A study of the bidimensional miscibility of ceramide and dioleoylphosphatidylcholine

Colloids and Surfaces, 65 (1992) 287-295

287

Elsevier Science Publishers B.V., Amsterdam

A study of the bidimensional miscibility of ceramide and dioleoylphosphatidylcholine F. B o n o s i , E. M a r g h e r i a n d G . G a b r i e l l i Department of Chemistry, University of Florence, Via G. Capponi, 9-50121 Florence, Italy (Received 12 N o v e m b e r 1991; accepted 7 F e b r u a r y 1992)

Abstract Some parameters related to the bidimensionai interactions between ceramide (CER) and dioleoylphosphatidyleholine (DOPC~ molecules, alone and in mixtures of different weight fractions, were deduced from the study of spreading monolayers at the air-water interface. Spreading isotherms were recorded using both 0.1 M NaC! (pH 5.6) and a phosphate buffer solution lpl I 7) as subphases in the 15-30°C temperature range. The bidimensional phases of the two components were obtained and their bidimensional miscibility was deduce£ as a consequence of positive deviations of the surface areas from the additivity relationship and by applying the pha,,e rule at the collapse pressure. Excess surface Gibbs energies, enthalpies and entropies of mixing were also computed. The results obtained showed that the two components were miscible under the experimental conditions at all temperatures investigated, using both the iqa+-ion-eontainingsubphase and lhe buffered one.

Keywords: Bidimensional mixtures; ceramide; dioleoylphosphatidylcholine; miscibility; monolaycrs.

Introduction Previous w o r k [1,2-1 from this laboratory showed that the study of spreading m o n o l a y e r s of lipids at the a i r - w a t e r interface can give useful information a b o u t the properties of these substances in different and m o r e complex molecular assemblies, such as black lipid m e m b r a n e s (BLMs), L a n g m u i r Blodgett (LB) films and vesicles, each of which can be considered as a m o d e l for biological m e m b r a n e s

[3]. In particular the bidimensional miscibility a m o n g different c o m p o n e n t s is i m p o r t a n t in defining the interactions in m e m b r a n e models. Furthermore, such i n f o r m a t i o n can be ~.xsed to u n d e r s t a n d the interactions a m o n g c o m p o n e n t s of biological m e m b r a n e s a n d to c o m p a r e different m e m b r a n e models. Correspondence to: G. Gabrielli, Dept. of Chemistry, University of Florence, Via G. Capponi, 9-50121 Florence, Italy. 0166-6622/92/$05.00

In this work, spreading m o n o l a y e r s of ceramide (CER) a n d dioleoylphosphatidylcholine ( D O P C ) alone a n d in mixtures at different weight ratios were studied at the a i r - w a t e r interface at 15, 25 and 30°C. C E R is the simplest sphingolipid, an important" class of m e m b r a n e lipids, playing an i m p o r t a n t role in molecular recognition processes. D O P C is a c o m p o n e n t of egg lecithin which is a phospholipid, a n o t h e r i m p o r t a n t class of lipids which are the m a i n c o m p o n e n t s of natural membranes. I n f o r m a t i o n a b o u t the interphasal properties such as molecular orientations of pure c o m p o n e n t s a n d their miscibility were obtained. T h e lipids were studied using two different subphases, a 0.1 M NaCI solution and a buffered solution at p H 7, since N a ÷ ions a n d neutral conditions are i m p o r tant in biological environments. T h e t h e r m o d y n a m i c parameters relating to the interphasal interactions between the two lipids

© 1992 - - Elsevier Science Publishers B.V. All rights reserved.

288

F. Bonosi et aL/Colloids Surfaces 65 (1992) 287-295

were then computed. A.,:tu~'d":'~the interphasal miscibility was, deduced a:~. I res~qt of the behaviour of molecuhr areas, as a f,~ction of the mixture composition, tn fact. if the: two components are immiscible or if they behave tike an ideal mix: ure, the following relations~oip is valid 1"4]:

A thermodynamic analysis of the parameters related to the interphasal miscibility was also performed, following the method of Goodrich I-7], which allows us to calculate the excess Gibbs energy of mixing AGmlx: ~2

(1)

A12 = x l , 4 t + . x 2 A 2

where A~z i~ the molecular area in the mixture monolayer at a fixed surface pressure rt, A I and Az are the molecular areas in the pure component monolayer at the same n, arm xl and Xz are the molar or weight fractions of the pure components in the mixture [5] s:uch that x~ + x2 = 1. Positi~,: or negative deviation from Eq n (1) indicates respectively the presenc: of repulsive or attractive interactions between .he two components in the mixed monolayers. The same relationship is followed by the compressibility modulus C[ 1, which is defined by (2)

C f I = _ A(drt/dA )r

Therefore from its behaviour as a function of the mixture composition, information about bidimensional interactions are again deduced. A further proof of the interphasal miscibility is obtained from the surface phase rule developed by Crisp [6], stating that when the film components are immiscible the collapse pressure does not vary with the mixture composition, i.e. F = C b + Cs - f b - f f

+ 3

(3)

where F is the number of degrees of freedom, C b and C" are the number of components in bulk and confined to the surface respectively, and fb and f~ are respectively the number of bulk phases and the number of surface pha~es in equilibrium with one another. The number 3 then refers to temperature, surface pressure and external pressure. Thus, at the collapse pressure, if we have two components at the air-water interphase, F=2

+ 2-a-f~+

3=4-ff

.

If the components are immiscible, f s = 2 and F = 2, i.e. the collapse pressure is constant.

AGmi x = N A

f

(A 12 - xl A1 - x2 A2~drc

(4)

where rt 1 and rc2 are two fixed surface pressures and N A is the Avogadro number. The enthalpic and entropic contributions to the excess Gibbs energy of mixing were then calculated, according to the Bacon and Barnes method 1"8], i.e. ASmix = - (ddGmix/dT),, - AAmix"

(dT/dT)

where AAmix= At2--xlA1 - - x 2 A 2 - 0 . 1 5 4 m N m - 1 K -1 and AHmix --- dGmix + TdSmi x

(5)

and d)'/dT=

(6)

Experimental The following substances were used. Ceramide (CER, Sigma Chemie), from bovine brain, contained a mixture of fatty acid residues, as determined by the supplier: H O - C H - C H = C H - ( C H 2)12-CH a

I

CH-NH-C=O

L's

CH2OH

where R had the following composition: C16:0, 1.9%; CI8:0, 32.3%; C20:4, 3.7%; C24:0, 10.9%; C24: !, 34.4%; others, 16.8%. Dioleoylphosphatidylcholine (DOPC, Sigma Chemie) was supplied as a chloroform solution (10 mg m1-1 ) with a purity of 99%. Chloroform (purity > 99%; Merck) was used as spreading solvent. NaCI (RPE, purity > 99.5%; C. Erba) was used in a 0.1 M water solution as the

F. Bonosi et al./Colloids Surfaces 65 (1992) 287-295

subphase at pH 5.6 (ionic strength, 0.1). The phosphate buffer (Titrisol, 0.026 M KH2 PO,, and 0.041 M Na2HPO,,, (Merck)) was used as the subphase at pH 7 (ionic strength, 0.149). The subphases were all prepared using doubly distilled water freed from colloidal and ionic impurities by a Milli-Q Organex System ultrafiltration apparatus (Millipore); the water had a resistivity greater than 18 M[~ cm. The n/A isotherms were determined with a Lauda Filmwaage FW2 apparatus, according to the Langmuir method. The surface pressure n was determined with an accuracy to less than 0.1 mN m - 1. After spreading the chloroform solution, we waited 10 min to allow for solvent evaporation before starting the continuous compression of the surface film at a speed of 7.7 mm min-1 The temperature was controlled with an Haake FK thermostat with an accuracy to + 0.01 °C. Results and discussion

Ao values in the range 40.4-43.5 A 2 mol-1 on the 0.1 M NaCI subphase and 49.5-51.6 A 2 mo1-1 on the phosphate buffered subphase were in good agreement with the value of 44.4 A 2 mol-1 found by Maggio et al. [9,10] on a 0.145M NaCI subphase. This Ao value is also quite similar to that shown by dipalmitoyiphosphatidylcholine (44 A 2 mol- 1) reported in the same papers; this is to be expected for two closely packed saturated hydrocarbon chains. This bidimensionai packing was also confirmed by the C-1 s, max values which were typical of a liquid condensed phase in the temperature range studied [!!]. The collapse pressure of CER, on both subphases, increased with increasing temperature, in agreement with previously reported data [12]. The collapse pressure variation as a function of temperature is related to the surface entropy at the collapse point, $~, because of the relationship (d~c/dT)A = S~

Pure components The ~z/A isotherms of CER on both subphases (Fig. !) showed a temperature dependence. The limiting area Ao (Table I) increased with increasing temperature, because of decreasing hydrophobic interactions between the hydrocarbon chains. The

80

1

289

elm NaCI 0.1 M

cIm

Phosphate butter

40

LL 6"'"6:~'"'6:6£'"6~." SUII~AClt~

(m'/mj)

Fig. !. ~t[A isotherms of CER on 0.1 M NaCI and phosphate subphases at 15°C (O), 25°C ( I ) and 30°C (&).

(7)

where ~c is the collapse pressure. The positive S~ value obtained for CER, on both subphases, indicated a lower degree of order in the collapsed phase compared with the bidimensional phase, and thus the collapse is an entropicaily favoured process. The comparison of the thermodynamic parameters reported in Table I showed that the change in pH influenced the surface behaviour of CER, with resulting expansion of the ~/A isotherms and decreasing Cs-1 values on going from pH 5.6 to pH 7, reflecting a weakening of lateral hydrophobic interactions. The ~/A isotherms of DOPC, on both subphases (Fig. 2), are very different from those of CER, since they are characterized by the presence of a liquid expanded phase. The limiting area Ao values ranged from i!4.8 to 135.7A2 molecule -~ on 0.1M NaCI subphase, and from 108.3 to 117.5 A 2 molecule -1 on the phosphate buffered one (Table2). This last finding was in good agreement with previously reported data [13:]. The Ao value increased on going from 15 to 25°C and

290

" B o n o s i et a l . / C o l l o i d s S u r f a c e s 05 {I,~92) 287-295

TABLE I Thermodynamic parameters of CER on 0.1 M NaCI (pH 5.6) subphase and on buffered subphase (pH 7) -I

Subphase

T 1"C1

Ao IN 2 mol- t)

n,,,,, (mN m - ~)

C,. m.',, (mN m - ~ )

0. I M NaCI

15 25 30

40.4 42.4 43.5

28.9 26.6 31.7

671.3 754.1 589.4

Phosphate buffer

15 25 3{|

49.5 50.5 51.6

30.9 35.3 42.0

498.0 576 552.6

presence of a c a r b o n - c a r b o n double bond in the alkyl residues of the molecule. The collapse pressure of D O P C decreased with increasing temperature, showing an opposite behaviour with respect to CER. The observed ~z~ value of 41 m N m -~ on the 0.1 M NaCI subphase at 25'~C is in good agreement with the value of 4 0 m N m - t at 24°C on pure water reported in Ref. [-14]. With decreasing pH we observed a mono!ayer expansion. D O P C is a zwitterionic molecule having a neutral charge at pH 7, while it carries a positive charge fraction at pH 5.6. Thus the m o n o l a y e r expansion at low pH is due to an increase in electrostatic repulsions between positively charged polar heads.

6O

A

A

x

DOPC

"'k.

DOPC buffer

a.

0.4,0

0,75

1.10

1.4fl).40

S'URFACl~ ~

0.75

1.10

1.45

(m~/m~l)

Fig. 2. n / A isotherms of D O P C on 0.1 M NaCI and phosphate subphases at 15~C {@), 2 5 : C (m) and 3 0 ' C (AL

was temperature-independent from 25 to 30°C. The incrc~se in A o and the lowering of the maxim u m of C ( t for D O P C , with respect to CER parameters, are indicative of a very different orientation at the a i r - w a t e r interface with a high tilting of the h y d r o p h o b i c interactions, owing to the

DOPC/CER mixtures We investigated D O P C / C E R monolayers at 0.3.. 0.4, 0.5 and 0.6 weight fractions and 15, 25 and 30°C on both subphases (Figs 3 and 4).

TABLE 2 Thermodynamic parameters of D O P C on 0.1 M NaCI (pH 5.6) subphase and on buffered subphase (pH 7) Subphase

T (JC)

Ao (A z m o l - t )

n~o~ (mN m - ~ )

-I C ...... (mN m " )

0.I M NaCI

I5 25 30

114.8 135.7 135.7

42.1 41.4 38.7

137.2 130.5 130.7

Phosphate buffer

15 25 30

108.3 I 18.8 I 17.5

40.5 40.4 38.7

121,7 I 15.3 I i 1.4

F. Bonosi et al./Colloids Surfaces 65 (1992) 287-295

291

r 48.

~k

DOPC/ClgR

48"

24

i.

24

o

O

~

\

DOPC/CER

48-

25OC

~

~! t~. z4

25°C

24.

o 4a-

DOPC/CER

48 -"

2A_

0.3

24

0.7

1.1

1.5

SUP,FaCE AREA(xnm/mg)

0 i,, 0.3

0.7

1.1

1.5

SU~ACE AREA (m~/ml)

Fig. 3. rr/A isotherms of the D O P C / C E R system on 0.1 M NaCI subphase at 15, 25 and 30"C, in the following CER weight fractions: 0 (.). 0.3 (Q), 0,4 (ms), 0.5 (A), 0.6 (4~) and I 1,).

Fig. 4. rr/A isotherms of the D O P C / C E R system on phosphate buffer subphase at 15, 25 and 30°C, in the following CER weight fractions: 0 (*), 0.3 ( 0 ) , 0.4 ( I l L 0.5 (A), 0.6 ( ~ ) and I (*).

The surface area variations as a function of m o n o l a y e r composition depend on temperature and on subphase. As regards the 0.I M NaCI subphase, at 15°C, we observed positive deviations from the additivity rule, while at 25 and 30°C we had a nearly additive relationship for low C E R contents (0-0.4 CER weight fractions) and again positive deviations for high C E R contents (Fig. 5). These results indicated that the two components are miscible with predominant repulsive interactions. Further evidence for the miscibility came from the plot of C~ ~ values against mixture composition (Fig. 6). The negative deviations from additivity, at all the surface pressures, indicated the presence of more expanded phases for the mixtures than for the pure components and the occurrence of repulsive interactions between them. The variation of collapse pressure with the m o n o l a y e r composition definitively confirmed the miscibility as a consequence of the surface phase rule application.

Once the miscibility between the two components was ascertained, a t h e r m o d y n a m i c analysis was performed. Figure 7 shows that AGmix values as a function of the mixture composition were always small in absolute value, thus indicating slightly interacting components. The ZIGmix values were always positive, with the exception of the mixture with C E R weight fraction equal to 0.4, which showed negative values of AGm~,,, thus indicating a slight stabilization with respect to pure components. The TASm~,, and Ail,..,ix values (Figs 8 and 9 respectively) were always positive, indicating that the main contribution to miscibility was entropic; this result is probably due to the presence of electrostatic repulsions between the polar heads of D O P C molecules. The only mixture in which negative value~ of ~4Sm~xand dHmix were observed was the one with CER weight fraction equal to 0.6, in which the previously discussed repulsive electrostatic inter-

F. Bom~si et al./Colloids Surfaces 65 (1992) 2 8 7 - 2 9 5

292

30°C

30°C 600"

1.00

.I; i

,I

300'

0.65

~0.30 " ~

.

t

/

I

1

/

/

/

.~°C

~

1.00 ~.~.~~... . ~~

0.65

0

0.30 15'C

15°C

000"

1.00

/ "J i

/

/

J

0.65

/

300'

/

0.:30

l

0.0

;

|

|

I

I

t

I

I

I

0.5

I

t

I

I

I

I

I

I

.0

XGa'T FRACTION (CER)

OT

/

J

.........

0.0

/

f

/

/

1

/

/

m_m." , .........

0.5

1.0

wemetr FRACTION(CER)

Fig. 5. S u r f a c e a r e a s vs C E R w e i g h t f r a c t i o n for t h e D O P C / C E R s y s t e m o n t h e 0.1 M N a C I s u b p h a s e at d i f f e r e n t t e m p e r a t u r e s : r~ = 4 n a N m - n (@); rt = 20 m N m - 1 { lil}.

Fig. 6. S u r f a c e c o m p r e s s i b i l i t y m o d u l i p l o t t e d a g a i n s t C E R w e i g h t f r a c t i o n for t h e D O P C / C E R s y s t e m o n 0.I M N a C I d i f f e r e n t t e m p e r a t u r e s : n = 4 m N m - t ( O ) ; n = 20 m N m - 1 (K.

actions between D O P C molecules were reduced by the presence of C E R molecules, thus inducing a decrease in the entropic c o n t r i b u t i o n and m a k i n g h y d r o p h o b i c interactions a m o n g the chains more important. As regards the buffered subphase, surface areas vs weight fraction plots always showed positive deviations from additivity (Fig. 10), greater than those observed on the 0.1 M NaCI subphase, indicating stronger repulsive interactions. This behaviour was confirmed by the negative deviations from the additivity of C£ 1 vs mixture c o m p o s i t i o n data (Fig. !1), a n d the surface compatibility between

D O P C and C E R was further d e m o n s t r a t e d by the variation of the collapse pressure as a function of C E R content. The t h e r m o d y n a m i c analysis of the bidimensionai mixture was performed as previously described. The AGmix, T,dSmix and dHmix values are shown in Figs 7, 8 a n d 9 respectively. The AG,,i~ values were always positive and greater than those observed for the 0.1 M NaCI subphase, thus confirming the decreased stability of this system. With regard to the enthalpic and entropic contributions, Figs 8 and 9 show that by increasing the C E R c o n t e n t at the a i r - w a t e r interface, TASm~ a n d dHmlx values

F. Bonosi et st.~Colloids S,rfaces 65 (1992) 287-295 NaCI

Phosphate buffer

NaCl 0.I M

1SiC

15'C

60-

~

5.0

~

M

0.1

_

Phosphate buffer

15'JC

15eC

21f,eC

ESeC

-20

|,5

A

293

-~'.0

2tl,eC

?~ee

5.0

-9O ,ISO'

~

-,'ml

-00

-2.0 =o'c

s#'c

•

50-

-P.O. ~ 0 J C

i S . 0

0.0 g,

i

~.

,

0.4 ,

i

w i

.

,

l

i

0.8 0.0 l

i

i.

,

0.4

T~CBTIeg~'HOH(CIR)

Q.B

Fig. 7. Excess free e n e r g y o f mixing, AGmix, as a function o f C E R weight fraction for the D O P C / C E R system, at different temperatures, on 0.t M NaCI and phosphate buffer subphases: ~=4mNm -I (O);~=20mNm -~ liB).

NaCI

0.1

Phosphate buffer

M

15eC

80"

50J

__.~

25eC

25eC

~ -1t0 -90 50

. _.___.~ , ~ ' - ~ • ' ~

-ZO -go

:l::,,.l,lls..,i,,.| 0.0• i.,,l:llll:.,,i.,. 0.~ 0.8 0.0 0,4 0+8 WI~GB'rrlucllom (CXR)

Fig. 8. TAS,,i~ as a function o f C E R weight fraction for the D O P C / C E R system, at different temperatures, on 0.1 M NaCI and phosphate buffer st, bphases: ~z = 4 m N m -~ ( 0 ) ; rr=20mN

~

e i

0.0

i

i

i

s

i

i

0.4

i

~ i

i

i

i

~

i i

0.8

i

i

0.0

i

i

it

i1

T

0.£

g [

B

0.8

Fig. 9. Excess free e n t h a l p y o f mixing, AHm~,, as a function of C E R fraction for the D O P C / C E R system, at different temperatures, on 0.1 M NaCI and phosphat: buffer subphases: 7r=4mNm -l (O);n=20mNm -t (g).

became negative, i.e. the enthalpic contribution to the bidimensional miscibility became more important, owing to the absence of electrostatic repulsions. Conclusions

-20 -90

i

a O I ~

m -i

(ram).

The results obtained allowed the following o n c l u s i o n s to be drawn. D O P C and CER showed bidimensionai miscibility at the air-water interface in the 1 5 - 3 0 ° C temperature range on both 0.1 M N a C | and pH 7 subphases. The miscibility is due to different main contributions depending on the subphase: (a) at pH 5,6 the most important contribution t o z l G m i x is entropic, o w m g to the electrostatic repulsions a m o n g charged molecules; (b) at pH 7 the most important contribution is enthalpic. At pH 7 the mixture spreading isotherms showed surface pressure values intermediate between those of the pure components, thus indicating that the condensing effect due to the higher pH value

F. Boltosi et al./Colloids Surfaces 65 (1992) . 8 7 - . . 9 5

294

420

30°C

1.00 /

260

/

I

/

t"

0.65

/

140

~o.3o

o

25,C

420

25°C ,/

"~280

i

/

f

0.65

/

/

J

~3

0.30

140

J

0 15°C

420

15OC

," /

/

/

/

1.00

/

f

280

,-I1~

..,,

,,.

/ /

140

o.o

. . . . . . . . . . . . . . . .

~mn'r

6.'5

i.o

FRACTION (CP-R)

Fig. 10. Surface areas vs CER weight fraction for the DOPC/ CER system on the phosphate buffer subphase at different t e m p e r a t u r e s : rc = 4 m N m - ~ ( 0 ) ;

rt = 2 0 m N m - ~ ( B ) .

o b s e r v e d in C E R m o n o l a y e r s also prevails in the mixtures. A recent s t u d y on the e l e c t r o n spin r e s o n a n c e line shape o f the d o x y l stearic p r o b e s inserted in DOPC/CER vesicles, s o n i c a t e d in a p h o s p h a t e buffer [-I 5], agrees with a surface c o m p a t i b i l i t y of the t w o lipids. In the s a m e w o r k , C E R a d d i t i o n resulted in an increase in the a v e r a g e size of D O P C vesicles, in a g r e e m e n t with the o c c u r r e n c e o f repulsive interactions o b s e r v e d in this study. This result can be c o r r e l a t e d with a g r e a t e r a v e r a g e distance b e t w e e n the molecule,;. T h u s it was c o n f i r m e d that the s t u d y o f mixed

o1" . . . . .

/

/ /

~ . . ~ .ff ,',,,,,

0.0 ~remB'r

/

/

/

0.65

0.30

/

f

/

7

//

f

/

,,,

0.5 EakC'~ON

Fig. I I . S u r f a c e c o m p r e s s i b i ' i t y

,, 1.0

(CER) moduli

plotted

against

CER

weight fraction for DOPC/CER system on the phosphate buffer subphase, at different temperatures: rt=4 mN m-~ (0); n = 2 0 m N m -I (Ul).

m o n o l a y e r s is i m p o r t a n t in o r d e r to predict p r o p e r ti,_ of m o r e c o m p l e x m o l e c u l a r assemblies. Acknowledgements

T h a n k s are d u e to the Italian M i n i s t e r o d e l r UniversitY. e della Ricerca Scientifica e T e c n o l o g i c a ( M U R S T ) a n d to Consiglio N a z i o n a l e delle Ricerche ( C N R ) ( P r o g e t t o 10, S o t t o p r o g e t t o 2). O n e o f the a u t h o r s (E.M.) t h a n k s C N R for a fellowship within the a b o v e p r o g r a m m e .

F. Bonosi et al./Colloids Surfaces 65 (1992) 287-295

References I

E. Margheri, A. Niccolai, G. Gabrielli and E. Ferroni, Colloids Surfaces, 53 (1991) 135. 2 P. Lo Nostro, A. Niccolai and C. Gabrielli, Colloids Surlhces, 39 (1989) 335. 3 J.H. Fendler, Membrane Mimetic Chemistry, Wiley, London, 1982. 4 G.L. Gaines, Insoluble Monolayers at the Liquid/Gas Interface, New York, 1966. 5 G.T. Barnes, J. Colloid interface Sci., 144(I) (1991) 299. 6 D.J. Crisp, Surface Chemistry Suppl. Research, Butterworths, London, 1949, p. 17. 7 F.C. Goodri~ + Proc. 2nd Int. Congress Surface Activity, Vol. !, Buttcrworths, London, 1957, p. 85.

295 8

K.J. Bacon and G.T. Barnes, J. Colloid Interface Sci., 67(I) (1978) 70. 9 B. Maggio, F.A. Cumar and R. Caputto, Biochem. J., 171 (1978) 559. I0 B. Maggio, F.A. Cumar and R. Car,~tto, Biochim. Biophys. Acta, 650 (198I) 69. il J.T. Davies and E.K. Rideal, Interracial Phenomena, Academic Press, New York, 1963. t2 G.D. Fidelio, B. Maggio and F.A. Cumar, Biochim. Biophys. Acta, 854 (1986) 231. 13 P. Tancrede, L. Parent, P. Paquin and R.M. Leblanc, J. Colloid Interface Sci., 83(2) (1981) 606. 14 N. Van Man, Y. Trudelle, P. Daumas and F. Heitz, Biophys. J., 54 0988) 563. 15 F. Bonosi, G. Gabrielli, E. Margheri and G. Martini, Langmuir, 6 (1990) 1769.