Effect of high electrolyte concentration on the cooperativity of the main phase-transition of DPPC

(1)

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Effect of high electrolyte concentration on the cooperativity of the main phase-transition of DPPC

P. Sapia, L. Sportelli

To cite this version:

P. Sapia, L. Sportelli. Effect of high electrolyte concentration on the cooperativity of the main phase-transition of DPPC. Journal de Physique II, EDP Sciences, 1994, 4 (7), pp.1107-1116.

�10.1051/jp2:1994190�. �jpa-00248032�

(2)

J. Phys. II France 4 (1994) l107-ll16 _JULY 1994, _PAGE l107

Classification Physic-s Abstracts

31.30L 64,70M 87.20

Effect of high electrolyte concentration

_on

the cooperativity of the main phase-transition of DPPC

P.

sapia

and L.

Sportelli (*)

Dipartimento

di Fisica, Laboratorio di Biofisica Molecolare, Universith della Calabria and Unita'INFM, 87036 Arcavacata di Rende (CS),

Italy

(Receii,ed 3 December 1993, ret,ised 28 March 1994, accepted 8

Ap;il

1994)

Abstract. we have

investigated

the effect _on the

cooperativity

of the

Pp.

- L~ main phase transition of multilamellae of

dipalmitoylphosphatidylcholine

IDPPC) of _a I _: I

electrolyte

_up to the concentration of 3 M in the dispersion ^medium. By

using

the ESR spectroscopy ^with ^the spin label partition

technique,

the _mean size of the cooperative unit, _v~ at the centre of the transition has been calculated according to the Zimm and Bragg (J. Chem Phys. 31 (1959) 526-535) theory of

cooperative transitions. By

increasing

the

electrolyte

concentration in the

dispersion

medium the

v~

value increases

suggesting

that the

lipid

molecules in the bilayers become more

tigthly packed.

Screening

of the electrostatic interactions between the dipoles _on the polar head of the DPPC molecules with increase of the

hydrophobic

interactions _as well _as modification of the _structure of

the water

layer

around the phosphocholine fragment of DPPCS _can

explain

the results obtained.

1. Introduction.

The role of ions in

determining

the _structure and function of

biological

membranes is well- known.

Owing

to this, the effects of ions _on natural membranes _as well _as on their related model systems ^have ^been

extensively investigated [1-8].

However, all studies

performed

^with

DSC, ESR, NMR, fluorescence and

X-ray

diffraction

techniques

have been concerned with

mono-, bi- and trivalent salts up ^to ^I M concentration in the

dispersion

medium. In addition, ⁱⁿ these

investigations

membrane model systems ^made

mainly

with

lipids carrying

one or more

charges

_per

polar

head,

giving

rise _to _a _net

charge density

_on the membrane surface, were used.

The results of these

investigations,

well-understood in the limit of the

Gouy-Chapman-Stern lipid

double

layer theory [9],

suggest two ways of interaction of ions with

phospholipid

membranes _a direct interaction with the

charge

on the

polar

heads of the

lipid

molecules and

an indirect one

consisting

in the induction of structural

changes

^of the interfacial water

layer.

Beside

this,

these studies have

suggested

that the interaction of the ions with the

bilayers

is

(*) To whom correspondence ~hould be addressed.

(3)

1108 JOURNAL DE

PHYSIQUE

II N° 7

limited to the

lipid/water interface, leaving unperturbed

the

hydrophobic

_core of the

bilayers.

On the contrary, very few _are the articles which

appeared

on the effects of

electrolytes

on

membrane model systems at concentrations

higher

than I M and _on the interaction between these and neutral

phospholipid

molecules _as

phosphatidylcholine

and

phosphatidylethanola-

mine

[6, 10-19].

From these studies it _comes _out that the action of different cations _on the

phase

transition features, I-e,,

width-height

and

phase

transition temperature, ^is _very ^small ^for

ionic

strength

less than I M and becomes _more marked at

higher

concentrations

[7].

Multilamellar and vesicle

mesophases

are affected in _a different _manner in the presence of the ions Na+ and Cl~ up ^to 3 M

[8].

In fact, the ionic

strength

^shifts

upward

the

Lp,

-

Pp,

and

Pp,

_-

L~ phase

transition temperatures ^of ^DPPC multilamellae and increases the orientational

degree

of order of the

lipids

in the

bilayers. Quite opposite

is the effect of the salt _on sonicated small unilamellar-vesicles _: both the molecular order and the transition temperature ^decrease.

Moreover, ⁱⁿ ^the case of multilamellae the

cooperativity

of the main

phase

transition results affected, too

[8],

To better understand the effects of ions _on the

cooperativity

of the

gel-to-fluid phase

^transition ^of ^DPPC

multilayers,

we have

performed

_an Electron

Spin

Resonance

(ESR) study

with the

spin

label

di-tert-butyl-nitroxide (DTBN)

and _a Differential

Scanning Calorimetry (DSC) investigation

of the multilamellar system ^when ^the ^Nacl concentration in the

dispersion

^medium ^is increased up ^to 3 M. Such _an

investigation,

^besides the

physical-

chemistry

interest is also of

biological

relevance since _a lot of bacteria

(Halophilic

bacterial

are

living

in extreme conditions of

pH

^and ionic

strength.

Well-known and

extensively investigated

is Halobacterium halobium which lives in _a medium

containing

_up to 4.3 M Nacl

[20, 21].

It possesses the

bacteriorhodopsin,

the

only protein

found in the

purple

membrane, _as

light

driven proton pump,

From both the

investigations

of the DTBN

partition

between the bulk

dispersion

medium and the fluid

hydrophobic

_core of the

lipid bilayers

and the calorimetric measurements, _a measure

of the

gel-to-fluid

transition

cooperativity, expressed

_as the _mean size of the

cooperative

unit,

v, as defined in the next

section,

has been determined. The

experimental

data show that with

increasing

the

electrolyte

concentration the

cooperativity

of the main

phase

transition of the

phosphocholine multilayers

increases. It is

suggested

that this is due _to _an increase in

hydrophobicity

of the

polar region

of the

lipid bilayers

with salt concentration,

2.

Theory.

2.I TRANSITION COOPERATIVITY. The

Pp,

-

L~,

I-e-, the ordered-to-fluid, main

phase

transition in DPPC multilamellae may be described in _terms of

growing

fluid

lipid

domains with temperature ^increase

[22, 23].

The

theory

of the

cooperative

helix-coil transitions

[24, 25] applied

to the ordered-to-fluid transition in the

phospholipid bilayers

_assumes that the

fluid domains of the

lipid

molecules _are

separated

from the ordered _ones

by

an « interfacial

region

_» constituted

by

the

lipid

molecules that

undergo

the transition. In other words, the

states that _a

lipid

molecule _can take up

during

the transition _are

essentially

three the ordered

state, s, the fluid state, f, and the interfacial

region

^between the ordered and fluid

phases,

^I.

The free

energies

of the molecules in these three _states _are

mainly

determined

by

the

following

contributions _: the internal energy

arising

from the rotational isomerisms of the

lipid

chains, the intermolecular _van der Waals forces between the chains, the electrostatic interactions

between the

polar

heads and the

configurational

disorder of the

lipid

chains. If _we define the

zero of the molecular free _energy _as the energy of _a molecule in the ordered _state, and

f~

_as that of _a molecule in the fluid _one surrounded

by

other molecules in the _same state, then the free energy of _a molecule in the interracial

region

_can be

expressed

as

f~

+

fj,

where

f~

^is ^the ^additional ^free energy

arising

from the mismatch in molecular

packing

between the ordered and fluid

lipid

domains.

(4)

N° 7 IONIC STRENGTH AND PHASE TRANSITION COOPERATIVITY 1109

It is this _excess of free energy,

resulting

from the mismatch in the molecular

packing

in the interfacial

region,

that

gives

rise to the

cooperativity

of the transition. In this respect, a

cooperativity _parameter

related _to this _excess of free energy may be introduced _to characterize the transition.

The mismatch in the molecular

packing

makes the interfacial _state

energetically

unfavorable.

So that

f~

^has the character of _an interfacial energy

tending

to reduce the extension of the interfacial

region, I-e-,

to reduce the number of molecules _at the

phases boundary,

hence

giving

rise _to the

cooperativity

in the system.

Let us now define the

degree

of transition, _@, as the _mean fraction of molecules in the fluid

state (n is the total number of

molecules) [22]

(n~)

0

=

(l)

and _~ _as the _mean fraction of molecules that

undergo

the transition, I-e,, the molecules in the interfacial state

J~ =

'~ (2)

The ratio

(Pf)

~

)

>

(3)

being

the _mean number of molecules in the fluid _state per interfacial molecule, _may be assumed _as _a _measure of the _mean size of the fluid

lipid

domains that exist at various stages ^of the transition.

Similarly,

the _mean number of molecules in the ordered _state per interfacial molecule will be

("s)

=

jj

~

(4)

From the above definitions of the free

energies,

_we define the

cooperativity

parameter,

«, aj the ratio of the

probability

of _an interfacial molecule to that of _a fluid _one

~- ^{~f,+ ffv(T}

~' ~~~~

~~

where k is the Boltzmann constant and T the absolute temperature.

It is

easily

_seen that the

cooperativity

parameter

depends only

_on the interfacial _excess of free energy

tr =

~'~~~

(6)

This parameter ^is ^related to the _mean size of the

cooperative unit,

_v, at the _centre of the transition. In

fact,

when T

=

T~,

the main

phase

^transition temperature,

( v~)

₌

( v~),

I-e-, the

mean size of the ordered and fluid domains _are

equal,

and

[22]

_:

" "

1"I)T~

~

' _~

l' ₍₇₎

~ W

(5)

ii lo JOURNAL DE PHYSIQUE II N° 7

Clearly,

_« is _an index of the

cooperativity

of the transition;

indeed,

the smaller

«, the greater ^the mean size of the

cooperative

unit

undergoing

the transition.

Furthermore, it has been shown that _near the transition temperature

T~,

it results

[22, 23]

_:

where

AH~

is the molar

enthalpy

of the main

phase

transition.

Equation (8)

shows that

0 has _a linear

dependence

_on I/T around

T~,

I-e-

0 (T

=

T~

)

= Cte

~ (9)

where

AH~

~

4 R

,$

^~~~

In this way,

by plotting

0 (as defined in

Fig.

I) _as a function of I/T, _a _may be determined from the

experimental

data.

By

means of

equations (7)

and

(10)

and the calorimetric value of

AH~

one gets v, the _mean size of the

cooperative

^unit.

3

m +

#

~

l m

11

~

i~

30 33 36 39 42 45

I

l'C

Fig. I. -Partition coefficient, P~, as a function of temperature ^of ^the spin label DTBN in DPPC multilayers in the absence of salt in the

dispersion

medium. In the inset _a DTBN-ESR spectrum ^is ^shown from which P~ H~/(H~ + Hw is calculated. The degree of transition o al (a ₊ h) is also defined for the main

phase

transition of DPPC.

2.2 ESR THEORY. The ESR spectrum ^of a

spin

label _can

generally

be described

by

the

following

effective

spin

Hamiltonian

[26-28]

_:

H

=

pHgS

₊ ITS

(11)

where the first _term _on the

right

_represents the Zeeman interaction between the

unpaired

electron

spin

S =1/2 and the

magnetic

field H, ^the ^second one represents ^the

hyperfine

(6)

N° 7 IONIC STRENGTH AND PHASE TRANSITION COOPERATIVITY _ii11

interaction of the electron

spin

with the

nitrogen

nuclear

spin

I

=

I,

p

is the Bohr magneton and g and T _are the g and

hyperfine

_tensor,

respectively.

In the _case of small molecules

undergoing rapid isotropic

motion with rotational correlation time _T~

~

10~~

s like DTBN does, the

anisotropy

in the _g and T _tensor elements is

averaged

out and the

spin

hamiltonian (I

I)

_assumes the

isotropic

form

H

= p_{go H- S-} ₊ To ^{I~ S~}

(12)

where

go =

1/3

Trg, To

=

1/3 TrT

(13)

The ESR spectrum, ⁱⁿ ^this case, consists of three

sharp

lines of

equal height

centered _at go and

spaced

of

To,

the

isotropic hyperfine coupling

constant.

When DTBN is dissolved in _an aqueous

lipid dispersion

it

partitions

between the aqueous

and the fluid

lipid phases.

The

corresponding

ESR spectrum ^is ^the

superposition

of two

isotropic

ESR patterns

(inset

of

Fig,

) _one

arising

from DTBN in the bulk

dispersion

medium and the other from the fraction of

spin

label in the fluid

hydrophobic

environment of the DPPC multilamellae, Since the fluid membranes have

higher viscosity

and lower

polarity

than water, the ESR spectra

coming

from the _two different environments have small differences both in the go and To ^values. ^This leads, at 10 GHz, _to _a

partial spectral

resolution _so that

only

the

high-

field line with mj = I is resolved

(inset

of

Fig. I),

The _measure of the

spin

label

signal

in the aqueous

phase, Hw,

and in the fluid

lipid

one,

H~,

allows _an estimate of the fraction of DTBN in the fluid

hydrophobic region

of the

bilayer,

by

means of the

partition

coefficient

[23, 28]

P~

=

H~/ (H~

+

Hw

) (14)

The

partition

coefficient

gives

a measure of the membrane

fluidity.

From the

plot

of the P

~

value _i's. temperature the

degree

of transition, 0

= al

(a

+ b ), where

a and b _are defined _as in

figure

I, is evaluated.

Clearly,

0 represents ^the ^fraction ^of

lipid

molecules in the fluid _state

[23],

3. Materials and methods.

Synthetic

1,

2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)

_was used _as obtained

by

Fluka. The

spin probe di-tert-butyl-nitroxide (DTBN)

was Aldrich

product

^stored at 4 °C in

ethanol solution, Nacl of reagent

grade

was from C.Erba. Distilled _water _was used

throughout.

DTBN-containing

DPPC

multilayer dispersions

were

prepared

as follows. The DPPC _was dissolved in ethanol and, after

evaporating

off the solvent with _a _stream of

dry nitrogen,

^the

thin

lipid

film _was

placed

for 12 h under _vacuum. The dried

sample

was then

fully hydrated

with _a 5 _x

10~~

_M _solution of DTBN in 10 mM,

pH

8.0

phosphate

buffer solution

(PBS)

containing

0, 1, 2 _or 3 M Nacl to obtain _a final DPPC concentration of 73

mg/ml

(0.01 M).

The

dispersion

was incubated for I h at 50°C, I-e-, above the main

phase

^transition temperature, ^and vortexed

[29].

The

multilayer samples

were sealed off in _a

glass capillary

and incubated _at 4 °C for 12 h before ESR spectra

running [30].

The ESR spectra were recorded with _a Bruker ER 200D-SRC spectrometer

operating

at

10GHz

equipped

with the ER4121VT Eurotherm temperature ^control ^unit

(ac-

curacy ^± 0.I

°C),

the ESP1600 Data

System

and the

TEjo~

standard

cavity.

All spectra were recorded with the

following experimental

_set-up _: 10mW microwave

power, 0.25

G~_~

field modulation

amplitude,

and

displayed

_as first derivative in

phase

(7)

1112 JOURNAL DE PHYSIQUE II N° 7

absorption signal,

100 kHz

magnetic

field modulation

frequency

_was used for

phase-sensitive

detection.

The DSC studies _were

performed

_on

samples prepared

_as for the ESR measurements, but

without

spin probe,

with the

help

of _a Setaram DSC-92

instrument,

which _uses Indium metal

as calibrant for temperature ^and energy.

Samples (20-30 mg)

were introduced into steel-cells and then sealed and heated _to the initial temperature ^for ^{I h.}

Thermograms

were

digitized

with

an IBM PS2-60 computer ^which ^allowed ^the determination of the main

phase

^transition

temperature ^and

enthalpy.

These

thermodynamic

parameters were studied _at the

heating

rate of 0,3 °C/min.

4. Results and discussion.

From the ESR spectra

(inset

^of

Fig, I)

the

partition

coefficient,

P~,

of DTBN which, as

known, ^is ^related to the membrane

fluidity

is evaluated. The

plots

^of

P~

as a function of the temperature ⁱⁿ ^the range 25-45 °C for the

electrolyte

concentration in the

dispersion

medium up to 3 M are

given

in

figure

2.

3 0 3 5 4 0 4 5

T

/

_°C

Fig. 2. Plots of P~ _as _a function of temperature ^of ^the spin label DTBN in multilayers of DPPC dispersed in media with different

salinity

_: (.j 0MNaCl~ (o) I MNaCI, (6) 2M Nacl and (Cl 3 M Nacl.

As _can be _seen, in absence of ions in the

dispersion

medium the

Lp,- Pp.

and

Pp

-

L~ phase

transitions of the

multilayers

of DPPC _occur _at 34,3 and 40.6 °C

respectively,

in

good

agreement ^with ^literature ^data

[9, 29] (Tab, I).

When Nacl is added _to the

phospholipid dispersion

the

pre-transition

temperature, T~, ^and ^the ^main one,

T~,

shift

upward

to 38.5 and 42.5 °C,

repectively.

Also the DSC

thermograms

ⁱⁿ

figure

4 show _an increase of both transition temperatures with the

pre-transition

more

upward

shifted than the main _one (Tab.

I).

In fact, in presence of

3 M Nacl it _occurs _at 41.5 °C. On the contrary, ^the

enthalpy

of both the pre- and main

phase

transitions remains

quite unchanged

within the

experimental

_errors

(Tab, I).

The determination of the

degree

of transition, 0, is shown in

figure

I, while in

figure

3 the 0- values _are

plotted

as a function of I/T for each salt concentration.

From the linearization of the

0(1/T)

_curves around the main transition temperature,

T~,

^the ^«-values are estimated and then the _mean size of the

cooperative unit,

_v, calculated.

(8)

N° 7 IONIC STRENGTH AND PHASE TRANSITION COOPERATIVITY 1l13

Table I, Pre-~ T~, ^and main,

T~, phase

transition temperature ^values ^deduced

from

ESR

and DSC measurements

for

DPPC multilamellae

dispersed

in media

containing increasing

Nacl concentration. The main

phase

transition

enthalpies,

the size

of

the

cooperative

unit,

v, and the _excess

interfacial free

_energy,

F~,

are also

reported.

[Nacl]

ESR DSC

mol

T/ T~

_T~

T~ AH~

_v ^F

(°C) (°C) (°C) (°C) (kcal/mol) (kcal/mol)

0 34.3 40.6 33.6 41.4 7,8 109 5.83

35.4 41.4 38.0 43.0 7.8 130 6,07

2 37,6 41.6 40,5 43.8 7,9 139 6,16

3 38,5 42.5 41,4 44,5 7,9 149 6,26

(*)

The accuracy of the transition temperatures ^is ± 0. I

°C,

of

AH~

is _± 0.2

kcal/mol,

of F~ ^is ± 0.05 kcal/mol and of the

cooperativity

_parameter _± 5.

e

16 3.17 3.18 19 3.20

1Ii i I

o~/

K~'

Fig.

3. Degree of transition, o, _as a function of I/T of DPPC multilayers

dispersed

ⁱⁿ media with different salinity (.) 0 M Nacl, (o) ^I ^M Nacl, Q 2 M Nacl and (D) 3 M Nacl.

The values of the latter parameter ^with ^salt concentration _are

given

ⁱⁿ ^table ^I. ^As can be _seen,

v increases from 109 in the absence of salt in the

phospholipid dispersion

to 149 in the presence of 3 M Nacl. It should be noted that the _v value found

by

_us in the absence of Nacl in the medium is lower than that

given

in reference

[25]

for the same system,

likely

due to a

different

degree

of

purity

of the DPPC used.

Nevertheless, the trend of the v-values indicates that the fluid

phase

^of ^the

lipid bilayers

is

energetically

stabilized

by

the presence of ions, Indeed, from

equations (5)

and

(6)

it follows that

f~

=

2

kT~ log (v 1) (15)

where

f,,

_as

already reported,

^is the

lowering

in the _mean free energy of _a

lipid

molecule in

passing

from the interfacial _to the fluid state. So that the observed increase in

cooperativity

as a

(9)

1l14 JOURNAL DE PHYSIQUE II N° 7

f

I

3M

~ O a Z W

30 35 40 45 50

T/°C

Fig. 4. DSC thermograms of DPPC multilayers

dispersed

in solutions with different salinity at pH ^8.0.

function of the I:I

electrolyte

concentration is indicative of

tighter

intermolecular

bindings, giving

a

negative

contribution _to the _mean free energy per molecule in the fluid _state with

respect to that in the interfacial _one.

In table I _are

reported

the values of the molar _excess free energy of the interfacial _state with respect to the fluid _one,

F,

=

N~ f~ (where N~

^is ^the

Avogadro

number), as obtained from

equation (15)

and

cooperativity

data. As _can be _seen,

by increasing

the ionic

strength

the

interfacial _state is

progressively

less convenient from _an

energetic point

of view _or,

correspondingly,

the fluid lamellar

phase

^of ^DPPC gets

increasing energetic stability

in presence of the salt.

An

explanation

of the above result _can be

given

in terms of _two mechanisms _contem-

poraneously occurring

at the

bilayer

interface. First, counter ions reduce the electrostatic

interactions between DPPC

dipoles by screening

them. In this way, the

polarity

of the

interracial _zone of the

bilayer

is

reduced,

with _a consequent ^increase ^{of its}

hydrophobicity

and

then of the attractive _van der Waals forces between the choline

segmental

_part of the

phospholipid

molecules,

giving

rise _to _a _more

cooperative Pp,-L~ phase

transition.

However, of the _two _counter

ions, I-e-,

Na+ and Cl~, the anion _seems to be _more effective than the cation in the interaction with the multilamellar

phase

^of ^DPPC. In fact, while the

chlorine ions _are able to screen the

positive charge

on the N+

(CH~)~

choline quaternary

ammonium group of the DPPC

polar head,

the

hydrated

Na+ _ones _cannot

easily

reach the

PO~ _zone of the

lipid bilayer

to

modify

the

hydrogen

bond network therein

existing,

as

they

do when the DPPC molecules form vesicles, I-e-, a

mesophase

with _a small radius of _curvature

(R

_m 100

I _compared

_with multilamellae

[8].

Besides this effect, the presence of the counter ions around the DPPC

dipole charges

could either alter the structure of the water

layer

or reduce the

degree

of

hydratation

of the

phospholipid polar

heads. The former effect should _concern the rearrangement ^{of the} structure

of the water clathrates around the

polar,

N+

(CH~

_)~, and

apolar, (CH~)~

,

regions

of the choline

fragments while,

the

de-hydration

the reduction of the number of water molecules

bound per DPPC

polar

head. A reduced

degree

^of

hydration

results, as known

[9],

in _an

upward

shift of both the

Lp,

-

Pp,

and Pp> -

L~ phase

^transition temperatures. This is

just

what is observed with the DSC measurements as a function of salt concentration

(Fig.

4 and

(10)

N° 7 IONIC STRENGTH AND PHASE TRANSITION COOPERATIVITY ills

Tab.

I),

_so that these results would support the latter mechanism of interaction.

Unfortunately,

at the present moment we have _no measurements, such _as _water proton ^self

diffusion,

to

support ^the modification of the

properties

of the bound _water _at the

bilayers-bulk

solution

interface. As _a consequence we cannot

clearly

decide which of the last _two

suggested

mechanisms is the most effective.

Likely,

both _act _at the _same time

giving, together

with the increase _at the

bilayer

surface of the attractive

dispersion

forces between less

charged polar heads,

_as end effect the observed increase in the

cooperativity

of the main

phase

transition of

DPPC

multilayers.

It is

noteworthy

that both mechanisms

suggested

lead _to the observed increase of the attractive

contribution, -F,,

to the energy of the fluid

phase

with respect to that of the interfacial _one.

The constancy, ^within ^the

experimental

_errors, of the main

phase

transition

enthalpy, AH~, which,

as known, is

mainly

related to the

melting

of the

hydrophobic acyl

chains

[9]

of

the

lipid bilayers

also supports ^the

hypothesis

that ions induce modifications which _are

essentially

localized in the interfacial

regions,

I-e-, in the

polar

zone of the DPPC

multilayers.

The

hydrophobic

_core of the

lipid bilayer

does not seem to be affected _so much

by

the

high

salt concentration,

Acknowledgments.

P, S. thanks the MURST for _a

fellowship.

^This work _was

financially supported by

CNR and

MURST research grants.

References

[1] Helm C. A., Laxhuber L., Losche M. and Mohwald H., Colloid Polymer Sci. 264 (1986) 46-55.

[2] Jacobson K. and Papahadjopoulos D., Biochemistry14 (1975) 152-161.

[3] Jaenigh F., Biophys. Chem. 4 (1976) 309-318.

[4] Trauble H. and Eibl H.. Proc. Nat. Acad. Sci. USA 71 (1974) 214-219.

[5] Trauble H., Teubner M., Woolley P. and Eibl H., Biophys. Chem. 4 (1976) 319-342.

[6] Bartucci R., Gulfo N. and

Sportelli

L., Biochim. Biaphys. Acta 1025 (1990) II?-121.

[7] Bartucci R. and L.

Sportelli,

Colloid Polymer ^Sci. ²⁷¹ (1993) 262-267.

[8]

Sapia

P. and

Sportelli

L., Colloid and Suifiac.es A Physicochemical ^and Engineering Aspe<.ts 72 (1993) 257-263.

[9] Cevc G. and Marsh D.,

Phospholipid

Bilayers

Physical Principles

and Models (Wiley, ^New York, 1987).

[10] Akutsu H. and Seelig J., Biochemistry 20 (1981) 7366-7373.

Ill Chapman D., Peel w. E., Kingston B. and Liiley T. H., Biochim Biophys. Acta 464 (1977 ^260- 275.

[12] Cunningham B. A., Shimotake J. E., Tamura-Lis W., ^Mastram T., Kwook W. M., Kaufman J. w.

and Lis L. J., Chem. Phys. Lipids 39 (1986) 135-143.

[13] Loshchiiova E. and Karvaiy B., Biochim. Biphys Acta 514 (1978) 274-285.

[14] Simon S. A., Lis J. K., Kauffman J. W. and MacDonald R. C., Biochim.

Biophys.

Acta 375 (1975) 317-326.

[15] Cserhati T. and Szogyi M., Chem. Phys. Lipids 34 (1983) 93-99.

[16] Lis L. J., Kauffman J. W. and Shriver D. F., Biochim Biophys. Acta 406 (1975) 453-465.

[17] Lis L. J., Parsegian V. A. and Rand R. P., Biochemistry 20 (1981) 1761-1770.

[18] Lis L. J., Lis W. T., Parsegian V. A. and Rand R. P., Biochemistry 20 (1981) 1771-1777.

[19] Tatulian S. A., Biochem.

Biophj's.

Acta 736 (1983) 189-195.

(11)

1116 JOURNAL DE PHYSIQUE II N° 7

[20] Stokenius W. and

BogomoIni

R. A., Ann. Rev. Biochem. 52 (1982) 587-616.

[21] Oesterhelt D. and Stokenius W.. Nature New Biol. 223 (197 II 149-153.

[22] Marsh D., Watts A. and Knowles P. F., Biochim. Biophys. Acta 465 (1977) 500-514.

[23] Marsh D., watts A. and Knowles P. F., Biochemistry 16 (1976) 3570-3578.

[24] Zimm B. H. and

Bragg

J. K., J. Chem. Phys. 31 (1959) 526-535,

[25]

Flory

P. J., Statistical mechanics of chain molecules (Interscience, New York, 1969).

[26] Nordio P. L.,

Spin Labeling. Theory

and

Application,

L. J. Berliner Ed. (Acad. Press, New York, 1976) _{p. 2.}

[27] Jost P. C. and Griffith O. H..

Spin

Labeling. Theory and

Application,

L. J. Berliner Ed. (Acad.

Press, New York, 1976) _p. 454.

[28] Marsh D., Membrane

Spectroscopy,

E. Grell Ed. (Springer, New York, 1981) _p. 51.

[29] Sackmann E.,

Biophysics~

w.

Hoppe,

W. Lohmann, H. Markl and H.

Ziegler

Eds. (Springer, New York, 1983) _p. 425.

[30] Marsh D., Biochemistry 19 (1980) 1632-1637.