corrected for the non-homogeneous line broadening caused by the unresolved (angular dependent) proton hyperfine splitting. We simply supposed that the proton hyperfine splitting was washed out by exchange interaction with oxygen present in samples as found by Kuznetsov et al. [39], but this was not proved experimentally for our model membrane systems. Furthermore, we have not corrected our data for misalignment of the bilayers which could also produce an angular dependent broadening of the hyperfine lines. We have not observed any significant asymmetry in the line shapes exceeding the experimental error which indicates that the bilayers were well oriented within 1°. This small misalignment will not effect B L coefficients, because the contribution of its broadening effect to the B L coefficients depends on the anisotropy of the motionally

TABLE III The angular linewidth coefficients

(roT).

Spin probe

L

EYPC + H20 BL

CL

BL

CL

16-DSA

0 2 4

-0.066 -0.048 0.032

0.083 0.069 -0.048

-0.042 -0.032 0.014

0.049 0.036 -0.018

12-DSA

0 2 4

-0.130 -0.082 0.086

0.204 0.108 -0.166

-0.125 -0.087 0.074

0.188 0.117 -0.128

EYPC + C6TMA + H20

240 TABLE IV The correlation times 70 and 72 (s) obtained from a strong collision analysis of the B L and CL angular linewidth coefficients using $33 values given in Table I.

Spin

Sample

probe 16-DSA

12-DSA

From B L

From CL

%" 101°

72" 109

72/'r0

70" 10to

EYPC + H20 EYPC + C6TMA + H20

3.0 2.1

10.6 8.4

35 40

3.8 2.1

EYPC + H20

4.9 4.9

4.4 6.3

9 13

6.5 6.4

EYPC + C6TMA + H20

averaged g tensor, which is rather small. However, the contributions to CL are dependent on the averaged A tensor which is highly anisotropic and thus they could be significant. Furthermore, we supposed that the nitrogen~ nuclear spin is quantized parallel to the magrietic field for all orientations y which simplifies the theory, but causes that the extracted CL coefficients are in a larger error than the BL coefficients [20]. Therefore, the data derived from the C L coefficients can be in a quite large error because of combination of several contributions and it is better to ignore them at this stage of spectra evaluation and to concentrate on the data derived from B L coefficients. The conclusions from % values are simple. The motion of an axis orthogonal to the doxyl group ring is faster in 16-DSA spin probe than in 12-DSA, as intuitively expected for the more fluid region of the bilayer center. Furthermore, the motion of the 16-DSA doxyl group is faster in the presence of C6TMA ion, as one may expect intuitively from the decreased 533 and Eg values (Tables I n l I and Figs. 1,2,5), and increased Pg values (Table II). The r 0 values of 12-DSA spin probe does not change in the presence of C6TMA ion, which also corresponds to the small difference in the 533 values for this spin probe. The ratio ~'2/% might indicate the asymmetry of doxyl group motion in the bilayer. This ratio is larger for the 16-DSA spin probe, and increases in the presence of CnTMA ion for both spin probes. However, it is difficult to comment further on this result, because the strong colli-

~'2" 10s

72/7o

m

m

5.5 --

85 --

sion model does not indicate how the correlation times should depend on the molecular structure or environment. Nevertheless, we would like to remind of increased rl value for 16-DSA spin probe (Table I), and of increased lateral packing of acyl chains detected by Raman spectroscopy [37] in the presence of C , T M A ions. In conclusion, we have found that the CnTMA ions affect the structure of the EYPC bilayers increasing the probability of gauche conformations (and decreasing the effective trans-gauche energy difference) in the hydrophobic core of the bilayer. This is accompanied by the decrease of the correlation time of the spin probe motion. These results are in accord with the molecular model of the EYPC-CnTMA interaction: The positively charged C , T M A polar group interacts with negatively charged EYPC phosphate and the C~TMA alkyl chain orients parallel to the EYPC hydrocarbon chains. The packing density of lipids is influenced due to lateral expansion of the bilayer and formation of free volume below the C~TMA alkyl chain end. The free volume is filled in due to the increased lipid chain transgauche isomerization. The free volume is largest for the shortest alkyl chain. As alkyl chain reaches the lipid chain length, the free volume decreases to zero. In contrast to our study performed on samples which were at less than limiting hydration, in model membranes prepared by hydrating the lipid in an excess of water and in biological membranes the free volume effect must be modulated by the partitioning of surfactants between the aqueous and lipid phases. As a result the final membrane perturbation is mini-

241

mal for both the shortest and longest alkyl chain lengths; this effect might be responsible for the well known cutoff in the biological activities of surfactants [27].

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