Eur. J . Biochem. 58, 133- 1 4 4 (1975)

Ion-Binding to Phospholipids Interaction of Calcium and Lanthanide Ions with Phosphatidylcholine (Lecithin) Helmut IIAUSER and Michael C. PHILLIPS

Biosciences Division, Unilever Research Laboratory, Colworth/Weiwyn, Hertl'ordshire Barry A. LEVINE and Robert 1. P.WILLIAMS

Inorganic Chemistry Laboratory, University of Oxford (Received April 9 /June 19, 1975)

Surface chemical and nuclear magnetic resonance (NMR) techniques have been used to study the interaction of CaZ and lanthanides with lecithins. With both methods positive reactions were detected at metal concentrations > 0.1 m M . 'H and "P high-resolution NMR spectra obtained with single bilayer vesicles of lecithin were invariant up to Ca2+concentrations of 0.1 M indicating that there is only a loose association between Ca2+ and the phospholipid. The weak interaction between Ca" and lecithin is confirmed by both surface chemical and NMR techniques showing that the packing of egg lecithin molecules present in bilayers does not change up to Ca2+concentrations of about 0.1 M. The packing was also independent of pH between 1 - 30. Contradictory results have been reported in the literature concerning the question of Ca2 binding to lecithins. The conflicting results are shown to have arisen from differences in the experimental conditions and differences in the sensitivity of the physical methods used by various authors to study Ca2+-lecithin interactions. An estimate of the strength of binding and molecular details of the interaction were derived using paramagnetic lanthanides as isomorphous replacements for Ca2+. From the changes in chemical shifts induced in the presence of lanthanides an apparent binding constant K A z 30 l/mol was calculated at lanthanide concentrations > 10 mM. Using surface chemical methods it was shown that this K A is up to 10 times larger than that for Ca2+ binding. The complete assignment of the 'H NMR spectrum of lecithin, including the resonances from the relatively immobilized glycerol group, was determined to derive molecular details of the cationlecithin interaction. From spin-lattice relaxation-time measurements and line broadening in the presence of GdC13 it is concluded that the cations are bound to the phosphate group and that this is the only binding site. The absolute proton shifts induced by paramagnetic lanthanides depended on the nature of the ion, but the shift ratios standardised to the shift of the 03POCH2 (choline) signal were invariant throughout the lanthanide series indicating that the shifts are purely pseudocontact. In contrast the "P shifts were found to contain significant contact contributions. These findings are consistent with a weak interaction and with the phosphate group being the binding site. The absolute shifts but not the shift ratios depended on the anion present indicating that the cation binding may be accompanied by binding of anions. Contrary to negatively charged phospholipids the interaction of lanthanides with lecithins was enhanced as the ionic strength was increased by adding NaCl. This was explained in terms of steric hindrance due to the extended conformation of the lecithin polar group. +

+

There is considerable interest in the mode of binding of cations to phospholipid bilayers for it is known ~-

Abbreviation. N M R , nuclear magnetic resonance.

that the stability of bilayers is affected by the presence of cations. A great deal of previous work carried out on this topic is partly contradictory. Even the question of whether the polar group of lecithin (phosphatidyl-

I31

choline). which is zwitterionic over the pH range 3 - 13 [ l ] , binds Ca" and other ions has been a matter of controversy. For instance. Shah and Schulmann [2,3] concluded from surface potential measurements that both saturated and unsaturated lecithins interact with 10 mM CaZ+and that the degree of interaction is in the order dioleoyl < egg < dipalmitoyl lecithin. Kimizuka and Koketsu [4] and Kimizuka rt rrl. [ 5 ] reported binding of "Ca" to monolayers and multilayers of egg. soy bean and dipalmitoyl lecithin. White and Lakshininarayanaiali [6] showed, by studying the distribution of. Ca' and synthetic lecithin in a twophase system, that Ca'+ was associatcd with the phospholipid. Bangham and Dawson [7] reported that the particles in unsonicated dispersions acquired a positive :-potential i n 0.01 M CaClz solutions. Trauble [8] measured the fluorescence of 1-anilino-8-naphthalenesulphonic acid incorporated into bilayers of dipalmitoyl lecithin and concluded that Caz+ is bound to the lecithin with a stability constant K = lo" M - ' . Dervichian [9], on the other hand, using a potentiometric titration method apparently showed that egg lecithin did not bind Ca2+ in the pH range 2-8. Rojas and Tobias [lo] and Hauser and Dawson 11 13 using surface radioactivity techniques reported that under certain experimental conditions there was no detectable interaction between Ca 2+ and pure egg lecithin at Caz+ concentrations of 0.1 mM and 0.1 pM. respectivcly. This experimental finding was confirmed by Santis and Rojas [I21 working with both pure dislearoyl and natural lecithins. One purpose of the present paper is to clarify this situation by comparing results obtained with two different physical techniqucs : (a) surface chemicnl and (b) nuclear magnetic resonance ( N M R ) methods. Two different lecithin structures have been used, lecithin monolayers and lecithin bilayers, respectively. The latter system as single-bilayer vesicles present in sonicated dispersions has been shown t o be useful for NMR studies because it gives well-resolved 'H. 13C and 3 1 P high-resolution N M R spectra [16,17]. Another purpose is to compare the binding of Ca2+ with that of lanthanide ions which gives details of the stoichiometry. strength of binding and the general nature of the binding site. This information is required for a subsequent paper which will give the full conlormationat analysis of the phospholipid polar group. The complete assignment of the 'H-NMR spectrum of phosphatidylclioliiie required for such an analysis is also given here.

Ion-Phospholipid Interactions

if necessary, purified by silicic acid chromatogrnphy so that when a large silica gel H plate was loaded with 0.5 mg of lecithin and run with CHC13/MeOH/7 M NHjOH (230/90/15. by vol.) as solvent, only a single spot was observed. The spreading solvents for the monolayer experiments were hexane, which was purified by passage tht-ough a column of activated alumina, and redistilled ethanol. 45CaCI, of specific activity 1000- 1600 Ciiinol were obtained from The Radiochemical Centre, (Amersham. U. K.). AnalaR grade NaCl and CaCI, were roasted at 500 T before use. Lanthanide nitrates were purchased from Koch-Light Ltd. Lanthanide chloride solutions in 'HzO were prepared as previously described [38]. All other chemicals were A. R. grade. The water used was distilled. deionized. distilled from alkaline permanganate under N, and redistilled in an all-glass apparatus. ' H 2 0 was used for the NMR experiments (approx. 99.7",, from Procheni Ltcl, Croydon. U. K.). Surfirc~~ Ciicniistr~~

The procedures used to obtain simultaneous nieasurements of surface pressure (7r) and surface potential ( A P-) 21s a function of surface area/molecule ( A ) at 20 f 1 -C have been described before 1131. Surface radioacti4ities ( A R ) werc measured with a gas-flow detector mounted above the air-water interface [ I I ] ; a calibration curve to convert surface radioactivity into surface concentration was constructed by two different methods (cf: [18]). Thc surface radioactivity nicthod is limited to low substrate concentrations of active material because at higher concentrations ( i .P . [45Ca2+]2 1 mM) the increase i n surface radioactivity over the high background count (at a given specific activity the background count increased linearly with 45Ca2+concentration) was negligible and beyond the sensitivity of our counting equipment. Also. because of a high background count and potential health hazards the original high specific activity of 1600 Cij mol had to be decreased gradually a t [Ca"] > 10pM. The absorbance of 1 ";, ( w h ) sonicated egg lecithin dispersions in water at 20 1 ' C was determined as a function of the concentration of Ca'+ at 520 ntn in a Unicam SP1800 spectrophotometer [32]. Theelectrophoretic mobilities of unsonicated lecithin liposomes at a concentration of 1 prn in water were measured at 25 f 0.5 ;C as a function of Ca" concentration in a cylindrical, horizontal electrophoresis cell [32]. N M R Metiiod.7

EXPERIMENTAL PROCEDURE icfu teriul.7

Egg yolk lecithin (Grade I ) was purchased from Lipid Products (South Nutfield, Surrey, U. K.) and.

The ' H NMR spectra were recorded either on a Varian XL 100-15 or a Bruker 270-MHz spectrometer both operating in the Fourier transform mode. With the Bruker instrunlent an Oxford Instrument Company superconducting magnet and for Fourier

H. Hauser, M . C. Phillips, B. A. Levine, and R. J. P. Williams

transform operations a Nicolet 1085 computer were used. Some ‘H spectra were also obtained on a 220 MHz Varian HR 220 instrument operating in the continuous wave mode. Tetramethylsilane and 3-(trimethylsily1)propane sulphonate were used as internal standards in C’HCI, and ’ H 2 0 solutions, respectively. Spectra obtained on irradiation at a frequency away from the observed resonances were used as the “blanks” during spin decoupling experiments. This eliminated any effects due to the Bloch-Siegert shift [39]. 31 P high-resolution spectra were obtained on a Bruker WH90 spectrometer operating at 36.436 MHz. (Me30),P0 was used as an internal standard. 31P wide-line NMR spectra were run on a Varian wideline NMR spectrometer operating at 8.13 MHz. All N M R experiments were carried out at 25 k 1 *C unless otherwise stated. Spin-lattice (Tl) relaxation times were determined using pulse Fourier transform techniques based on a 180 - 7 - 90 sequence [36].

135

450

r

350 -

10.~

10.~

b*’I

10‘’

10.’

loo

(MI

Fig. 1. Effect of Ca’+ on the su~fucrpoientialA V ofegg lecithin monolayers. The film pressure was 18 mN/m corresponding to an area of about 0.7 nm2 per moleculc. The subphdse was distilled water pH 5.5. The vertical bars represent the spread of 3 - 6 experiments

O

RESULTS Surface Chemical Techniques

Curves of 71 versus A for egg lecithin were obtained on distilled water and substrates containing NaCl and CaCl,. No significant change in the 71 - A curve was observed over a concentration range of [Ca”] = 10 nM to 0.1 M. The n - A curve typical for unsaturated lecithins (e. g. egg lecithin) has been reported before [13]. Surface potentials ( A V) were measured at an area of 0.7 nm2/egg lecithin molecule, characteristic of the molecular packing in liquid crystalline bilayers, over the range [Ca”] = 10 nM to 1 M. In these experiments, an appropriate amount of egg lecithin was spread on CaCl, solutions (pH 5 . 5 ) and the movable barrier adjusted to give a theoretical area/molecule of 0.7 nm’; 71 was 18 k 1 mN/m. At CaZt concentrations > 10 pM, a linear relationship with an average slope of 17 mV per 10-fold change in [Ca”] was obtained when d V was plotted as a function of log [Ca”] (Fig. 1). In Fig.2, the surface rddiOaCtiVity is plotted as a function of Ca2 concentration. When lecithin was spread on subphases containing [Ca”] < 0.1 mM a depletion of CaZt in the interface was observed. A similar Ca” depletion in the interface was observed with monolayers of docosyltrimethylammonium bromide. At Caz+ concentrations > 0.1 mM a slight positive adsorption of 45CaZ+was measured. As mentioned above, the adsorption experiments with radioactive 45Ca2 could not be carried out with any reasonable accuracy at CaZf concentrations > 1 mM. The electrophoretic mobility of unsonicated lecithin liposomes became slightly positive at Ca2+ concentrations > 0.1 mM (Fig. 3). Above a CaZt concentration of 1 mM the turbidity of a sonicated lecithin

-2.0

1 . roo 10+

10.~

lo3

[@I

10.’

(MI

Fig. 2. The surjace radiouctivity o f 4 ’ C u 2 + udsorbed on egg lecithin monoluyers undrr conditions described .for Fig. I and recordrd us a junction of CaZf concen~utirin.The specific 45Ca2+activity in the experiment was 25 Ci mol-’. The vertical bars represent the spread of 3 - 6 experiments

+

10.~

10.’

10.~

loo

baI(MI Fig. 3. Electrophoretic mobi1it.v (pmls) per unit field ( V / c m / as u ,function of Ca2+ concentration. Experiments were carried out at 25

0.05 ”C.(A-A)

Egg lecithin; (A-A)

dioleoyl lecithin

+

dispersion containing small ( z 30 nm diameter), spherical “single bilayer” vesicles began to increase and when [Ca”] = 0.1 M precipitation became rapid (Fig. 4).

136

Ion-Phospholipid Interactions

NUCLEAR MAGNETIC RESONANCE.

Assignnzen t When sonicated under certain experimental conditions previously described [14,15] aqueous dispersions of egg lecithin contain all lipid in form of small, spherical vesicles surrounded by a single bilayer. These vesicles lend themselves to NMR studies for they give good high-resolution spectra to which the lipid molecules contribute to 100 7 % [16].

*

1

P

0.8

-0.2

'

I 10'~

lo-&

10.'

10.~

loo

[Caz'l (MI

Fig. 4. Turbidity measured as ahsorbancr at 52Onm of 1 "/, ( w t v ) sonicated egg lecithin dispersions in water as a~firnctionof Ca' concentration +

The assignment of the 'H-NMR spectra of 1,2-sndilauroyl-3-phosphatidylcholine (Table 1) and 1,2sn-dimyristoyl-3-phosphatidylcholinein C'HC1, is consistent with that of egg phosphatidylcholine in C2HC13described before 1161 and the assignment of egg lysophosphatidylcholine in C'HCI, (Table 1 ) is very similar to that of phosphatidylcholines. On the basis of chemical shift values and resonance intensities and by comparison with spectra obtained from solutions of phosphatidylcholine in C'HCl3, it is immediately possible to assign all the hydrocarbon chain resonances of both sonicated aqueous dispersions (Table 1, CJ [16,40]). However, the assignment of the resonances from the polar groups of these lipids is complicated for some signals are rather broad and obscured by sharper signals. The assignment of these groups was made therefore on the basis of chemical shift and intensity data, pH titration, titration with diamagnetic LaC1, and through the use of homonuclear and heteronuclear spin-decoupling. Fig. 5 is a 'H-NMR spectrum of egg phosphatidylcholine. It differs significantly from the spectrum of phosphatidylcholine in C'HCI, [16]. The relatively sharp signals at 6 = 4.28 ppm, 6 = 3.69 ppm and 6 = 3.25 ppm are assigned to choline protons, the 03POCH,, the CH,N and the N(CH,): groups respectively, in agreement with previous work [16]. Integration shows that the signals are due to 2.2 and 9 protons and irradation

Table 1. Chemical shlfts of phosphatid~kholinesand related compounds Chemical shifts of compounds dissolved in 'HzO are the average f standard deviation of at least 6 runs and expressed in ppm downfield from an internal 3-(trirnethylsilyl)propane sulphonate standard. Thc chcrnical shifts of compounds dissolvcd in C'HCl, are the average of 2- 3 experiments. The assignments of dilauroyl phosphatidylcholine in C2HCIz are the same as those of egg phosphatidylcholine in C'HC1, [lh]. The synthesis and purity of the saturated lecithins is described clscwhcre [22] Signal

Chemical shift 6 of dilauroyl lecithin in C'HCI,

egg lysoleci- dilauroyl thin in lecithin in C'HCl, 'H,O

dimyristoyl lecithin in

egg lecithin in 'HzO

'H20

egg lysolecithin in 'H,O

phosphorylcholine pH 1.8

phosphorylcholine pH 5.5

s,-glycero3-phosphorylcholine

PPm ~

Terminal CH, (CHZ). HC=CH HzC-C=C CH2-(C=C)z CH2-C-CO CH, - CO CH,OP(gl yceride) CH-OCO CHZ-OCO

0.89 1.25-1.28 5.32 2.01 2.80 1.60 2.29

0.88-0.90 1.25

3.94 5.185 4.10

3.93

CH,OP(choline) 4.30-4.33 CH,N 3.81 N(CH,), Outside, cJ [47] 3.36 N(CH,), inside

~~~

~~

0.88 k0.01 0.88kO.01 1.28 *0.01 1.28f0.01

1.98 1.59 2.295

1.57 kO.01 1.57+0.01 2.38 kO.01 2.38+0.01

~~

....

~

~~~

0.885k0.01 1.27f0.01 5.30 kO.01 2.02 f 0.01 2.82 k 0.01 1.57k0.02 2.38k0.01

0.85+0.01 1.26k0.004 5.32 f0.02 2.00 0.01

4.02 f0.02

3.90 f 0.01

4.41 f 0.02

4.01 f 0.01 4.1 1 k 0.01 4.28 k 0.005 4.29 3.65k0.005 3.65

4.16 3.58

4.35 3.68

3.21+0.005

3.21

3.23

*

1.57k0.01 2.36k0.005 3.93 3.93 3.68

5.31 kO.01 5.31 f 0.01 4.04 4.30-4.33 3.71-3.82

4.295 kO.01 4.29+0.01 3.69 fO.01 3.68k0.01

4.28 kO.01 3.6Yf0.01

3.27-3.32

3.245k0.005 3.24k0.01 3.23 3.20

3.25k0.01 3.23

3.21

~~~~

H.Hxuser. M. C. Phillips. B. A . Levine. and R . J. P. Willianis

I37

2

I$

0

I

I

I

0

II I 0

,i

A

k

B.lPpm

*

Fig. 5. .?70-1tfH: proton IVM R .rpec.tr~rtn($ so~ricuteclc'gg pl~osphutic/~~lc~/ioli~i~~ di.rpcr.cion (20 nigiml = 0.0267 M I in 'H,O I rionrinol pH 5.51. Dispersions were made up and sonicated as described previously 11-1- 161. The insert shows an expanded spectrum. The signal a t d = 0 pprn is froin the internal ?-(triinethvlsily1)propanesulphonate standard (TSS)

of the O,POCHz group at 6 = 4.28ppm causes the collapse of the-CHzN signal at 6 = 3.69 ppm. The spectra of dilauyrojl phosphatidylcholine at 30 \-'Care similar to the spectrum of Fig. 5 except that the signals from the polar group are somewhat broader. particularly the signal at 6 = 4.02 ppm. The reason for this is probably molecular packing which is tighter with the saturated lipids. Heating the sample to 55-60 - C causes narrowing of the resonances between 6 = 44.30 ppm and the spectral region between the water peak and the N(CH.3)3resonance thus obtained closely resembles that ot' egg phosphatidylcholine at room teniperat ure. This leaves the glycerol group of phosphatidylcholine to be assigned. In the spectra of the saturated phosphatidylcholines the single proton at 6 = 5.31 ppni can be fairly confidently assigned to the glycerol proton (CH-OCO) on the basis of comparison with the spectra in chloroform and nuclear Overhauser experiments. Fig. 6 shows that the chemical shifts of the signals from the polar group of egg phosphatidylcholine d o not change with pH between pH 1 - 10. At pH values < pH = 1 downfield shifts are observed with the signals at 6 = 4.28 ppni (choline), (5 = 4.02 ppm, S = 4.41 ppm and 6 = 3.69 ppni (choline). The magnitude of the shift changes 46 is in the following order: A 6 at 4.02 ppm > A 6 at 4.28 ppm > A 6 at 3.69 ppm > Ad at 4.41 ppm. At LaCI, concentrations of 0.25 M (pH 7.0) the diamagnetic shift changes observed are very closely similar to those in Fig.6 both with respect to sign and magnitude. As the pK value of the phosphodiester

3.5

t

2 N(CH&

outside

N (CH3)3 inside

3 .O 0

1

2

3

4

5

6

7

8

9

10

11

12

PH

Fig. 6. pH deprnel~nceyf rht. proton .vhi/is (!/ u sonicutc~dt i i ~ p c w i o n01' r ~ ~ p l i o s ~ / i u t i i ! i ~ l c in l i o' H l i ,~Oi ~. Only ~ the resonances from the polar group are plotted. no shift changes being observed for the signals from the hydrocarbon chains. Thc N(CH, 10 mM. Under optimal experimental conditions it was possible to demonstrate an increase in surface radioactivity and a positive adsorption of Ca2’ to lecithin monolayers at [Ca”] > 0.1 mM (Fig.2). However. at [Ca”] > 1 mM the experimental error did not permit the determination of the surface concentration of Ca2+ with any reasonable accuracy. Fig.? shows that when [Ca”] I 0.1 mM the surface radioactivity remained below background levcl indicating that there was a depletion of Ca2- in the interface. In this respect lecithin behaved like a positively charged monolayer with a positive surface or ipotential. This result is possibly due to the lecithin (-)

(+)

PO, - N dipole being orientated perpendicular to the bilayer plane as postulated by Phillips v t a/. [31]. The surfaco potential A V did not change on addition of calciuni when [Ca”] < 10 pM.Thc linear increase in AV of 17 mV per 10-fold increase in Ca2+ concentration when [Ca2+] > 10 pM has been interpreted [3] in terms of binding of Ca” to the phosphodicster group of lecithin. However, A V is a complcx function of several interdependent contributions which cannot be determined individually. For this reason A V data are difficult .to interpret in terms of a molecular model. The uncertainties involved in interpreting A Y log [Ca2’] plots are obvious when these relationships tor triolein and phosphatidylserine are compared. Triolein which has no phosphate group gave a slope of 14 mV and phosphatidylserine, which is known to bind Ca2+ strongly. gave a slope of 23 mV per 10-fold increase in Ca2 concentration. Since triolein monolayers did not bind 45Ca2+at all, the meaning of the A V changes with increasing [Ca”] is not clear. Fig.4 shows that when [Ca”] 2 0.1 M sufficient binding occurs to cause a n increase i n turbidity of

H . Hauser.. hl. C . Phillips. B . A. Levine. and R . J . P. Williams

sonicated egg lecithin dispersions and eventual precipitation. The N M R measurements are in agreement with weak binding at these concentrations. In sunimary. the changes observed with surface chemical techniques when the Ca' concentration is high enough indicate that there is an interaction between lecithin and C a 2 + ,but the interaction is weak. The conflicting results reported in the literature are due to differences in the experimental conditions and also to differences in the sensitivity of the methods used by various authors.

143

the shift changes A d induced in the "C-NMR spectrum by DyCI,. From what was said above the "binding constant" for the interaction with C a 2 + will be < 30 I, mol.

+

N M R Stritlics o f ' M ~ ~ t a l - P h o s ~ h n t i ~ ~ ~ l cIn. hfrructions oline The interaction of phospholipids with Ca" is of great biological interest, but unfortunately the common 'OCaZ+ isotope has no nuclear spin or paramagnetic electron and hence does not lend itself to NMR studies. Indeed, no effect was observed on the 'H-NMR spectrum up to Ca2+ concentrations of 0.1 -0.3 M. Similarly, C a Z f had no measurable effect on the 31P-NMR spectrum. However. details of the ion-lipid interaction can be derived from the perturbation of the 'H, 13C and 31Phigh-resolution spectra by paramagnetic lanthanides used as isomorphous replacements for Ca' [29]. In order to compare the interaction of C a 2 + with that of lanthanides their relative efficiencies in increasing the absorbance at 520 nm of a 1 ',,sonicated dispersion of phosphatidylserine and in displacing 4 5 ~ + ~adsorbed 2 to monolayers of dicetyl phosphate were determined (for experimental details see [32]). About 10 times more CaCl' than EuCI, or NdCl, was required to increase the absorbance to an arbitrary value of 1 and to displace half of the adsorbed radioactivity indicating that the interaction is mainly electrostatic so favouring the trivalent lanthanide. The quantitative aspects of the nietal phospholipid interactions will be discussed in a subsequent paper (H. Hauser, B. A. Levine, M. C. Phillips & R. J. P. Williams, unpublished). Shift changes induced in proton [18,24,41-451 and 3'P-NMR [23] spectra of phosphatidylcholine by lanthanide ions have been reported from several laboratories. The shift changes d S of the three different proton signals of the choline group induced by various lanthanide ions (Fig.10) can be used to derive the stoichiometry and the binding constants for the reaction : lanthanide + phosphatidylcholine (lanthanide-phosphatidylcholine). The cations form a 1 : 2 complex with the phospholipid; the binding "constant" is dependent on the metal concentration at concentrations < 10 mM, (details of the analysis will be given in a subsequent paper mentioned above). A similar binding was derived from +

Binding Site' Binding studies with the lanthanide cations enable the definition of the metal binding site. The shift induced. corrected for any diamagnetic effects, is the sum of the contact (through-bond) and the pseudocontact (dipolar) perturbations. Since the contact shift contribution decreases rapidly through a series of bonds only the atoms of the immediate coordination sphere of the metal ion would show a detectable contact shift. The experimental finding that the proton shift ratios were independent of cations means that the induced shifts must be purely pseudocontact in origin [46]. However, the shift of the 31Presonance suffers an appreciable contact shift contribution as is seen from the variation of the shift magnitude for different lanthanides (Table 2). This immediately points to the phosphodiester group as the binding site on the lecithin which is confirmed by the observed relaxation data (Fig. 12, Table 3). The greater degree of line broadening of the 3 1 Presonance induced by Gd" relative to that observed for any of the proton signals means that the phosphorus is nearer to the metal than any other group, since the induced relaxation is r - 6 dependent. This is also the conclusion reached from the relative diamagnetic shifts observed with La3+.In a subsequent paper (mentioned above) we shall show that binding of the lanthanide ions is due to the phosphate group only. From the known assignments of the proton spectra (given above) and the effect of the metal ions on the individual resonances it is clear in an immediate qualitative sense that the protons of the carbons bound to the phosphate are nearest to the metal ion. The observation that the absolute shifts depend on the anion is an indication that the trivalent lanthanide ion may be accompanied by anions binding to the lipid polar group. The overall size and net charge of the anion would affect the binding properties of the metal and thus the magnitude of the shift at a fixed metal ion concentration. This explanation is consistent with electrophoretic mobility (i potential) measurements (Fig.3). In the absence of ions the electrophoretic mobility of unsonicated egg lecithin liposomes is close to zero. In contrast to the other parameters ( c f : Fig. 1-4, 10- 12) which rise continuously with increasing ion concentration, the ipotential becomes slightly positive at [Ca"] > 0.1 mM and remains unchanged on further increases t o about 0.1 M. This is observed with both egg lecithin as well as dioleoyl lecithin and can be explained by postulating that Ca2+ interacts together with co-anions so that there is no significant alteration of the net charge of the lipid interface.

144

H. Hauser, M. C. Phillips, B. A. Levine, and R. J. P. Williams: Ion-Phospholipid Interactions

The ion-exchange properties of phosphatidylserine have been reported by several groups [lo, 11,28,30,37] and Ca2+ bound to that phospholipid exchanges readily with an excess of Na’ or K’. In contrast, Na’ seems unable to displace lanthanides from the lecithin polar group even when present in a 200- 300-fold excess. With such an excess of Na’ the shift changes are even enhanced. A possible explanation of this phenomenon is that the “extended” conformation of the lecithin polar group [31] provides an energy barrier opposing the approach of cations to the phosphate group. The interaction energies of divalent and multivalent ions, but not monovalent ions, with the phosphate group are sufficient to overcome this barrier.

REFERENCES 1. Bangham, A. D. & Dawson, R. C. M. (1959) Biochern. J . 72,

486 492. 2. Shah, D . 0.& Schulman, J. H. (1965) J . Lipid Res. 6, 341 - 349. 3. Shah, D. 0.& Schulman, J. H. (1967) J . LipidRes. 8,227-233. 4. Kimizuka, H. & Koketsu, K. (1962) Nuture (Lund.) 196,995996. 5 . Kimizuka, H.. Nakahara, T., Uejo, 11. & Yamauchi, A. (1967) Biochim. Biophp. Acta, 137, 549- 556. 6. White, M . S. & Lakshininarayanaiah, N. (1969) Currents in Modern Bio1og.y 3, 39 -44. 7. Bangham, A. D. & Dawson, R . M. C. (1962) Biochim. Biophys. A c I ~ , 5 9103-115. , 8. Trluble, H. (1971) Nurur~i~issen.sctiu~ien, 58, 277 - 284. 9. Dervichian, D. G. (1956) in Biochemical Problems of’ Lipids (Popjik, G. & Le Breton, E., eds) pp. 3- 13, Butterworths, London. 10. Rojas. E. & Tobias, J. M. (1965) Biochim. Biophys. Acru, 94, 394-404. 1 1 . Hauser, H. & Dawson, R. M. C. (1967) Eur. J . Biochem. 1, 61 - 69. 12. Santis, M. & Rojas, E. (1969) Biochim. Biophys. Acta, 193, 319-332. 13. Peltauf, F., Hauser, H. & Phillips, M. C. (1971) Biochim. Bioplzjs. Acra, 249, 539- 547. 14. Hauser, H. 0. (1971) Biochem. Biophys. Res. Commun. 45, 1049- 1055. 15. Hauscr, H. & Irons, L. (1972) Hoppe-Seyler’s Z . Phj3sio/.Chcm. 353, 1579- 1590. 16. Finer, E. G., Flook, A. G . & Hauser, H. (1972) Biochim. Biophys. Acru. 260,49 69. 17. Penkett, S. A., Flook, A. G . & Chapman, D. (1968) Chem. Ph.ys. Lipids, 2, 273 - 290. 18. Hauser, H. h Phillips, M. C. (1973) in Pror. 6th I n t . Congr. on Surfim Activity, vol. 2. 371 -380, Carl Hanser Verlag. Munich. 19. Papahadjopoulos, D., Nir, S. & Ohki, S. (1972) Binchim. Biophs.s. Acta, 266, 561 - 583. -

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H. Hauser. Laboratoriuni f i r Biochemie der E.T.H., Universitltstrasse 16, CH-8006 Zurich. Switzerland

M. C. Phillips, Unilever Research Laboratories, The Frythe, Welwyn, Hertfordshire, Great Britain AL6 9AG B. A. Levine and R. J. P. Williams, Department of Inorganic Chemistry, University of Oxford, South Parks Road, Oxford, Great Britain OX 1 3QU