Low frequency and diffusive motion in aligned phospholipid multilayers studied by pulsed NMR

Chemistry and Physics of Lipids, 27 (1980) 151-164 © Elsevier/North-Holland Scientific Publishers Ltd.

LOW F R E Q U E N C Y AND DIFFUSIVE M O T I O N IN A L I G N E D PHOSPHOLIPID M U L T I L A Y E R S STUDIED BY PULSED N M R B.A. CORN-ELL and J.M. POPE* CSIRO Division of Food Research, North Ryde, N.S.W. 2113 and *University of New South Wales, Department of Physics, P.O. Box 1, Kensington, N.S.W. 2033 (Australia)

Received September 13th, 1979

accepted January 5th, 1980

Measurements of T~, Tt,, T~o and the FID are reported for protons in aligned stacks of Egg Yolk Lecithin (EYL)-D20 multilayers. From the ~ot dependence of T~o and the ~o0dependence of T~ the two dispersive regions of the spectral density function were identified. The lower frequency region has a centre angular frequency of 2.2 x 105rad/s and the upper frequency region a centre angular frequency of 4 x 107rad/s. From the orientation dependence of T~p,T~a and the FID, the lower frequency dispersion is assigned to fluctuations in the packing density of the hydrocarbon chains in the plane of the bilayer, and the upper frequency region to the translational diffusion of the lipid molecules. Based on a similar approach to one previously described, a dittusion coefficient of 1.4 × 10-s em2/s is calculated for the EYL molecules in the bilayers.

Introduction The technique of nuclear magnetic r0sonance (NMR) has been used extensively in the study of the dynamic state of phospholipid molecules in aqueous lamellar dispersions. (See for example refs. 1----6). T w o approaches are c o m m o n l y adopted. T h e first is to study the reduced static in.teractions and their relationship to the spatial anisotropy of the motion. The second is to study the nuclear relaxation processes and their relationship to the timescale of the motion. The f o r m e r approach has been particularly useful in m e a s u r e m e n t s of 31p and 2H on native and deuterated phospholipids (see for example refs. 7--9). T o date virtually all spin-lattice relaxation measurements on multilamellar phospholipid dispersions have been restricted to the protonic groups. Most of these m e a s u r e m e n t s have b e e n m a d e on unaligned solutions of lipid [10~12]. Fung and Martin [13] have reported a measurem e n t of Tm in aligned phospholipid bilayers in which T~ was found to be independent of the orientation of the sample to the field direction. In addition several studies, have been reported of the motion of the hydrocarbon chains in related soap-water systems. (See for example refs. 14---16). T w o reports are available of the phospholipid protonie spin lattice relax-

151

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B.A. Cornell and J.M. Pope, Pulsed NMR studies o[ phospholipid multilayers

ation time in the rotating reference frame (Ttp). Salisbury et al. [10] have measured T~p at H1 fields of 4 and 8 x 10-aT over a range of temperatures and hydrations for the protons in dipalmitoyl phosphatidylcholine (DPPC). Local T~ minima were observed at temperatures up to 100°C indicating the presence of reorientation rates for some of the protonic groups at 2.13 × 105 rad/s and 4.27 × 105 rad/s respectively. A recent NMR study of hydrated DPPC has shown an H~ dependence of TI~ in the range 1.6 to 25 × 10-4 T. This result has been interpreted as arising from the diffusive motion of the phospholipids with a jump rate of order 1.4 × 106/s [17]. The only reported measurement of the relaxation time in the local dipolar field for hydrated lecithin bilayers, T1D, is that by Charvolin and Rigny [2]. T~Dwas found to be significantly shorter than the values of Ttp reported elsewhere for similar samples (see above) although the corresponding decay was much longer than the observed FID. A more recent study of anhydrous DPPC by Gilboa [18] has also shown a Txt) which is far shorter than T1 at 60 and 16 MHz. In this paper we present a consistent set of measurements of T1, TI~ and T~r, for the protons in samples of hydrated EYL aligned between glass slides. From these results it will be shown that the distribution of correlation times extends down to the local field region. By comparison with a similar study by Charvolin and Rigny [14] of the soap-water system, C~2K--D20, such low frequency modes of reorientation are to be expected and are assigned to co-operative motions of the hydrocarbon chains. Since the proton FID obeys a timescale dependence of the form 1(3 cos2 4,-1)[ -~ as a function of the relative orientation ~b of the bilayer normal to the main magnetic field direction, the lowest frequency motion must at all times maintain the reduced static dipolar interactions parallel to the bilayer normal. We suggest that this effect is due to fluctuations in the local density of molecular packing. As a result, the intermolecular dipolar interaction, although projected onto the bilayer normal by the rapid translational diffusion of the hydrocarbon chains, is fluctuating in magnitude. The frequency dependence of our Tt measurements is assigned here to the contribution to the spectral density function made by the translational diffusion of the lipid molecules. Using the approach of Burnett and Harmon [19] and Fisher and James [17] we estimate a diffusion coefficient of 1.4 × 10-s cm2/s for EYL at 25°C.

Materials and methods

Egg yolk lecithin was prepared using the technique of Singleton et al. [20] and its purity checked by thin layer chromatography (TLC) using a 65 : 25 : 4 solvent of CaCl3, MeOH and H20 respectively and iodine vapour to visualise the spots. Some samples were prepared from EYL purchased from Sigma Chemicals Inc. which was similarly checked by TLC. Stacks of aligned

B.A. Comeli and J.M. Pope, Pulsed NMR studies of phospholipid multilayers

153

E Y L multilayers were prepared by drying down an approx. 5 - - 1 0 m g quantity of E Y L from a CHCI3 solution onto a series of 20--30 thin glass slides. The dimensions of the slides were 6 mm × 20 mm × 0.1 ram. The slides with their coating of E Y L were left overnight in a vacuum chamber with a potential vacuum of approx. 10-6 mm Hg. Measured quantities of D20 were then syringed onto the E Y L coating of each slide and the slides stacked to form a sandwich of some 30 layers. As each slide was added to the stack it was gently rubbed back and forth relative to the remainder of the stack to aid in the alignment of the E Y L on the glass surface. The degree of alignment was apparent not only from the angular dependency of the FID but also by optical microscopy between cross polarisers. The stacks were stored under N2 in the presence of a known vapour pressure of D20. The degree of hydration of the E Y L was established by pumping the samples to constant weight following the N M R experiment. The results quoted here refer to samples with approx. 6 ~ 1 0 D 2 0 molecules per E Y L molecule. A b o v e approx. 3 molecules of water per lecithin molecule the N M R properties of the protonic groups of the lecithin molecule appear insensitive to further hydration. Increasing the level of hydration beyond n ~> 20 water molecules per lecithin molecule did however cause a progressive loss of alignment. The N M R measurements were made using a commercial Bruker SXP 4-100 high power pulsed N M R spectrometer. Unless labelled to the contrary all measurements reported here were taken at an ambient temperature of 25 ___I°C. The E Y L stacks were mounted inside a glass tube which could be rotated about an axis perpendicular to Ho and set to a particular angle ~b with a reproducibility of <0.1 °. The measurements of the FID, T~ and T~p were made using the conventional pulse sequences. T1D was estimated as described in the results section from a Jeener and Broeckaert [21] 90~ - ~-~45~0 - ~'~- 45~0 pulse sequence.

Results

The FID, T~D and T~p were all found to be dependent on the orientation angle 4' of the normal to lipid stack relative to the applied magnetic field. T h e FID has been shown to vary as a ~unction of orientation according to a timescale of the form 1(3 c o s 2 4~ - 1)1-1 [22]. Separate measurements have been made at short times, immediately following the recovery of the amplifier chain at the completion of the 90* pulse, on the dependence of the slope of the FID on the orientation angle 4~. Allowing for the correction of approx. 0.7 ° in the mosaic spread of the sample stack the same 1(3 cos 2 4~ - 1)1-1 relationship was found to hold. Tit, was measured at two values of ~'1 near the value which provided the

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B.A. Cornell and J.M. Pope, Pulsed NMR studies of phospholipid multilayers

T -1 (sec.-1) 1D

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1 I I I 0-5 I( 3 cos2o - ~)1 ~.s 2.0 Fig. 1. The orientation dependence of the dipolar relaxation time T~D. In order to emphasise the difference between the orientation dependence of the FID and TtD, the gresent graph is in the form Ti~t versus 1(3cos2~k- 1)[. Were the same dependency on ~k to hold, we would expect a straight line with a positive slope extrapolating to the origin (see text). 0

0

m a x i m u m free induction signal following the second 45 ° pulse. T h e angular d e p e n d e n c e of TtD is s h o w n in Fig. 1. This plot is in the f o r m Tt-D1 versus ](3 COS2 ~k -- 1)[. If T~D o b e y e d the s a m e angular d e p e n d e n c y on $ as does the F I D we would expect a straight line with a positive slope passing t h r o u g h the origin. Clearly this is not the case as T~D p l a t e a u s at approx. 32 ms for 0 . 8 ~> 1 3 c o s 2 ~ - 1 ] > 0 . T h e d e c a y of the signal following the second 45 ° pulse in a J e e n e r and B r o e c k a e r t s e q u e n c e was, within e x p e r i m e n t a l accuracy, a single c o m p o n e n t e x p o n e n t i a l as s h o w n in Fig. 2. A t short values of ~2 ~ ~'~.it was difficult to r e a d the a m p l i t u d e of the F I D following the s e c o n d 45 ° pulse owing to the p r e s e n c e of an e c h o which occurs after the s e c o n d 45 ° pulse a n d o v e r l a p s the F I D . T h e e s t i m a t e of T~D was o b t a i n e d t h e r e f o r e f r o m the F I D a m p l i t u d e at values of ~'2 ~" ~'t. T~p was m e a s u r e d as a function of b o t h o r i e n t a t i o n ~k and the radio f r e q u e n c y field strength H~. A n e x a m p l e of the loss of a m p l i t u d e of the F I D following the spin locking pulse as a function of the locking pulse d u r a t i o n is

B.A. Comell and ZM. Pope, Pulsed NMR studies o[ phospholipid raulalayers

155

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shown in Fig. 3. The decay is not a single exponential function of the locking pulse duration as it contains both a small raIiidly decaying component and a major component which decays more slowly. The values of T b quoted here refer to the more slowly decaying component as shown in Fig. 3. The dependence of T b on the value of tot = yHt is shown in Fig. 4. At the magic angle ~b = 54.7°, T b was apparently independent of H~. At ~b = 0 ° however, T b showed a strong dependence on H~ over the initial 2 x 10 -4 T (tot ~ 5 x 10~rad/s) and a slower dependence up to a value of approx. 60 × 10-4T (tot ~-1.5 x 10~ tad/s) at which T b was independent of orientation. As the residual dipolar interactions within the sample are of order 2 x 10-4 T it is felt that the shorter values of T b at spin locking fields, H t of < 2 x 10-4 T

156

B.A. Cornell and J.M. Pope, Pulsed NMR studies of phospholipid muitilayers

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Fig. 3. Decay of the FID following the spin locking pulse as a function of the locking pulse duration. reflect the inability of the spin locking field to suppress the dephasing of the spins due to the local fields. The variation of T~, as a function of orientation angle ~ is similar in form to that for T~D in Fig. 2. T~, does not obey a simple 1(3 cos 2 4~ - 1)1 relationship as does the FID. Measurements were also made of T1 at to0/2~r = 4, 8, 30 and 60 MHz. These are shown together with the T~D and T~ data in Fig. 4 in the form of an approximate spectral density function for the average motion of the proton-containing groups in the frequency range 10~ to 109rad/s. The frequency dependence of the T ~ 1 data is that of to~ = ~,H~ obtained from the duration of a 90 ° pulse of the same R F field strength.

Discussion The relaxation data for oriented EYL-DzO bilayers presented in Fig. 4 are consistent with there being at least two contributions to the frequency dependence of the relaxation times in the angular frequency range 1~---

B.A. Cornell and J.M. Pope, Pulsed NMR studies of phospholipid multilayers

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Fi~. 4. ~ ¢ appro~at¢ ~ a l dCnsi~ run,ion in the f o ~ of the relaxa~on rat~ T~ (&), T ~ (O) at ~ T,-~ ~ ) at the marc ~ ¢ and T~' (@) = a f u n ~ . of ~¢ appropriate an~lar fr~ucn~. ~ ~ ran@¢ ]~ < = < ]~/s the ~,ti~uo~ c ~ ¢ for ~ ~ = ~ c ~ o~ys the ~latio=hip T ~ = r~(~ = ~ , ~ ~0) + 2h=~,d(] + ~ = ~ , ~ ) whe~ h = 2.4 x l~Is a.d ~,L= 0.~ x ~0-~ s.~ ¢ = d¢~ndea= of the relaxation~t¢ in the range ]~ < = < ~Is ist ~ ~adual for thiscl~ o( f~qucn~ d¢~ndcn~. ~is is seen by ~ m p a d ~ n ~ the dotted cu~e which d¢~s the cxpr¢~ion T i -~ = 2h:~'~/(1 + ( t o o l ' S )

where h = 2.2 x lO~/s a n d ¢~ = 0.25 x 10 -7 s.

109/s. When the sample stacks were oriented at the magic angle relative to the main magnetic field, the low frequency contributions to relaxation (to < 106 rad/s) were eliminated. In contrast, the form of the spectral density function above to = l&rad/s appears to be independent of the orientation angle. It is significant that T~D and the extrapolation of T1, for tot-*0 obey a similar dependence on the orientation angle 4~ and tend to similar times at any particular orientation. This suggests that a common relaxation mechanism contributes to both measurements and that no additional contributions to relaxation occur at reorientation rates below to~ = 103 rad/s. The increase in T1,

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B.A. Cornell and J.M. Pope, Pulsed NMR studies of phospholipid mulalayers

seen below tot = 104rad/s is assigned to a loss of spin locking during the measurement. This latter effect is most marked at $ = 0 °. The centre frequencies of the two dispersive regions seen at ~b = 0 ° in Fig. 4 are 2.2 x 10~rad/s and 4 × 107 rad/s. The upper centre frequency (tou) is estimated as shown in Fig. 4 from the frequency corresponding to Tt7 = T i7 (~b --- 54.7 °, tot--*0)/2. The high frequency plateau of Tt is neglected and the spectral density function is assumed to asymptote to zero at frequencies significantly above 109 rad/s. The centre frequency of the low frequency dispersion is taken as that corresponding to Ti2 = [T?2(~b = 54.7 °, tol "--)'0) + T{al((~ = 0 °, tol "-')'0)]/2. It is interesting to note that despite the existence of a frequency dependence for Tip well into the local field region of the spectral density function, the time scale of the FID obeys a 1(3 cos2 ~b- 1)l-t dependence on ~b. This shows that the motion contributing to the gO 1 dependence of Tip must maintain the proiection of the net motionally averaged dipolar interactions parallel to the bilayer normal. As this component of the motion is not itself sufficiently rapid to result in a projection of the dipolar interaction onto the bilayer normal the observed angular dependence of the FID must arise from an additional motion with reorientation rates at higher frequencies. This may be used as a guide to the type of motion which gives rise to this component of the spectral density function. In particular it is unlikely that the translational diffusion of the lipid molecules in the plane of the membrane could occur in a manner which at every instant holds the long axis of the lipid molecule perpendicular to the plane of the membrane. Although a gap exists in the spectral density function between the upper limit of Ttp (tol = 4 × 106 rad/s) and the lower limit of T~ (tOo= 2.5 x 107 rad/s) the higher frequency dispersion appears to occur over a relatively wider range of frequencies. As shown in Fig. 4, attempts to fit the to dependence of the relaxation data to expressions of the form Ti~ ~

2 h 2'rCL 1 + 4(tot'/'cL) 2

and 2h2~'c~ Ti~ ~ 1 + (too~) 2

B.A. Cornell and J.M. Pope, Pulsed NMR studies of phospholipid multilayers

159

where 1 T~L= - - ,

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and h is a constant dependent on the strength of the residual dipolar interaction, have been successful for the to~ dependence of Tt~ but clearly do not predict the to dependence of T~. Fisher and James [17] have recently reported a study of the to~ dependence of T~. These authors studied coarse dispersions of the lipid and thus were unaware of the superposition of T~ decay curves from which their results were obtained. However there is qualitative agreement with the ~o~ dependence of T~ reported here. Fisher and James have interpreted their data using a treatment reported earlier by Burnett and Harman [19]. According to this approach, on the assumption that intermolecular interactions are important in contributing to the measured relaxation,

1/T~p = Co~[~ + 1/T~ where C is a negative constant and for three dimensional isotropic tumbling is given by

C = -~/~T4h2~'N/2OD~/~. For two dimensional diffusion this is modified by Fisher and James to

C = - 3 ~ f ' ~ y ~ h ~ ' N / 4 0 D ~n. Measuring the value of C from a plot of T~p versus to ]/2 and substituting for the constants, a value for the self diffusion coefficient D may be obtained. Based on such an analysis these authors obtained values for D of order 2--5 x 10-9 cm2/s for DPPC. Furthermore by assuming a diffusive process involving thermally activated jumps between lattice sites separated by 8 A they calculate a correlation time ~'c associated with this motion of between 2.3~7.3x 10-7s. However a calculation of the corresponding values of to = 1/~'c, at which such motions would be expected to contribute to a change in level o f the spectral density function, shows that they cover a range an order of magnitude higher than that over which Fisher and James observed a dispersion of the T~o data. We suggest therefore that the frequency dependence seen in the Tto measurements reported here and by Fisher and James is not due to a diffusive motion. Indeed if the jump distance associated with diffusion were smaller than 8 A, as appears probable, the

160

B.A. Cornell and J.M. Pope, Pulsed NMR studies of phospholipid multilayers

correlation time would be shorter and the region of frequency of the spectral density function effeeted by such a motion even higher. An alternative approach is to assign the frequency dependence in the T1 region to the diffusive motion. Figure 5 shows a plot of T~ 1 v e r s u s to 1/2 taken from Fig. 4. Using the straight line region of Fig. 4 below to~-< 1 and the constants quoted tiy Fisher and James we calculate a diffusion coefficient of 1.4 x 10-8cm2/s *. Using o~u~-1/zc, to calculate the correlation time of this motion (0.25 x 10-7 S) and the expression ~r = 4X/~-~zcu, we obtain a jump distance for the elementary diffusion step of ~3.5/~. This calculation is self consistent in that the ~'cuemployed here is the correlation time of the actual dispersive component of the spectral density function assigned to the motion under study. Furthermore the extrapolation of the straight line portion of Fig. 5 gives a value of T[ ~ of the same order as T ~ (~b = 54.7 °) and Tip (~b = 54.7 °, ~o~--*0). Clearly the variation in the relaxation rate in the range 106 to 10Srad/s is very much slower than that predicted by a simple (2hzc)/(1 + (a~OZc)~) relationship, but fits an ~o~/2 dependence well. The calculated values of D and ~r are in good agreement with estimates made elsewhere [23--25]. Petersen and Chan [26] have also deduced the presence of a component of the lipid motion with a correlation time of order 10-7 s. It is interesting to note that these authors show that this motion causes the long axis of the hydrocarbon chains to undergo large (approx. 55) angle rotations away from the bilayer normal. This form of motion may well be associated with the translational diffusion of the lipid molecules (see below) and is in agreement with the relaxation data reported here. If indeed the lipid diffusion contributes to the dispersion of the spectral density function in the region of the T~ measurements it remains for us to account for the motion contributing to the frequency dependence of T~p. In an NMR study of potassium laurate-D20, Charvolin and Rigny have reported the presence of a similar low frequency component for the dynamics of the potassium laurate chains. These authors suggest an interpretation based on the work by Rouse [27] and more recently Agren [28] in which the hydrocarbon chain is thought to undergo slow intramolecular reorientations due to random additive combinations of the flexing of the individual carbon-carbon bonds. In the case of the potassium laurate system, correlation times as long as 0.3 x 10-Ss are observed and assigned to this effect. The dependence of Tto on deuteration as reported by Fisher and James and the orientation dependence of the FID, T~, and T~p reported

*In calculating D, Fisher and James assumed that conversion of the Burnett and Harmon theory for 2-dimensional rather than 3-dimensional diffusion involves a simple modification of the constant C, by a factor 2%/~]3V~. A similar assumption for the dependence of Tl on ¢o0

involvesa factor (2X/~- 1)/(3X/~- 1).

B.A. Cornell and .I.M. Pope, Pulsed NMR studies of phospholipid mul~ilayers

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here, suggest however that the origin of these low frequency rates of reorientation is a modulation of the inter-chain, reduced, dipolar interaction. Charvolin and Rigny comment on the need for co-operation between the motion of the hydrocarbon chains possessing the lower frequency modes of reorientation, however n o theory exists to predict such a contribution to the relaxation. The orientation dependence of the FID shows that the motion effective in projecting the dipolar interaction onto the bilayer normal is faster than that contributing to the T~, dispersion. Provided the reodentation of the chain long axes described by Petersen and Chan is both symmetrical and associated with a fast translational diffusion, the correlatioa time of order 10-7s assigned by these authors to this motion is sufficient to project both intra and inter molecular dipolar interactions onto the bilayer normal. If in addition localised fluctuations in the packing density of the lipid chains modulate the interchain spacing, the magnitude of this interaction will vary resulting in the low frequency contribution to relaxation seen in Fig. 4. When the lipid planes are at the magic angle to the main magnetic field,

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B.A. Comell and J.M. Pope, Pulsed NMR studies of phospholipid multilayers

these static interactions are virtually eliminated and thus the interchain contribution to relaxation below to ~ 1.5 × 10-6 rad/s is lost. The angular dependence of T1D and TI~ seen in Figs. 1 and 4, therefore results from the angular variation of the residual intra and thus inter molecular dipolar interactions (both projected along the chain axes). At the magic angle these make a negligible contribution to relaxation compared to the residual isotropic component T~2 (~b = 54.7 °, tol-o0). Away from the magic angle these anisotropic contributions increase, becoming comparable with the isotropic component. From the approximation T~(to~-~0) ~ 2h2rc we may calculate interaction strengths of 2.4 × 103/s and 2.2 × 10~/s corresponding to the lower and upper dispersive regions respectively. From this it may be seen that only a small fraction of the inter chain dipolar interaction is left following the averaging resulting from the translational diffusion. The existence of these low frequency modes of reorientation is consistent with the improved resolution seen by Chapman et al. [29] for EYL dispersions spun at the magic angle at spinning rates of order 103/s. It is interesting to note that for similar modes of diffusion the product D r is approximately constant. We may use this to test the relationship between the diffusion process in the single chain soaps and the double chain phospholipids. Charvolin and Rigny [14] have reported a value of D for the potassium laurate soap system of order 10 -6 cm2/s. Using this value of D for the soap systems and the values of D and ~" measured in the present study for lecithin bilayers, if diffusion in both systems occurs by a similar mechanism we expect a correlation time for diffusive jumps in the soaps of order 10-9s as indeed is reported by Charvolin and Rigny. The slower diffusion coefficient of the phospholipids is presumably due to the need for the diffusive jumps of both chains to be co-operatively involved in the translation of the whole molecule. The difference between the spectral density functions of the two chain and single chain cases seems therefore to be a shift to higher frequencies of the jump rates associated with the diffusion of a single chain. The low frequency correlation times, however, appear to be very similar at 0.35 × 10-5 s for C 1 2 K - - D 2 0 and 0.23 × 10-5 s for EYL-D20. A possible mechanism for diffusion which can also account for the large angle excursions of chain segments away from the bilayer normal proposed by Petersen and Chan (see above) involves the formation of double g a u c h e (g÷tg-) kinks in the chains which, once formed, propagate up the chain resulting in a diffusional displacement of the whole molecule. While the correlation time associated with such kink formation might be expected to be of order 10 -9 S for a single chain, the necessity for two similar kinks to form simultaneously for diffusion to take place in the lecithins can account for the much longer correlation time of 10-7S reported by Petersen

B.A. ~-nell and ZM. Pope, Pulsed NMR studies of phospholipid muMiayers

163

and Chan and observed in the present study. Such motions will indeed carry the section of the chain involved in a kink through angles approx. 55° away from the bilayer normal. Following the completion of this work Kimmich and Voight [30] have reported a series of spin-lattice relaxation measurements taken from nonaligned dispersions of dipalmitoyl lecithin in D20. These data are shown to be consistent with a defect diffusion model in which kinks form within the hydrocarbon chains and diffuse between both layers of polar head groups at the lipid water interface. This approach has been shown to be consistent with both the temperature dependence of T~ in the range 0 ° to 60° and the frequency dependence of Tt in the range 10~ to 10SHz, for hydrated dipalmitoyl lecithin. Deviations from the predictions of the model occur at lower frequencies which is in agreement with the ~ot dependence of Tip seen in the present study. Kimmich and Voight have assigned the low frequency dispersion of T~ for dipalmitoyl lecithin in the fluid phase to the overall tumbling of the multilamellar bilayers. The dependence of T~p on deuteration, as reported by Fisher and James, and the observation of a low frequency dispersion in aligned samples of lipid where overall tumbling cannot occur both suggest that the origin of this low frequency motion stems from an intermolecular effect as discussed above. The T~ results described here are in good agreement with those obtained by Kimmich and Voight although in this report we have applied the interpretation proposed by Fisher and James.

Conclusions Two types of motion appear to be important in determining the form of the "all proton" spectral density function up to angular frequencies of order 109 tad/s: (a) translational diffusion of the lipid molecules in the plane of the membrane; (b) localised fluctuations in the packing density of the hydrocarbon chains. Based on the measurements reported here the translational diffusion coefficient is approximately 1.4 x 10-Scm2/s. If this diffusion is describing an elemental jump process, the average jump rate is of order 4 x 10T/s, for a jump distance of approx. 3.5 ~. The correlation time for the density fluctuations is approximately 0.23 x 10-5 s.

Acknowledgements One of us (J.M.P.) wishes to acknowledge financial support for this project from the ,Australian Research Grants Committee.

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B.A. Comell and J.M. Pope, Pulsed NMR studies oJ:phospholipid muMlayers

The authors gratefully acknowledge the assistance of Mr G.W. Francis for his h e l p in w r i t i n g t h e c o m p u t e r p r o g r a m s n e c e s s a r y f o r this s t u d y .

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