Interfacial properties of the antifungal iturins on various electrolyte solutions

Interfacial Properties of the Antifungal Iturins on Various Electrolyte Solutions RI~GINE MAGET-DANA, *,1 LAURENCE T H I M O N , t FRANt~OISE PEYPOUX,t AND MARIUS PTAK* *Centre de Biophysique Moldculaire, C.N.R.S., 1A, avenue de la Recherche Scientifique, 45071 Orleans Cedex 2, France; and t Laboratoire de Biochimie Microbienne, Universitd Lyon L 43, boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France Received May 6, 1991; accepted August 21, 1991 We have studied the interfacial behavior of antifungal iturins (principally baciUomycin F, mycosubtilin, and iturin A) in solution and spread as monomolecular films on aqueous phases containing various chloride salts. The presence of electrolytes lowers the interfacial concentration (surface excess) of iturin solutions. The compression isotherm curves are not superimposable, and for all the c o m p o u n d s the stability of the monolayer is higher on KC1 as attested by the transition pressure (for the liquid-expanded part) and by the collapse pressure values. The reduction of the molecular free energy of spreading AG,, that measures the affinity of the polar cycle of the lipopeptide for the aqueous phase components, is m a x i m u m on KC1 in the case of mycosubtilin. The energy of compression AGe, that measures the intermoleeular forces between the film forming molecules, is generally higher on KC1. The compression leads to more ordered structures when mycosubtilin and iturin A are spread on KC1. W h e n spread on CaC6, iturin A and bacillomycin F molecules can adopt only two kinds of arrangements, as shown by the convergence of the LE parts of their isotherms. These data are interpreted as interactions between iturins and ions and, more precisely, as discriminating interactions between iturins and the various cations with a special role for K +. © 1992AcademicPress,Inc. INTRODUCTION

Iturins form a family of special biosurfactants possessing antifungal activity. This biological property is related to an increased K + permeability of the cytoplasmic membrane of target cells (1) and depends on the primary structure of the peptide cycle (Fig. 1 ). The activity is dramatically reduced by the methylation of the D-Tyr 2 residue (2) or greatly increased by the sequence inversion of the two adjacent D-Ser 6 L-Asn 7 residues as in mycosubtilin when compared to iturin A (3). All the iturin compounds studied induce ion conducting pores (4-6), assumed to be related to the formation of aggregated structures in the lipid membranes ( 7, 8). In this paper we have studied the effect of electrolytes on the behavior of iturins in aqueous solutions or spread

as monolayers at the air-water interface, a situation quite similar to their state when they reach the membrane of target cells. This was done in an attempt to correlate the ion conducting properties of iturins to the change of some of their physico-chemical parameters, in the presence of ions. MATERIALS A N D M E T H O D S

All the iturin compounds were prepared and purified according to Ref. (9) for iturin A, Ref. (2) for MeTyr-iturin A,2 Ref. (10) for mycosubtilin, and Ref. ( 1 1 ) for bacillomycin F. Pure water was obtained from a Millipore (Milli-Q) apparatus. The saline solutions (0.1 M LiC1, NaC1, KC1, and CaC12) were treated 2 Derivative of iturin A methylated on the D-Tyr 2 residue.

J To w h o m correspondence should be addressed. 174 002 1-9797/92 $3.00 Copyright© I992 by AcademicPress,Inc. All rightsof reproduction in any form reserved.

Journal ojColloidandIme(face Science, Vol. 149.No. 1. March I, 1992

175

INTERFACIAL PROPERTIES OF ITURINS A M I N O ACID R E S I D U E S COMPOUND

L1

D2

D3

L4

L5

D6

L7

Iturin A

Asn

Tyr

Asn

Gin

Pro

Asn

Ser

Iturin C

Asp

Tyr

ASh

Gin

Pro

Asn

Set

Mycosubtilin

ASh

Tyr

Asn

Gin

Pro

Ser

Asn

Bacillomycin L

ASp

Tyr

Asn

Set

Gin

Ser

Thr

Bacillomycin D

Asn

Tyr

Asn

Pro

Glu

Set

Thr

Bacillomycin F

Asn

Tyr

Asn

Gin

Pro

Asn

Thr

C]O--Lc,H A s n - - O " r y r - - D l s n * /2 L Gl~ / CH-(CH )n-OH-( CH ) 8 - C H 3 2 I 2 I CH I 3 N H _ _ L Ser__D A s n _ L Pro

/z c14 iso c15

FIG. 1. Primary structure of iturins: (a) peptide sequences of the various compounds of the iturin family;

(b) iturin A formula.

with charcoal in order to remove any amphiphilic impurity. Monolayer experiments were carried out using a Langmuir film balance system previously described (12). The equilibrium spreading pressure was determined at constant area (20 cm 2) by depositing small amounts of iturin on the aqueous surface until no further increase in surface pressure was observed (13). For isotherm (Tr-A curves) measurements, iturins were dissolved in the following solvent mixture: 3 pyridine / cyclohexane / ethanol/hexafluoroisopropanol/hexane (56:13:13:10:8 by vol.) and then spread on the saline subphase with a 50-tzl Hamilton microsyringe. The rate &compression was 0.05 n m 2 molec -~ rain -] . 3 We failed to find a solvent mixture for the charged compounds: iturin C, bacillomycin L, and bacillomycin D.

The trough was thermostated and the temperature of the subphase was maintained within +__0.2°C. The reproducibility of the mean molecular area was within +2%. For determination of CMC values, the surface tension of iturin solutions was measured with a Kriiss tensiometer using the procedure o f d u Noiiy with a platinum ring. Iturins were dissolved in a 2 - m M T r i s buffer, pH 8.0, containing in some experiments NaC1, KC1, or CaCI2 (0.1 M). RESULTS AND DISCUSSION

Critical Micellar Concentration In Fig. 2, the surface tension is plotted versus the logarithm of iturin concentration. The CMC value is determined as the intersection point of two linear segments. The results are presented in Table I. For the three compounds Journal of Colloid and Interface Science, Vol. 149, No. 1, March 1, 1992

176

MAGET-DANA ET AL

Q "3

70.

E E 60.

Z

5O.

Iturin A ( 1 0 - S M )

it W

• ~AD~

~o~ ° ~ Temperature (*(2)

FIG. 2. Critical micellar concentration determination: (a) surface tension as a function of concentration for iturin solutions ( T = 25°C) (Or) in 2 m M Tris buffer pH 8, (A) + 0.1 MKC1. (b) CMC of iturin A solutions in 2 m M Tris buffer pH 8 as a function of temperature.

studied (iturin A, mycosubtilin, and bacillomycin F ) the CMC values are in the same range (4 to 6 × 10 -5 M). This result is not surprising according to the similar structure of these compounds. Among the chloride salts only KC1 affects significantly the CMC values: it decreases the CMC values of mycosubtilin and bacillomycin F by about 20 and 25%, respectively. The change of the CMC with addition of an electrolyte to a nonionic surfactant can be expressed by the equation log CMC = - K s C + log (CMC)c=0, where Ks is a constant which depends on the salt and the solution studied and C is the electrolyte concentration ( 14, 15). The calculated Ks values in the presence of KC1 are -0.33 for iturin A, 0.93 for mycosubtilin, and 1.33 for bacillomycin F. Various interpretations have been proposed for the observed effects of electrolytes. Mukerjee (15) explained the lowering of CMC in terms of salting out of the hydrocarbon moiety, but in our systems the acyl chains of iturins have the same nature and have comparable length. A more plausible qualitative interpretation seems to be the one of Beecher (16): that the micelle of the nonionic surfactant is not truly nonionic but posJournal of Colloid and Interface Science, Vol. 149, No. I, March 1, 1992

sesses a net charge due to a specific ion interaction. Then results would agree with an interaction of K ÷ with mycosubtilin and bacillomycin F. We can assume that in the premicellar region, the solution of iturin is ideal. Then, from the slope of the decreasing segment of the (3, - log C) plot, we can calculate Pmax, the interfacial concentration (surface excess) by application of the Gibbs equation of adsorption ( 17 ), rmax =

--

1 / R T ( d j , / d In C),

and the surface area per molecule is given by a = l/r

....

We can see (Table I) that the presence of electrolytes greatly decreases the interfacial concentration ofiturins, hence the surface area per molecule increases. This may be related to an electrostatic repulsion between iturin molecules in the hypothesis of a cation interaction. The change in the standard free energy of a monomer, when associated in micelles, is given by AGm = R T In CMC. Table I includes values of the heat of micelle formation ~ / m , which is given by the relation (18) AIIm = - RT2 d( ln CMC ) / dT.

An example of the variation of CMC with temperature is shown Fig. 2(b). In the case of iturin A, the CMC increases with temperature. Spreading of Iturins

The equilibrium spreading pressure (~re) of iturins on pure water are shown Fig. 3. For all the compounds tested ~rechanges linearly with temperature. Bacillomycin F forms the more stable monolayer in the temperature range studied, then mycosubtilin and MeTyr iturin A, respectively. For bacillomycin F, the slope

INTERFACIAL PROPERTIES OF ITURINS

?

?

~

X

X

x

~

~"

+1 ,r'q I

,o +1 ~ ,o +

c5 +1 ...

+1

+1

+l

I

I

dTre/ dT is slightly positive, suggesting that the

+

I

,% e.q

+[ +1 +1 +l +1 +1 +1 +1 +l +1 +1 +1

z I

I

I

I

]

I

I

I

I

I

I

I

<

gr~ oi

+1 +i +l +1 +1 +1 +1 +1 +1 +1 +1 +1

+1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1

+1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +t +1

r)

~z

rT~

=

8

=

177

monolayer is in the solid form (13). The same variation is observed for mycosubtilin until the break in the slope at a position possibly correlated with the melting temperature: Tm = 25°C ( 19, 20). No change of 71e (between 15 and 30°C) in the case of MeTyr iturin A is observed. For iturin A, iturin C, and bacillomycin D, dTrddThas a positive value related to a positive entropy change ASs = A~dTr~/dT (Ae is the monolayer area per molecule at 7r,) during the spreading process. This positive entropy change indicates that the structure of iturin monolayers is less ordered than the bulk solid phase in which the intermolecular forces are strong. Bacillomycin D begins to spread at only 30°C, and this spreading temperature Ts (20) is much higher than in the case of iturin C (Ts ~ 5°C) or iturin A (Ts - 15 ° C). These results indicate that the chainchain interactions are important in bacillomycin D arrangements, whereas iturin A monolayers interact strongly with the aqueous phase (21 ). We have calculated the free energy of spreading AGs = -AeTre (Ae estimated from the isotherm curves, see below). Factors influencing monolayer formation and then AGs values include (a) the energy required to break away from the bulk state; (b) the affinity of the polar part of the molecule for the subphase components (water and ions); and (c) the interactions between the hydrocarbon chains of the molecules oriented as the monolayer. For a given lipopepfide, (a) and (c) are identical whatever the chloride salt used in the subphase. Then the &Gs values only reflect differences in the affinity of the peptide cycle for the subphase, i.e., for the various cations. Figure 4 shows the temperature dependence of AGs recorded when iturins are spread on different saline subphases. For all the compounds, AGs values varied greatly according to the nature of the cation in the subphase. For mycosubtilin the decrease of AGs is the more important with KC1. We also observe a discontinuity in the (2xGs-T) plot between 15 and 20°C without change in the slope. A simJournal of Colloid and Interface Science, Vol. 149, No. I, March 1, 1992

178

MAGET-DANA ET AL A

--O-

.%.--

1'0

'

2'0

~

JL 130

Temperature( ° C ) FIG. 3. Equilibrium spreading pressure of iturins on pure water as a function of temperature: - • - Bacillomycin F; - • - Mycosubtilin;- 0 - BacillomycinL; - • Baeillomycin D; --©-- MeTyr iturin A; --0-- Iturin A; - - D - - Iturin C.

ilar behavior is found for iturin A and MeTyr iturin A spread on KC1. However, for iturin A the AG~ values above 20°C are much lower. The discontinuity observed corresponds to a lower affinity of the peptide cycle for the KC1 subphase caused by a change in the arrangement of iturin monolayers. The AG~ values for the spreading of bacillomycin F on KC1 and CaC12 solutions do not vary with temperature. Below 20°C they are lower than those obtained for the spreading on pure water.

temperature of the mycosubtilin monolayers spread on KCl: the molecular area A0 extrapolated at ~- = 0 varies from 1.05 + 0.05 n m : at 10°C to 1.55 + 0.08 nm 2 at 30°C, which traduce an increase of about 50% (only 16% on water). At the same time, the compressibility coefficient in the LE part of the isotherm {3o = - 1 / A o ( d A / d T r ) T v a r i e s from 9 N -~ m at 10°C to 14 N -1 m at 30°C, whereas it varies only from 7 to 8 N - 1m on water. This increase in/30 in the presence of KC1 is related to weaker chain-chain interactions. Such an expansion is observed also in the case of baciIlomycin F and MeTyr iturin A but to a lesser extent. To the contrary we observe a contraction of the iturin A monolayers spread on KC1. On CaC12, expansion of the mycosubtilin monolayers with temperature is rather weak ( + 16%), whereas the compressibility coefficient/30 shows an increase of 78%: from 9 N -z m at 10°C to 16 N -1 m at 30°C. An important feature on CaC12 is the convergence of the LE part of both the iturin A and bacillomycin F isotb, erms: at 0.94 n m 2 and 4.5 m N m -~ for

Temperature ( °

C )

a

b

_10. / ~

-0"

" I

.01

Compression Isotherm Curves JS.

The compression isotherm (~--A) curves of iturins obtained at 10°C on various saline subphases are compared in Fig. 5. For a given lipopeptide, isotherms are not superimposed, although the general shape is conserved. If we compare these isotherms to those obtained on water, we notice an expansion of the iturin monolayers in the presence of salts, except for MeTyr iturin A. In this case the isotherm on water is near the one obtained on KC1. The collapse pressure ~-c, which gives an indication of the stability of the monolayer, is always m a x i m u m on KC1 and m i n i m u m on CaC12. However, 7re is higher on pure water. As seen in Fig. 6, in all cases 7re decreases with temperature. We observe a great expansion with Journal of Colloid and lnteiJace Science, Vol. 149, No. 1, March 1, I992

o _20.

E

"3

d

d

<1-1o

1~

.

2.o

,

3,0

_1o 1,o

,

-20J

ix

I

~o

,

~o

J --~,----4r----4:~"

-20J

FIG. 4. Temperature dependence of the free energyof spreading on 0.1 M chloride salts: (&) KC1, (El) NaCI~ (©) LiC1, (0) CaC12, (~) pure water. (a) mycosubtilin; (b) iturin A; (c) bacillornycinF; (d) MeTyr iturin A.

INTERFACIAL PROPERTIES OF ITURINS

179

- ~ ( a.% )

40.

30-

20.

~

KCl

20.

E

CaCla~,, ~

Z 10. o P

ID

30.

10.

.

o'.5

40-

~ 30O3

~%*~

.............. .,i.~

(d)

40-

~KC'

c,,

Ac At Ao 30.

20-

10-

~

0'.5 I'TCt......... , ! 1 [

....... %

KCI

CIJ "L..,. CaC I 2 / Li

,o_

NaCI~ ~ \\" ,'X CaCl~/ ~ ~ ,

lo.

0'.~

Molecular Area (nm2)

0.5

1

FIG. 5. Isotherm curves ofiturins on various saline subphases: (a) mycosubtilin;(b) itufin A; (c) MeTyr iturin A; (d) bacillomycin F. The concentration of salts is 0.1 M," the temperature is 10°C; ---, isotherm curves obtained on pure water. (c) inset: schematic representation of an isotherm with indication of the parameters used in this study.

iturin A and 0.87 n m 2 and 20 m N m -1 for bacillomycin F. Then, under compression, molecules spread on CaC12 may adopt only two definite orientations (or conformations): the initial one at ~r ~ 0 and a final one, without a continuous pathway. Ca 2+ would then be a structural element of the monolayers. The compression isotherm curves present a transition zone attributed to a change in the molecular arrangement at the air-water interface. This transition zone is marked by an inflection rather than a plateau. The temperature dependence of 7rt is shown in Fig. 7. We note a general negative shift of ~rt with temperature. This decrease suggests that the transition does not correspond to a thermotropic phase transition similar to the one observed for lipid monolayers. It could rather be a transition from a monolayer state to some kind of oriented bilayer state (different from the collapsed

state). In this way the behavior of iturin monolayers is close to that of polypeptides (22). It can be seen that 71"t > 7r e when iturins are spread on water and below 20°C. For mycosubtilin and bacillomycin F, a-t > 7re on the whole range of temperature whatever the chloride salt on the subphase. Inversely ~rt < ~re when iturin A monolayers are spread on chloride salts ((Tre-T) plots on chloride salts not shown). Then, except for this latter case, the iturin monolayers at 7rt are not in a thermodynamic equilibrium at temperatures below 20°C. For mycosubtilin we observe differences in the 7rt values ( K + > N a + , (Li +) > Ca 2+) in addition to similar dTrt/dT values. This result suggests that the orientation (or conformation) of the peptide cycle differs on the various cations (21 ). When iturin A is spread on KCI, the (~rt-T) plot presents a discontinuity between 15 and 20°C that may Juumal of Colloid and Interface Science, VoL 149, No. 1, March I, 1992

180

MAGET-DANA ET AL 1o

a

b

2O 40

40. 30

30-

30-

20-

20. 10

10.

10.

0.5

1

o'.s

E

Z

10 15

4O.

40.

0

3o.

3O.

30 25

30

15 20

¢~ 20.

25

20-

10_

~ 10_

0.5

1

t

11.5

0.5

1

40.

40.

3O

30.

20.

20.

10.

10.

0'.5

~

l'.s

h 0.5

Molecular Area (nm 2) FIG. 6. Isotherm curves of iturins at different temperatures. Column (a), mycosubtilin; column (b), bacillomycin F; column (c), iturin A; column (d), MeTyr iturin A. From top to bottom: pure water subphase, 0.1 M KC1 subphase, 0.1 M CaClz subphase.

correspond to a change in the interfacial arrangement of the molecules. For the four compounds studied, 7rt is the highest when they are spread on KC1, indicating that K + ions stabilize the LE state of monolayers.

Thermodynamics of l~5"lmCompression The free energy of compression AGo = -

7cdA represents the work required to

-

I

Journal of Colloid and Interface Science, Vol. 149, No. 1, March 1, 1992

compress a monolayer from a state where no intermolecular contacts occur (A~ is the molecular area at the lift-off of the isotherm) to a c o n d e n s e d - - b u t not collapsed--state (A2 = 0.2 n m 2 for iturin A, 0.3 n m 2 for M e T y r iturin A, 0.4 n m 2 for mycosubtilin, and 0.44 n m 2 for bacillomycin F) (23). 2xGcmeasures the intermolecular forces in the film and depends on both the hydrophilic peptide cycle and the acyl chain part. For a given lipopeptide spread on saline subphases, the differences in the AGe

INTERFACIAL

PROPERTIES

OF ITURINS

C

d

,lo 152o

40 30

181

~

20.

2

0

3040

~1 5

25

20.

25

10

10.

10.

"i

0:5

E Z

40, ~

1

~

0:5

40.

O) 30. ¢/) ~0 20. n

20.

(9 10-

10 15 10.

30_ 2O

o15

o'.s

1

40,

40_

30.

30.

20.

10 15

10 15

20. 10.

10_

Molecular Area

~5

(nm2)

FIG. 6--Continued

values depend only on the interaction of the peptide cycle with the various cations. Figure 8 shows that the ( A G ~ - T ) plots are generally linear and vary with the cation in the subphase. Examination of the AG~ values indicates that it is generally easier to compress an iturin monolayer spread on water than spread on chloride salts (except MeTyr iturin A). Higher AG~ values observed on KC1 reflect higher intermolecular cohesion in the film. From the slope of (AGc-T) plots one can deduce the sign of the entropy of compression

ASc = - d ( A G e ) / d T and then of the enthalpy AHc = AGc + T A S k . The slope is positive

when iturin A is spread on KC1 and when mycosubtilin is spread on KCI or LiC1. Then AS~ and the calculated values of A//~ are negative, directly related to a more ordered arrangement of the monolayer under compression: the molecules become more closely packed. In all the other cases the slope is negative, giving positive values of AS¢ and A//c corresponding to a disorganization of the iturin monolayers under compression. Journal of Colloid and Interface Science, Vol. 149, No. l, March 1, 1992

182

MAGET-DANA ET AL a 35_ 35.

\,~

b

\

%

a0.

20.

¢=

20

o_

i

L

15.]

t

,

1'0

2'0

20.

.~--~

~zX~ZX

"El......

'

2'o

'

3'o 10

Temperature

~ / v ~

~__

,5.

" ~ : ........... 8 1'0

3'0

d

,0.1-<>~.___4~_

5

'

c

I

'~

t

" 1'0

zX

~,_

" : ~'=,-t~-.-.-@" '

2'0

'

fro

(°C)

FIG. 7. Temperature dependence of the transition pressure of iturin compounds spread on 0.1 M chloride salts: (A) KC1, (O) LiC1, ( n ) NaC1, ( 0 ) CaC12, (*) pure water. (a) mycosubtilin; (b) bacillomycin F; (c) iturin A; (d) MeTyr iturin A.

Iturin A monolayers show an interesting behavior: on water d(kGo)/dT is greatly negative, and positive on KC1 only. The presence of K + in the subphase is an important factor of order. Furthermore, on KC1 the (AG~-T) plots present a discontinuity for mycosubtilin, iturin A, and bacillomycin F. For the two latter compounds, 2xG~values are much lower for T > 20°C: the intermolecular cohesion in the film is then weaker.

layer subphases greatly modifies the interface parameters. They induce a drop in the surface excess concentration of iturin solutions. The analysis of the compression isotherm curves clearly indicates a dependence of the various characteristic data upon the nature of the cation in the subphase. The presence of calcium ions induce a particular behavior of bacillomycin F and iturin A monolayers with a convergence point on the isotherms. We observe a general expansion of the monolayers, especially pronounced when mycosubtilin is spread on KC1. The thermodynamic parameters suggest a sudden structural change with temperature when iturins are spread on KC1 and suggest an affinity of mycosubtilin monolayers to K +. Then from all our data, we can reasonably say that iturins interact with alkaline cations, especially with K +. This is in agreement with previous experiments showing that the presence of electrolytes greatly modifies the H - N M R spectrum of iturin A. The amide proton chemical shift of D-Tyr 2 varies first with the presence of electrolytes and second with the nature of the ?.0`

a

b

25.

20. A

~

o

....g] _o................. 15,

'

ca

~

1~

.O->
'

1'o

2~

We already knew that minor changes in the primary structure lead to some differences in the conformation of iturins (24, 25 ). The results herein show that these structural changes induce differences in the surface properties of iturins: surface excess concentration of iturin solutions, equilibrium spreading pressure and organization at the air-water interface vary from one compound to another. The presence of electrolytes in iturin solutions or in monoJournal of Colloid and Interface Science, Vol. 149, No. 1, M a r c h 1. 1992

3~

v

c CONCLUSION

"

<3

d

"

20.1

iO ............ " ~ \.

! - ~ - ~

15

[]

'

2'o

..,

-

[]

3'o

1'0

2'0

a'o

Temperature (° C ) FIG. 8. Temperature dependence of the energy- of compression ofiturin monolayers spread on 0.1 Mchloride salts: (A) KC1, (O) LiC1, ([~) NaC1, (<)) CaC12, (*) pure water. (a) mycosubtilin; (b) iturin A; (c) bacillomycin F; (d) MeTyr iturin A.

INTERFACIAL PROPERTIES OF ITURINS

cation (D. Marion, personal communication ). A possible perturbation of the hydrogen bond between this NH group and the nearest carbonyl group could lead the latter to bind a cation. The implication of the D-Tyr 2 residue would explain the peculiar behavior of MeTyr iturin A compared to the other compounds. The cation binding ability of iturins leads to the hypothesis that, in addition to forming conducting pores in membranes, iturins can also act as cation carriers. REFERENCES 1. Besson, F., Peypoux, F., Michel, G., and Delcambe, L., Biochem. Biophys. Res. Commun. 81, 297 (1978). 2. Peypoux, F., Besson, F., Michel, G., and Delcambe, L., J. Antibiot. 32, 136 (1979). 3. Besson, F., Peypoux, F., Michel, G., and Delcambe, L., aT.Antibiot. 32, 828 (1979). 4. Maget-Dana, R., Ptak, M., Peypoux, F., and Michel, G., Biochim. Biophys. Acta 815, 405 (1985). 5. Maget-Dana, R., Heitz, F., Ptak, M., Peypoux, F., and Guinand, M., Biochem. Biophys. Res. Commun. 129, 965 (1985). 6. Maget-Dana, R., Ptak, M., Peypoux, F., and Michel, G., Biochim. Biophys. Acta 898, 1 (1987). 7. Maget-Dana, R., Harnois, I., and Ptak, M., Biochim. Biophys. Aeta 981, 309 (1989). 8. Maget-Dana, R., and Ptak, M., Biochim. Biophys. Acta 1023, 34 (1990).

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Journal of Colloid and lnterface Science, Vol. 149, No. 1, March 1, 1992