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Extraction of Cytochrome c in Sodium Dodecylbenzenesulfonate Microemulsions Claude Jolivalt, Michel Minier, and Henri Renon* Centre de Recherche en Proc6d6s de Transformation de la MatiBre, Ecole des Mines de Paris, 60, Boulevard Saint-Michel, F-75006 Paris, France
The partition of cytochrome c between an aqueous phase in equilibrium with a microemulsion composed of sodium dodecylbenzenesulfonate, butanol, and decane was studied. By adjusting the pH and the ionic strength in the aqueous phase, cytochrome c can be quantitatively extracted in the microemulsion as long as the protein concentration in the organic phase is less than the initial concentration of micelles. Above this limit, an intermediate phase, containing part surfactant and part cytochrome c, is located between the aqueous and microemulsion phases. The role of electrostatic interactions in the protein partitioning is shown by varying the composition of the phases (pH, salinity, surfactant, and cosurfactant concentrations) and then discussed. Extraction depends on the relative values of the pH of the aqueous phase and the p l of cytochrome c, the size of the micelles, and the protonation of acidic residues. Cytochrome c can be recovered in an aqueous phase by adding butanol in the microemulsion phase. In such conditions, cytochrome c retains 90% of its initial activity, as measured before the extraction step.
Introduction Extraction in reversed micelles is a new method for the recovery of proteins from an aqueous solution. It is a twophase separation method based on the same principles as classical liquid-liquid extraction used industrially for metallic ions or antibiotics. The difficulty in applying this method to protein solutions comes from the lability of these molecules, which are denaturated by organic solvents. For these reasons, reversed micelles provide a favorable environment: on a macroscopic scale, proteins are solubilized in an organic phase, but on a local scale, they are expected to be located in the aqueous core of the micelles: they interact with the shell of charged surfactant and are preserved from denaturation. Up to now, most of the reversed micellar systems investigated involved ionic surfactants. Luisi’s group studied AOT microemulsions, with special attention given to the activity of the enzymes, both when solubilized in the aqueous core of the micelles (Barbaric and Luisi, 1981; Luthi and Luisi, 1984)and after recovery in a bulk aqueous phase (Leser et al., 1986). Hatton and co-workers also studied mostly AOT microemulsions. They have shown that lysozyme, cytochrome c, and ribonuclease A can be separated on the basis of their p l (Goklen and Hatton, 1987). Woll et al. (1989) first developed the concept of bioaffinity, adding a cosurfactant, octyl 8-D-glucopyranoside, to the microemulsion. In such conditions, the partitioning of concanavalin A, which is known to form a specific complex with this molecule, is much enhanced, while the extraction of other proteins is not affected by the modification of the composition of the organic phase. Increased selectivity for avidin extraction was also achieved by using a biotinylated cosurfactant (Coughlin and Baclaski, 1990). Recently, Paradkar and Dordick (1991) achieved the extraction of glycoproteins bound to concanavalin A. Dekker e t al. (1986,1989,1990)studied the extraction of a-amylase in a micellar system formed with methyl-
* Author to whom correspondence should be addressed. 8756-7938/93/3009-0456$04.00/0
trioctylammonium chloride, a cationic surfactant, in octane/octanol. The authors focused on the extraction process of the protein and modeled the mass transfer. The interpretation of the experimental results of extraction is based on some very general concepts, such as the electrostatic interactions between the surfactant and the protein inside the micelle or the exclusion of the protein occurring when the size of the micelle is smaller than that of the protein. Some attempts to quantify these effects have been published recently (Rahaman and Hatton, 1991; Bruno e t al., 1990; Bratko et al., 1988). It seems that no specific interaction between proteins and AOT is involved. The primary purpose of this work is to provide experimental data for the extraction of proteins in reversed micelles formed with an anionic surfactant different from AOT, a sodium alkylbenzenesulfonate (SDBS),which could be used at a technical grade in order to extend the possible applications of reversed micelles to protein extraction.
Experimental Section Chemicals. Sodium dodecylbenzenesulfonate (SDBS), n-decane (quality purum, purity >98 5% 1, benzethonium chloride (Hyamine 1622,purity >98%),dimidium bromide (purity >95%), and disulfine blue were obtained from Fluka and used as received. Although nominally with a C12 alkyl chain, SDBS was composed of a mixture of species with different alkyl chain lengths, C12 being the most abundant. SDBS also contained 17wt 5% NazS04, titrated by a lead acetate precipitation method. n-Butanol was obtained from Sigma (purity >99 5% ) and was used without further purification. Horse heart cytochrome c (type VI) was obtained from Sigma. BCA from Pierce was used for protein assay. Extraction Procedure. The organic phase was prepared in a 15-mL glass tube by weighing SDBS, butanol, and decane. Then an aqueous solution of cytochrome c was added, the pH of which was fixed by adding an appropriate volume of concentrated HC1 or NaOH solu-
0 1993 American Chemical Society and Amerlcan Instltute of Chemical Engineers
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Table I. Typical Winsor I1 System for the Extraction of Cytochrome c components
SDBS butanol decane water and salts total
initial composition 0.3 g crude powder 0.6 g 3.4 g 5.67 g [NaCl] = 1 mol/dma 10 g
microemulsion composition at equilibrium 0.117 mol/dm3 9.4 vol % 80.1 vol % 63.5 g/dm3 [ci-I = 0.52 m0i/dm3 in the water pool V = 5.6 cma
tion, taking into account the influence of the sodium salt initially present in the crude surfactant solid. The volumes of both aqueous and organic initial phases were chosen so that at equilibrium the volumes of both phases should be equivalent (around 5.5 mL). The mixture was stirred on an orbital table at 250 rpm for 15 min at 25 “C and then centrifuged for 10 min at lo00 rpm, at the same temperature, in order to accelerate the separation of the phases. The final pH of the aqueous phase was measured. Titration. The protein concentration was estimated spectrophotometrically using an extinction coefficient of 88 600 M-’ cm-l in the aqueous phases and 78 000 M-’ cm-1 in the microemulsion phase at 408 nm for the oxidized form. Absorption spectra were recorded on a HewlettPackard 8452A diode array spectrophotometer. The activity of cytochrome c was estimated by its capability to be reduced by a sodium ascorbate solution at pH 7 in a 0.1 M phosphate buffer. The concentration of ascorbate was always at least 100 times that of the protein. The addition of 10 vol 7% ethanol to the microemulsion produced phase splitting. The upper phase contained decane, ethanol, and butanol, whereas the lower phase was a complex mixture of alcohol, surfactant, water, and all cytochrome c. It was then possible to measure the cytochrome c concentration in the lower phase using a colorimetric method derived from Biuret (BCA titration kit). The volume of the aqueous phase was derived from the concentration of SDBS obtained by titration, and the concentration of cytochrome c in the microemulsion was obtained by mass balance. That method was used to confirm the results obtained by spectrophotometry. The SDBS concentration, both in the microemulsion and in the aqueous phase, was determined by titration with benzethonium chloride in a biphasic system: SDBS forms a pink chloroform-soluble complex with the dimidium bromide. When benzethonium chloride is added, this equilibrium is shifted toward the formation of a complex benzethonium/dodecylbenzenesulfonate. Chloroform solution becomes less and less colored. After the equivalent point has been reached, the benzethonium chloride forms a blue, water-solublecomplex with disulfiie blue. Crude surfactant from Fluka contains 83 w t 95 alkylbenzenesulfonate, as determined by this method. Decane in the microemulsion and butanol in both phases were determined by gas chromatography. Chloride titration in both phases was performed on a Metrohm potentiograph using a silver electrode. Water in the organicphase was measured by a coulometricmethod (after Karl Fisher) using a titrator Tacussel *Aquaprocesseur”.
Results and Discussion The composition of the extraction system was chosen in order to obtain a biphasic equilibrium between a water in oil (W/O)microemulsionand an aqueous phase in excess, that is, a Winsor I1 system. Much work (Kahler et al., 1983; Lascaux et al., 1983; Baker et al., 1984; Guest and Langevin, 1986; Bahri et al.,1987;Ashan et al., 1991) has been published on alkylbenzenesulfonatessince Schechter
excess aqueous phase composition at equilibrium
4 2 0
10
I
8
11
9 PH
Winsor 14
lo pl cytochrome c
\
-
0
\
12
Figure 2. Influence of pH on cytochrome c extraction. Initial aqueous phase: [KCl] = 0.82 M1tcytochrome cl = 18.2 pM. Microemulsion composition at equilibrium: [SDBS] = 0.12 M/[butanol] = 9.2 vol %/decane/[HzOl = 65 g/L. of the micelles, which could lead to the phase splitting observed, while the concentration of micelles remains very close to that calculated from eq 2 in a protein-free system. Effect of pH. Cytochrome c is a 12.4-kDa molecule containing 12 acidic and 24 basic residues, with a rather high isoelectric point (around 10.6). When the pH is changed, the overall charge of the protein, determined by the protonation of its residues, is modified as are electrostatic interactions with negatively charged SDBS. Extraction of cytochrome c in microemulsions is driven directly by these interactions as shown in Figure 2. Experimental assays were carried out with an initial cytochrome c concentration of 18.2 pM in the aqueous phase, which prevented any problem of phase splitting or precipitation, as see in the previous section. At a pH higher than the PI, repulsive electrostatic interactions prevented the extraction of the protein. Between the PIand pH 8, the protonation of some of the basic residues at the protein surface allowed a partial extraction. When the pH was less than 8, cytochrome c was quantitatively solubilized in the microemulsion. From these experimental resulh, it can be deduced, following conclusions of Kelley et al. (1991)in AOTmicroemulsion, that the extraction is driven by electrostatic interactions between the protein and the surfactant. Effect of Salt Concentration, The ionic strength of the aqueous phase is another parameter which influences the extraction of cytochrome c. The ionic strength was adjusted by varying the KCl concentration in the initial aqueous phase. The lower limit was set by the transition from a Winsor I1 to a Winsor I11microemulsion ([KCl] = 0.54 M). The upper limit was [KCl] = 2.45 M. Above this concentration, the decrease of the protein solubility induces precipitation from the aqueous phase. The initial aqueous phase contained only potassium chloride, but sodium sulfate brought by the crude surfactant increased the pH, which had to be controlled at a value of 9.6 in the aqueous excess phase at equilibrium. In these conditions, at low KC1 concentration (around 0.6 M but above the WII/WIII transition limit), cytochrome c was completely extracted (Figure 3). The increase in KC1 concentration led to the decrease of cytochrome c concentration in the microemulsion. A t [KC11 = 2.2 M, no cytochrome c was extracted. All of the protein initially in the aqueous phase remained in the excess aqueous phase, without precipitation. A decrease of the water concentration in the micellar phase accompanies the increase of KC1 concentration (Figure 4). As seen above, the water concentration in the micellar phase depends on the cation type (Figure 4). At a given salt concentration, the microemulsion contains more water
10 0.5
1
1.5
2
Initial chloride concentration in the aqueous phase. Y
2.5
Figure 4. Influence of chloride and associate cation concentration on water concentrationin the microemulsion: (H) NaC1; ( 0 )KCl. Microemulsion composition at equilibrium: [SDBS] = 0.12 M/butanol concentration varies with KC1 concentration from 7 to 10 vol %/decane. Aqueous phase at equilibrium: pH 9.6.
when formed with NaCl than with KCl. As a consequence, micelles are larger when the counterion is sodium rather than potassium, at a given salt concentration. If we suppose that the transition from Winsor I1 to Winsor I11 takes place when the radius of the micelles reaches a critical value, rc*,this result explains why the WII/WIII transition is observed at a higher salt concentration for NaCl than for KC1. According to eq 1, r, varies from 67 to 23 A when the KC1 concentration increases from 0.54 to 2.2 M. A t [KCll= 2.2 M, one protein volume is still only 50% of the volume of the water core of one empty micelle [the radius of cytochrome c is around 18 A (Sheu et al., 198611. The size exclusion effect as developed by Goklen (1986) for alcohol dehydrogenase in AOT microemulsions cannot provide, in this case, a suitable explanation for the nonextraction of cytochrome c: empty micelles are large enough compared to the protein size, even a t high ionic strength, to host one cytochrome c molecule in their water core. Studies of KCl concentration effects were also achieved at lower pH. A t pH values below 6, cytochrome c was totally extracted regardless of the salt concentration (in the Winsor I1 field), which confirms the theory that the steric hindrance is not the relevant effect when changing KC1 concentration.
Biotechnol. hog., 1993, Vol. 9, No. 5
400 Cylochrome c extraction. percent
Chlo e concentration in the r a t e r pool. M
9 T
2'52
T
1.5
0. 0
5
05
1 1.5 2 2.5 Chloride Concentration in the aqueous phase, M
15 20 25 Butanol In microemulsion, percent volume
30
3
Figure 5. Distribution of chloride ions between micellar water pools and the aqueous phase:).( NaC1; ( 0 )KC1. Microemulsion composition at equilibrium: [SDBS] = 0.12 M/[butanol] = 9 vol %/decane. Aqueous phase at equilibrium: pH 9.6. The PKa values of the acidic residues on a polypeptidic chain are around 4 (Stryer, 1981),but it was reported that deprotonation may be hindered inside the reversed micelles (Bardez, 1987). The pKacould be higher inside the micelles than in the excess phase. Following that hypothesis, it can be assumed that, when the pH in the excess aqueous phase was around 5, the acidic residues of cytochrome c (mostly aspartic and glutamic) were still protonated. The only charges on the protein would be on basic, positive residues, and the local electrostatic interactions with the surfactant molecules should favor extraction. In such conditions, cytochrome c could be extracted, even at high ionic strength. At pH values between 5 and 9, cytochrome c is still positively charged overall: 19 lysine residues are distributed on the protein surface (Vos et al., 19871, and 9 of the 12 acidic residues are clustered; the local repulsive interactions between the negative cluster and the surfactant shell tend to prevent extraction. Cytochrome c is only partly solubilized when micelles are large enough that distance attenuates repulsive interactions. The enhancement of the screening effect of the electrostatic interactions can also be due to the increase of the ionic species concentration in the micelles. Titration of both water and chloride ions in the microemulsion yields an evaluation of the concentration of chloride ions in the micellar water pool, as shown in Figure 5. When the KC1 or NaCl concentration is increased in the initial aqueous phase, the concentration of chloride ions in the water pool of the micelles increases. But chloride concentration inside the micelles was about one-half that in the bulk phase, with this proportion remaining constant in the whole range of KC1concentrations studied. According to Leodidis and Hatton (1989), chloride ions are excluded from micelles because of their size. Neither Na+ nor K+was determined in this work but their concentrations are likely to vary as the chloride concentration, leading to the assumption that the ionic strength inside the micelles increases when the salt concentration increases in the bulk aqueous phase. Effect of Butanol Concentration. The overall proportion of butanol in the system was varied from 4.8 to 17 vol % The pH of the aqueous phase was adjusted a t pH 7 by adding concentrated HC1. Two NaCl concentrations were studied 0.92 and 1 M. The increase of the total concentration of butanol in the system led to an increase of the butanol concentration in the organic phase from 5.2 to 28 vol %. Figure 6 represents the percentage of extracted cytochrome c as a function of the butanol concentration in the microemulsion. For [NaClI = 0.92 M, no influence of butanol concentration was found: all of the protein initially in the aqueous phase was extracted. For [NaClI = 1 M in the aqueous phase, the extraction
.
10
Figure 6. Influenceof butanol on cytochromec extraction.Phase compositions at equilibrium: microemulsion, [SDBS] = 0.12 M/decane; aqueousphase,pH 7;( 0 )[NaCl] = 0.92M, (m) [NaCl] = 1 M. of cytochrome c decreased from 100% for less than 15 vol % butanol to only 50% for 28 vol % butanol. Direct measurements showed that the water concentration decreases when the butanol concentration increases until the butanol concentration is 20 vol % in the organic phase. Above this limit, the opposite tendency isobserved. According to the geometric model (eq l),the minimal radius for the micelles with increasing butanol concentration would be around 35 A for [NaClI = 1M. SDBS micelles remained larger than the protein, which could explain why no steric effect was found at any butanol concentration. Electrostatic interactions, which were shown to be predominant in the extraction of cytochrome c, depend on both the global composition of the micellar core and its geometric features. The determination of the butanol concentration in the interfacial area, followingthe method developed by Guest and Langevin (1986), shows that an increase in the butanol proportion in the organic phase induces an increase in the molar ratio of butanol/SDBS for up to 20 butanol molecules per SDBS molecule for 28 vol % butanol in the organic phase. In such proportions, the cohesion of the interfacial film is supposed to fail. Because one of the known functions of alcohol molecules in microemulsionsis to decrease the rigidity of the interface (De Gennes and Taupin, 1982), the micelle boundaries become very imprecise,disturbing the interactions between the ionic species. The decrease in cytochrome c extraction at [NaClI = 1 M in the aqueous phase, with increasing butanol concentration, could thus be a direct consequence of the screening effect of the butanol molecules in the interfacial area. However, complementary experimental measurements could help to explain why no influence was found at [NaCll = 0.92 M. The titration of chloride ions in the water pools showed that their concentration decreased from 0.75 to 0.25 M as the butanol concentration increased in the . agreement with the microemulsion from 5 to 25 ~ 0 1 % In salt concentration dependence proposed in the previous section, it seems that this exclusion of chloride ions counterbalances the butanol destructuration effect at 0.92 M NaCl concentration to keep protein extraction complete. The decrease of the solubilization of cytochrome c in microemulsion while increasing the butanol proportion was used to perform the desextraction of the protein. Typically, 3 mL of a microemulsioncontaining cytochrome c was diluted with 1mL of butanol and contacted with a fresh aqueous solution at pH 7 containing 2 M NaC1, so that a Winsor I1 microemulsion should be formed at equilibrium. In such conditions, cytochrome c was quantitatively recovered in the aqueous phase within less than
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15 min and retained 90% of its activity, compared to the initial protein solution before the extraction step.
Dekker, M.; Van't Riet, K.; Weijers, S. R. Enzyme recovery by liquid-liquid extraction using reversed micelles. Chem. Eng.
Conclusion
Dekker, M.; Van't Riet, K.; Bijsterbosch, B. H.; Wolbert, R. B. G.; Hilhorst, R. Modeling and optimization of the reversed micellar extraction of a-amylase. AIChE J. 1989,35(2),321-
It was shown that a SDBS microemulsion could be an appropriate micellar system for the extraction of cytochrome c. The extraction performance and the microscopic structure of the microemulsionare closely correlated since partition studies have shown that cytochrome c can be quantitatively extracted in the micelles, as long as the protein concentration in the microemulsion is less than the concentration of the micelles themselves. From pH and ionic strength studies, it was demonstrated that electrostatic interactions are the main driving forces of the solubilization process. When the size of the micelles is large enough, the repulsive electrostatic interactions between deprotonated carboxylic groups on the surface of the protein and the anionic surfactant can be attenuated by a shell of water molecules, allowing quantitative extraction at pH values lower than the PI. When the micelles are small, i.e., at high ionic strength, cytochrome c can be extracted when the pH is low enough for the acidic residues of the molecule to be protonated. The size of the micelles,calculated from a rough geometrical model, was always larger than the size of the protein, up to the limits of the accessible experimental conditions within a Winsor I1 domain. Other investigations are needed before extraction in SDBS microemulsions can be proposed as a possible candidate for industrial application, particularly the application of the system to the selective extraction of a wide set of proteins, which is now underway in this laboratory. Literature Cited Ashan, T.; Aveyard, R.; Binks, B. P. Winsor transitions and interfacial film compositions in systems containing sodium dodecylbenzenesulphonateand alkanols. Colloids Surf. 1991, 52,339-352.
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Baker, R. C.; Florence, A. T.; Tadros, T. F.; Wood, R. M. Investigations into the formation and characterization of microemulsions. I. Phase diagrams of the ternary system water-sodium alkylbenzenesulfonate-hexanol and the quaternary system water-xylene-sodium alkylbenzenesulfonatehexanol. J. Colloid Interface Sci. 1984,100(2),311-331. Barbaric, S.;Luisi, P. L. Micellar solubilization of biopolymers in organic solvents. 5. Activity and conformation of a chymotrypsin in isooctane-AOT reverse micelles. J. Am. Chem. SOC.1981,103,4239-4244.
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Kelley, B. D.; Rahaman, R. S.; Hatton, T. A. Salt and surfactant effects on protein solubilization in AOT-isooctane reversed micelles. In Organized assemblies in chemical analysis. Vol. 1: Reuersed micelles; Hinze,W. L.,Ed.; JAI Press: Greenwich, CT, 1991. Lascaux, M. P.;Dusart, 0.; Granet, R.; Piekarski, S. Propribtes superficiellesd'hexadecylbenzene sulfonates de sodium ramifies & l'interface eau-air. J. Chim. Phys. 1983,80(7-8),5-10, Leodidis, E. B.; Hatton, T. A. Specific ion effects in electrical double layers: selective solubilization of cations in AOT reversed micelles. Langmuir 1989,5,741-753. Leser,M.; Wei, G.; Luisi,P. L.; Maestro, M. Application of reverse micelles for extraction of proteins. Biochem. Biophys. Res. Commun. 1986,135 (2),629-635.
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Accepted May 18,1993.
