Langmuir 1993,9, 2865-2860
2855
Structural Study of AOT Reverse Micelles Containing Native and Modified a-Chymotrypsin F. PitrB,tP*C. Regnaut,s and M. P. Pileni'PtJ Laboratoire SRSI, Universitk P. et M. Curie, batiment F, 4 place Jusieu, 75005 Paris, France, CEN Saclay DRECAM, SCM, 91191 Gif sur Yvette, France, and Facultk des Sciences, Universitk de Paris 12 Val de Marne, Avenue du Gknkral de Gaulle, 94010 Cr6teil Cedex, France Received April 4, 1993. In Final Form: July 14,1993@ The synthesis of partially hydrophobic a-chymotrypsinis performed in AOT reverse micelles to covalently bind hydrophobic molecules on the surface of the a-chymotrypsin. Fluorescence experiments indicate that the modified a-chymotrypsin is anchored on the inner surface of the host reverse micelle. The structures of the microemulsions of empty or filled reverse micelles are investigated by small-angleX-ray scattering, dynamic quasi-elastic light scattering, and conductivity measurements. The corresponding intermicellar interactions are analyzed in terms of the adhesive hard sphere model. It is shown that neither the presence nor the change in the location of the a-chymotrypsin modifies the intermicellar interactions and structure.
I. Introduction A microemulsion is a thermodynamically stable mixture of water, oil, and surfactant where water and oil are separated by a monolayer of surfactants. Due to the amphiphilicnature of the surfactant, numerousdisordered or partially ordered phases are formed depending on temperature and concentration.' The ternary phase diagram for water/isooctane/AOT (sodium bis(2-ethylhexyl) sulfosuccinate)shows a large zone where the reverse micellar phase takes place. In this liquidlike phase, the ratio of water concentration to surfactant concentration (w = [H201/[AOT]) determines the size of the reverse micelles. At w = 15, the water pool radius r, increases linearly with w. This linear relation can be explained by a simple geometricalmodel: which assumes that the area per surfactant molecule is constant, that all the surfactant molecules participate in the, interface, and that the microemulsion is monodisperse. The droplet concentration, [RMI, at a given water content, w,is proportional to the water volume fraction, 4,, according to the relation [RMI = 34,/4~,3
If the aqueousvolume fractionis large enough,an aggregate of macroscopic dimensions appears corresponding to a percolation transition. The percolation threshold corresponds to the maximum of the permittivity and to the onset of the c~nductivity.~ The effective intermicellar potential depends on the temperature, on the solvent,and on the size of the dr0p1et.a.~It has been shown that this potential can be well represented by a hard sphere core potential plus a very short range attractivetail,6i6the range of which does not exceed 10% of the diameter of the
* To whom correspondence should be addressed at the Universitk P. et M. Curie. + Universitk P. et M. Curie. t CEN Saclay, DRECAM,SCM. 8 Universite de Paris 12 Val de Marne. e Abstract published in Advance ACS Abstracts, October 1,1993.
(1) For a general survey we: (a) Surfactants in Solution; Mittal, K., Lindman, B., Ede.; Plenum: New York, 1984; 1987. (b) Physics of Complex and Supermolecular Fluids; Safran, S . A., Clark, N. A., Eds.; Wiles New York, 1987. (2) Pileni, M.P.; Zemb, T.;Petit, C. Chem. Phys. Lett. 1986,118,414. (3) Van Dijk, M.A. Phys. Rev. Lett. 1986, 9,55,1003. (4) Huang, J. S.J. Chem. Phys. 1985, 82, 480. (5) Lemaire,B.;Bothorel, P.; Row, D. J. Phys. Chem. 1983,87,1023.
droplet. All the collisions are not elastic, and some of them are efficient so that an exchange process between two reverse micelles takes p l a ~ e . ~This - ~ property favors chemical or enzymaticreactions obtainedby mixing reverse micellar solutions separately containing the reagents.lO Microemulsions have the ability to serve as hosts of macromolecules, in particular enzymes.l&l3 The solubilizationof enzymes in organic solventscould provide many opportunities for the development of new biocatalysis synthetic reactions in the organic phase. The incorporation of enzymes inside a microemulsion can greatly alter the host reverse m i ~ e l l e s . ~Such J ~ modifications might be used in the future for biotechnologic applications of the reverse micelles. Previously, it has been shown that cytochrome a hydrophilic peripheralmembraneprotein with a high isoelectric point, takes place on the inner reverse micelle surface17and modifies the reverse micellar surface in such a way that it increases the attractive part of the intermicellar potential16 and lowers both the percolation onset and the consolute point. These modifications which are due to the attractive electrostatic interactionsbetween the protein and the polar head group of the surfactants do not occur in systems containing nonionic surfactants. In order to understand more precisely which surfactant-protein interaction can modify the intermicellarpotential and extend local perturbations (6) Huang, J. S.;Safran, S. A.; Kim, M. W.; Greet, G.;Kotlarchyk,M.; Quirke, N. Phys. Rev. Lett. 1984,53,592. (7) Eicke,H. F.; Shepherd,J. C. W.; Steinmann,A. J. Colloid Interface Sci. 1976, 56, 168. (8) Atik! S . S.;Thomas,J. K. Chem. Phys. Lett. 1981, 79,351. (9) Robinson, B. H.; Toprakcioglu,C.; Dore, J. C.; Chieux,P. J. Chem. SOC.,Faraday Trans. 1 1984,80,413. (10) Structure and reactivity in reuerse micelles; Pileni, M. P., Ed.; Elsevier: New York, 1989. (11) Luiei, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 947,209. (12) Larsson,K.M.;Adlercreutz,P.;Mattiaeaon,B.Biotechnol.Bioeng. 1987,30, 81. (13) Verhaert, R. M. D.; Hilhorst, R.; VermuB, M.; Schaafema, T. J.; Veeger, C. Eur. J. Biochem. 1990, 187, 59. (14) Chatenay, D.; Urbach, W.; Nicot, C.; Vacher, M.; Waks, M. J . Phys. Chem. 1987,91,2198. (15) Aduances in membrane fluidity, 2nd ed.; Aloia, R. C., Curtain, C. C., Gordon, L. M., Eds.; Liss, A. R., Inc.: New York, 1988. (16) Huruguen,J. P.; Authier, M.; Greffe, J. L.; Pileni, M. P. J . Phys.: Condens. Matter 1991,3,865. (17) Petit, C.; Brochette, P.; Pileni, M. P. J. Phys. Chem. 1986, 90, 6517.
0743-7463/93/2409-2855$04.00/00 1993 American Chemical Society
2856 Langmuir, Vol. 9,No.11,1993 to a larger scale, we study here the influence of the location of the enzyme on the intermicellar structure. We choose a weakly charged liydrophilic enzyme, a-chymotrypsin, and modify ita hydrophilic character by binding hydrophobic molecules on ita surface. Such a modification induces a strong affinity of the enzyme with the internal surface of the reverse micelles. The a-chymotrypsin is a widely used hydrophilic spherical enzyme (r = 21.5 A) in the pharmaceutical industry. Ita enzymatic activity in reverse micelles has been widely studied these last few years.1gm It takes place in the aqueous core of the host reverse micelle."^^^ The present paper is organized as follows: In the ExperimentalSection are reported the method of synthesis of the modified a-chymotrypsinby usingreversemicelleszz and the fluorescenceexperiments from which the location of the a-chymotrypsin in the host reverse micelle is deduced. In section I11the small-angle X-ray scattering, quasi-elastic light scattering, and conductivity measurementa are reported. They are analyzed in terms of intermicellar interaction by using the adhesive sphere model which is convenient for this class of microemul~ i o n s The . ~ ~case of unfilled reverse micelles which serves as the reference is consideredfirst. Then the same analysis is applied to the case of the filled reverse micelles with native or modified a-chymotrypsin.
11. Experimental Section 1. Materials. AOT was obtained from Sigma;a-chymotrypsin and isooctanewere obtained from Fluka They were used without further purification. 2. Experimental Apparatus. The conductivity measurements were performed by using a platinum electrode and a Tacuesel CD 810 instrument. The small-angle X-ray scattering (SAXS)experiments were performed at LURE (Orsay) on the D22 diffractometer. The wave vector range in the present investigation is
The quasi-elastic light scattering (BELS) measurements were performed with an argon ion laser (514.5 A), focused at the center of the &"-diameter cylindrical sample cell. The autocorrelation functions were obtained with a 136-channel Brookhaven digital correlator. They were measured at 45O from the direction of the incident beam. The fluorescence experiments were performed with a SPEX fluorolog-2 device (Xe lamp, 150 W). The high-performanceliquid chromatography (HPLC) separation was performed on a reversed-phase column, Cd, Nucleosil-Silica, 5 pm, lo00 A (150 X 4.6 i.d.). The two mobile phases A and B contained 0.1% TFA in water (A) and 90% 2-propanol and 10% water (B).The flow was 0.7 mL/min. A Waters W E system with a Waters 991 photodiode detector system was used for the analysis. The enzymes dissolved in a mixture made of 90% A and 10% B were injected onto the equilibrated column (90% A and 10% B) and eluted using the two following linear gradients (3 min to reach 55% A and 45% B followed by 16 min to reach 30% A and 70% B). 3. Preparation Mode of Filled Reverse Micelles. First, we prepared a micellar solution with a f i e d w ratio. Then, the solution was poured into a vessel containing the lyophilized enzyme. The concentration of the enzyme was determined by UV spectroscopy. The volume of the enzyme was negligible (18) Martinek, K.; Levashov, A. V.; Klyachko, N. K.; Berezin, I. Biochem. Biophya. Acta 1981,657, 277. (19) Ruckenstein, E.; Karpe, P. J. Colloid Interface Sci. 1990, 139, 409. (20) BN, R.; Walde, P. Eur. J . Biochem. 1991,199,96. (21) Fletcher, P. D. I.; Robinson, B. H.; Tabony, J. J. Chem. SOC., Faraday Trans. 1 1986,82,2311. (22) Levaehov, A. V.; Kabanov, A. V.; Khmelnitsky, Y. L.; Berezin, I. V.; Martinek, K. Dokl. Akad. Nauk SSSR 1984,278,246. (23) Regnaut, C.; Ravey, J. C. J. Chem. Phys. 1989,91, 1211; Errata 1990,92,3250.
Pitrg et a1.
I
H
H-C-0-C-CI
+ oCT-h'
-
UCT-N-C--O-!--H
+
H'+ C1
H
Figure 1. Acylation reaction of the amino groups of the a-chymotrypsin by the fluorenylmethyl chloroformate. 12
6
I
I \
2 *-
270
3W
I 330
WAVELENGTH (nm)
0
i: * 4
G 0-r-
WAVELENGTH (m)
0
270
300
330
WAVELENGTH (nm)
Figure 2. Absorption spectrum of the FMOC-a-chymotrypsin from the HPLC peak. Absorption spectra of native a-chymotrypsin (inset A) and FMOCCl (inset B). Table I. Kinetic Parameters 9, and k,t of a-ChumotruDsinsa
native chymotrypsin 5.9 2.5 7.3 1.5 FMOC-chymotrypsin a Hydrolysis of N-glutaryl-L-phenylalanine p-nitroaniline at 30 "C in Tris buffer (0.05 M, pH 8.0) + 2% (v/v) methanol. compared with the volume of water which was added to the micellar solution (w = 40). 4. Chemical Modification of the a-Chymotrypsin by Using AOT Reverse Micelles. It has been previously shown that reverse micelles can be used to covalentlymodify enzymes.= We applied this method to increase the hydrophobic character of a-chymotrypsin. The chromophore which is covalentlybound to amino groups of a-chymotrypsin is the 9-fluorenylmethyl chloroformate (FMOCCl). The chloroformate group reaction with the amino groups is reported in Figure 1. The native enzyme is solubilized in a borate buffer (pH 9.5; 0.1 M)solution and is injected into the microemulsions. The water content is fixed at w = 20. The chromophore FMOCCl, previously dissolved in isooctane, is added to the micellar solution. The ratio of reagent concentration to enzyme concentration is equal to 20. The acylation reaction is performed at 10 OC. The reverse micellar solution remains clear. After 20 min, the solution is poured into a 10-fold volume of cold acetone (-10 "C) in order to separate the enzyme from the AOT molecules and from free reagent. This solution iscentrifugated at 5000 rpm. The precipitate is dispersed again in 100 mL of acetone and centrifugated again. After removingacetone, water is added to the precipitate. The solution is dialyzed at 10 OC for 12 h to remove the residual borate salt. The remainder is lyophilized. The formation of FMOC-a-chymotrypsin is tested by reversedphase HPLC. The absorption spectrum corresponding to the HPLC peak is characterized by a principal maximum centered a t 266 nm with shoulders and secondary maxima at 280,290, and 298 nm (Figure 2). By comparing the absorption spectra of the native a-chymotrypsin (inset A, Figure 2) and the readant FMOCCl (inset B, Figure 21, it must be concludedthat on average 3.5 FMOC groups are bound to a-chymotrypsin.
Structural Study of AOT Reverse Micelles
0
0.04
4 (A-9
0.08
0
~
0.04
Langmuir, Vol. 9, No.11, 1993 2867
0.08
gL
I
q (A9
Figure 3. Experimental (thick lines) and simulated (thin lines) SAXS spectra at polar volume fraction 4 = 8%without (A) and with (B)polydispersity (a = 0.2). The respective activities of native and modified a-chymotrypsins (Table I) are compared by measuring the Michaelis constant, Kmrand the catalytic kinetic rate constant, kat, of the hydrolysis of N-glutaryl-~-phenylalanine?~ The respective K, of the native and modified a-chymotrypsins are rather similar. However,the kat constantof the FMOC-a-chymotrypsin is lower than the kat constant of the native enzyme. This could be due to the fiation of some reagents on the terminal amino group on isoleucine.26 Nevertheless, the FMOC-a-chymotrypsin retains about 60% of ita initial activity after the modification process.
111. Results and Discussion 1. Variation of the Attractive Interaction Potential of the AOT Reverse Micelles with the Polar Volume Fraction. The scattered X-ray intensity,l(q), is measured as a function of the scattering wave vector, q defined by q = ( 4 r / X )sin(612) where B and X are the scattered angle and the wavelength of the X-ray beam. The intensity is expressed as I(q) = m q ) S(q) where n is the number of reverse micelles per unit volume. The form factor P(q)corresponds to the internal structure of the aggregates while the structure factor S(q) corresponds to the structural arrangement of the aggregates under the volume exclusion and other intermicellar interactions. In dilute solution, the structure factor is approximately equal to unity. Thus, the scattered intensity gives the form factor. In this work, such a form factor is assumed to be represented by a homogeneous sphere model:
0
0.04
0.08
q (A-9 q (A’) Figure 4. Experimental (full lines) and simulated (dashedlines) intensity of SAXS spectra at various polar volume fractions [$ = 8% (1); I$ = 16% (2); 4 = 27% (311 of empty reverse micelles (A) and of reverse micelles containing one a-chymotrypsinper micelle (B).
AOT polar head volume26is equal to 200 A3, the polar volume fraction, $, is related to the water volume fraction, &, by the following expression: $p
= q5w
+ 0.12[AOTl
Assuming that the suspensions are monodispersed, the experimental intensity at low q is compared in Figure 3A with the form factor defined above. Good agreement between the experimentaland simulated data is observed. However, a small divergence between these two curves can be noticed at high q values. We attribute this discrepancy to the size polydispersity of the reverse micelles. By using a Gaussian with u as the root-meansquare deviation from the mean size, ( r) ,the intensity is evaluated as
Good agreement between the experimentaland simulated curves is now obtained also for large values of q (Figure 3B). At a high polar volume fraction, the deviation of the structure factor from 1must be taken into account. In Figure4A, the scatteringpatterns obtained at variouspolar volume fractions are reported. In order to calculate the structure factor, we assumed that the intermicellar POtential is an adhesive sphere potential which is the limit of a square well potential defined by27 VmO -- lim A(r)
k,T
A+
where and ps = 0.22 where v is the polar volume, p = 0.35 ~ - o A are - ~ the reverse micelle electronic density and the solvent electronic density, and rpis the polar radius of the reverse micelle. Such a model leads to an overestimation of 3% of the polar radius with respect to the core shell model (electron densities of water pw = 0.33 e-.A4 and of the polar head pp = 0.52 e-.A4, rp- r, = 3.5 A). However, such a refinement does not significantly modify the determination of the intermicellar attraction from smallangle data analysis. Therefore, in the homogeneoussphere model, the polar radius is the sum of the water core radius and the AOT polar head diameter. Assuming that the (24) Erlanger, B. F.; Edel, F.; Cooper, A. G. Arch. Biochem. Biophys. 1966,115, 206. (25) Oppenheimer, H.L.;Labouesse,B.; Heas, G. J.B i d . Chem. 1966, 241,11,2720.
A(r) = + a for O < r < d
A(r) = In [12rAl(d + All for d < r < d A(r) = 0 for r > d
+A
+A
where d , A, and r1are, respectively, the diameter of the particles, the diameter of the square well range, and the stickiness. Using the Percus-Yevick (PY) an analytical expression of the intermicellar structure factor S(q)is obtained23and can be written as (26) Brochette, P. These de Doctorat de l’Universit8 Paria VI, 1987.
(27) Baxter, R J. J. Chem. Phys. 1968, 49, 2770. (28) Baxter, R. J. A u t . J.Phys. 1968, 21, 663.
Pitre et al.
2858 Langmuir, Vol. 9, No. 11, 1993 Table 11. Values of the Radius, Root-Mean-Square Deviation u, Stickiness d ,and Depth of the Intermicdlar units), at Selected Polar Volume Potential E (M' Fractions # = &a
9 (%) 117
8 71 0.2 2.5
EIkT
-1.9
radius (A) U
a
15 70
0.18 1.6 -1.55
27 65
39 64
0.18
0.16
0.7
0.35 -0.55
-0.9
Reverse micelles without enzymes at w = 40 and T = 22 "C.
Table 111. Values of the Radius,Root-Mean-Square Deviation u, Stickiness T-I, and Depth of the Intermicellar Potential E (M' units), at Selected Polar Volume Fractions 4 = &at w = 40and T = 22 39 15 27 9 (7%) 8 64 72 69 radius (A) 72 0.17 0.18 0.18 U 0.19 1.4 0.7 0.4 117 2.5 -0.6 -1.45 -0.95 EIkT -1.9 a Reverse micelles fiied by one a-chymotrypsin.
S(q) = [QW Q(-q)l-' where
d
E
4l
0
+ B(X)eiX and
40
50
n 0
P "
2
"
4
"
6
"
8
1
'
I
0
@ (%) Figure 6. Diffusion coefficient versus particle volume fraction, 4, of empty reverse micelles (open squares) and reverse micelles containing one a-chymotrypsin (fiied squares).
X = qd/2
1 -r34 - h4(l - 4)l-sinXx 1-b 4 3i-[1+ 24 - X4(1- 411 -1-4 and where the adhesion coefficient h is given by
30
0 @) Figure6. Stickiness, r1versus volume fraction at 22 O C and w = 40. Reverse micelles without enzymes (open squares) and containing one a-chymotrypsin per micelle (fiied squares).
34
Q(q) = 1
20
10
0
-
h=6[z+-] 1 -[36[j+&l24 (1-4)
noticeable trend is the pronounced decrease of r l ,which indicatesthat the intermicellarattraction vanishes (Figure 5). In the dilute media, the pair potential strength can be deduced from the average diffusion coefficient, D,measured by dynamic quasi-elasticlight scattering (DQELS). Figure 6 shows the variation of D versus 4 obtained from our QELS measurements. At small volume fractions, the D versw 4 dependence is linear:
D = Do(l + ad) The hard sphere volume fraction is given by
4 = nrd3/6 The PY expression of S(q) is in good agreement with the We must however availableMonte-Carlosimulation.23~2B~30 mention that the PY approximation has some weakness for the monodispersesticky sphere model which is reduced in the case of polydisperse spheres and for values of 7 not too small when compared to unity.31 These two assumptions are reasonably verified in the present study since the polydispersity of the reverse micelles is about 20% (Table 11)32and the values of 7 obtained are of the order of unity. The fits to the SAXS curves at w = 40, at high polar volume fractions (>15% 1, were obtained by varying the hard sphere diameter, d, and the stickiness 7-l. It was found that d correspondswithin a few percent to the polar diameter of the reverse micelle, 2rP Therefore, d = 2rp is taken in all the fits. The SAXS data are reasonably well fitted for a given sticky parameter, 7-1 (Figure 4A). Estimations of the polydispersityobtained by considering the high q region are reported in Table 11. According to this table, both r, and u are rather independent of the volume fraction 4 = t$p. On the contrary, the most (29) Seaton, N. A.; Glandt, E.D. J. Chem. Phys. 1986,87,1786. (30)Kranendonk, W. G. T.; Frenkel, D. Mol. Phys. 1988,64,403. (31)Stell, G. J. Stat. Phys. 1991,63, 1203. (32) Hilfiier, R.; Eicke, H. F.; Sager, W.; Steeb, C.; Hofmeier, U. Ber. Bunsen-Ges. Phys. Chem. 1990,94,677.
DO and a are, respectively, the diffusion coefficient at infinite dilution and the second virial coefficient. The latter is'the sum of the well-known second osmotic virial Coefficient and other terms which take into account hydrodynamic effects.33 Assuming that the particles are spherical, the hydrodynamical radius Rh is gven by the Stokes-Einstein expression
Do = kT/6uqRh where k, T,and q are, respectively,the B o l t " constaut, the temperature, and the shear viscosity of the solvent. According to these DQELS experiments,Rh is equal to 86 A. Taking into account the length of the alkyl chains of the AOT, this radius is in good agreementwith the previous determination by SAXS. According to Felderhofs approach33applied to the adhesive sphere model, the second virial coefficient can be related to the sticky parameter. One obtains the following relationship: a = 1.56 - 1.027-l
From the DQELS data we obtain 7-1 = 4.3. This value agrees very well with the value obtained by extrapolating the SAXS curves in the low 4 range (Figure 5). Instead of the adhesivespheremodel,we can consider an equivalent square well model with a standard width of 5 A corre(33)Felderhof, B. U. J. Phys. A: Math. Gem 1978, 11, 929.
Langmuir,Vol. 9,No. 11,1993 2859
Structural Study of AOT Reverse Micelles "I
I
0 - 0 0
10
20
30
40
50
0 (%o) Figure 7. Adhesion coefficient, A, versus volume fraction,4 at 22 O C . AOT reverse micelles w = 40 (open squares),coated silica spheres (open circles), and the discrete solvent model (croeses). sponding approximately to the length of overlap of the AOT chains.= Table I1 shows that the well depth is reduced by a factor of 4 from 4 = 8% to 4 = 39%. The values and the decreaseof T-obtained ' in the present study at w = 40are very closeto those obtained by Robertus and c o - w ~ r k e rat s ~w~=~35 ~ on the same system from a very careful analysis which includes in addition the chain length and the hard sphere diameter as free adjustable parameters. According to these researchers "such intriguingdecreasesof the attraction when increasingvolume fraction can be related to the expulsion of the solvent molecules from the surfactant layer and to the corresponding change in the composition of the interfacial layer". In our view, the nature of such changes is not yet clear because in suspensions with rigid interfaces, one expects that expelled solvent effeds will produce an increaseof the attraction between the soluteparticleswhen the polar volume fraction increases. Such a behavior is effectivelydeduced from the SAXS experiments on silica spherescoated by octadecylchainssuspended in benzene.37 This behavior agreeswith the discrete solvent model where a microemulsion is considered as a mixture of small solvent spheres and large solute s p h e r e ~ . 3According ~*~~ to the PY theory, one findsthat the effectivestickinessbetween hard solute^^^^^ is given by A = W1/[(l - 42)(1- 42+ 24J1
(2)
where 41 and 42 are, respectively, the volume fractions of the solvent and of the solute particles. Such an effective stickiness of purely entropic origin agrees reasonably well with the experimentson silicaspheres but clearlydisagrees with the trends observed in the AOT reverse micelles in isooctane as shown in Figure 7. Therefore, an alternative explanation of the particular attraction versus the polar volume fraction dependence in AOT reverse micelles could be found by considering thermal fluctuations of the micellar surface. According to Helfrich?l the thermal fluctuations in lamellar systems induce repulsiveinteractions,resultingin the mutual steric hindrance of the undulating films. In reverse micelles at a large volume fraction and fixed w,the averagedistancebetween the micelles is considerably (34) Huang, J. S. J. Chem. Phys. 1985,82,481. (35) Robertus, C.; Joosten,J. G.H.;Levine,Y.K.J. Chem.Phys. 1990, 93,7293. (36)Robertus, C.; Jooeten, J. G.H.; Levine, Y. K. Phys. Reo. 1990,42, 4820. (37) de Kruif, C. G.;Rouw, P. W.; Briels, W. J.; Duita, M. H. G.; Vrij, A.; May, R. P. Langmuir 1989,5,422. (38) Vrij, A.; Jamen, J. W.; Dhont, J. K. G.;Pathmamanoharan,C.; Kops-Werkhoven,M. M.; Fijnaut, H. M. Faraday Discuss. Chem. SOC. 19a, 76, 19. (39) Heno, Y.; Regnaut, C. J. Chem. Phys. 1991,95, 9204. (40)Heno, Y.; Regnaut, C. C. R. Acad. Sci. 1992,315,163. (41) Helfrich, W. Z. Naturforsch. 1978,33a, 305.
10
20
30
50
40
9 (%I Figure8. Conductivity, A, of the reverse micellar solution versus volume fraction, 4, at 22 O C (open symbols) and 35 O C (fiied symbols), in the absence (squares)and in the presence of one native a-chymotrypsin per micelle (triangles)and two modified a-chymotrypsins per micelle (circles).
3M)
320
340
wavelength (om)
360
300
320
340
360
wavelength ("In)
Figure 9. (A) Fluorescence spectra of the FMOC-a-chymob rypsin in aqueous (dashed line)and in AOT reverse micelle (full line)solutions. (B)Fluorescence spectra of FMOCClin isooctane (fullline) and aqueous solutions (dashed line).
reduced. This favors the face to face configuration of the large droplets which could be viewed as fluctuating planes as in Helfrich's model. Althoughthe attractive interaction potential is greatly reduced at high volume fractions, the mean distance between droplets is reduced so that the percolation phenomenon effectively occurs even if the interaction is weak.42 As a matter of fact, an increase in the conductivityby severalorders of magnitude is observed at 4 = 40% (Figure 8). 3. Structure of the Reverse Micelles Filled by the a-Chymotrypsin Derivatives. 3.1. Average Location ofthe Enzyme Derivatives in AOT Reverse Micelles. The location of the FMOC-a-chymotrypsin is determined by a comparative analysis of the fluorescence spectra of the FMOCCl reactant and of the FMOC-a-chymotrypsin, In the aqueous solution, the FMOCl and the FMC-achymotrypsin of similar spectra (Figure 9) are identical and characterized by two peaks centered at 305 and 315 nm. In pure isooctane, the FMOCCl spectrum is characterized by one maximum centered at 305 nm and a shoulder at 315 nm. Spectra with the same peak and shoulder are observed for the pure reactant FMOCCl as well as for the FMOC-a-chymotrypsin in the reverse micellar media (Figure 9). Moreover, no changes in the fluorescence spectra were detected by varying w from 10 to 40. Therefore, these fluorescence data indicate that the FMOC-a-chymotrypsin is located at the inner oilwater interface, contrary to the native enzyme which is located at the center of the water pool." 3.2. Comparative Structural Studies of the Unfilled and Filled Reverse Micelles. A t w = 40,the maximum numbers of native a-chymotrypsinand modified a-chymotrypsinper droplet, are, respectivelyone and two. This increase in the average of FMOC-a-chymotrypsin per droplet confirms the affinity of FMOC-a-chymotrypsin for the interface. The SAXS measurements have been performed on the sample microemulsions filled with the native a-chymotrypsin under the same previous volume fraction, temperature, and w values. The results are analyzed following (42) Heyes, D. M. J. Phys.: Condens. Matter 1990,2, 2241.
Pitrk et al.
2860 Langmuir, Vol. 9, No. 11, 1993 Table IV. Values of the Radius, Root-Mean-Square Deviation u, Stickiness 7-l) and Depth of the Intermicellar Potential E (PT units), at Selected Polar Volume Fractions 6 = & , a t w = 40, T = 22 without chymotrypsin
4 (%)
8
radius(&
70 0.2 2.7 -2
d
ll7
EIkT a
23 67 0.18
0.9 -1.1
two FMOC-chymotrypsins per micelle 8 71 0.22 2.7 -2
23 69 0.18 1 -1.2
Reverse micelles filled by two FMOC-a-chymotrypsins.
the model of section 111.1. The comparisonwith the SAXS data on empty micelles clearly shows (Table IV) that no significant change in the water pool radius, in the polydispersity, in the stickness, or in the depth of the intermicellar potential occurs. Thus, the solubilization of a-chymotrypsin in AOT reverse micelles, at w = 40, does not induceany observable change. Such an invariance is also confirmed by the DQELS experiments (Figure 6). In the case of FMOC-a-chymotrypsin, similar investigations have been performed at w = 40and various polar volume fractions. As indicated above one has on average two enzymes per micelle in the case of FMOC-a-chymotrypsin so that the structure of the microemulsion is analyzed under this assumption. Since the solubility of the FMOC-a-chymotrypsin is larger than the solubility of the native enzyme, the structure of the microemulsion has been studied by assuming two enzymes per micelle. Table IV shows clearly that neither the size and the polydispersity nor the stickinessis modified by the change of the location of the enzyme. In addition,we also performed comparativeconductivity measurements: one at 22 OC and another at 35 OC (where the stickiness is larger) on the three systems (Figure 8). The conductivitycurves and the percolation onset are very similar in the three cases. Only a minor reduction of the percolation threshold can be noticed at 35 "C from the unfilled to the filled reverse micelle case, while no change due to the location of the enzyme is apparent. Therefore, the temperature dependence of the intermicellar interaction is almost insensitive to the location of the enzyme.
IV. Conclusion In order to explore surfactant-protein interactions at the reverse micelle interface, SAXSexperimentshave been
performed on microemulsions of AOT reverse micelles in isooctane in the presence of different a-chymotrypsins with different surfactant-enzyme interactions. These experiments have been analyzed in terms of the sticky sphere model which models the intermicellar potential. In AOT reverse micelles such an interaction is mainly characterized by a decrease of the effective stickiness of the droplets as the volume fraction is increased at a constant water to surfactant concentration ratio. This specific decrease which has been equally observed when solvents other than isooctane are ~ s e d cannot ~ ~ pbe~ fully ~ explained by the expelled solvent effect as in suspensions with a more rigid interface such as silica spheres coated with polymersdispersed in benzene.% From the sensitivity of the intermicellar potential in AOT reverse micelles to the micellar interface, one expects that such a potential would also be sensitive to the presence and the location of the enzyme close to the micellar interface. In the case of the native a-chymotrypsin, no modification of this potential is observed from the analysis of SAXS data, nor from the additional information given by the DQELS and conductivity measurements. This behavior and the location at the center of the reverse micellel' indicate that the micellar surface is not perturbed. By binding hydrophobic molecules on the surfaceof the a-chymotrypsin,we forced a-chymotrypsinto interact with the surfaceof the host reverse micelle. SAXS experimenta and conductivity measurements do not suggest that a modification of the intermicellar potential occurs due to the location of the modified a-chymotrypsin, contrary to the case of cytochromec, where the location at the micellar surface produces an increase of the attractions.16 Similar datahave been obtained in our laboratory by using several ribonucleaseu derivatives instead of a-chymotrypsin.The fact that modified a-chymotrypsin does not induce significant changes in the intermicellar potential can be explained by the nature of the surfactant-enzyme interactions. Indeed, the modified enzyme interacts with the micellar interface by means of hydrophobic forces, while the cytochrome c is bound to the reverse micellar surface by means of strong attractive electrostatic forces. Thus, in the case of modified a-chymotrypsin the micellar interface appears as weakly perturbed. (43) Caesin, G.; Pileni, M. P. To be submitted for publication. (44) Michel, F.; Pileni, M. P. Langmuir, in press.
