Minimal AOT Concentration of Binary Protein Component in Reverse Micellar Extraction

0960±3085/97/$10.00+0.00 q Institution of Chemical Engineers

MINIMAL AOT CONCENTRATION OF BINARY PROTEIN COMPONENT IN REVERSE MICELLAR EXTRACTION K. NAOE, M. TAMAI, M. KAWAGOE, M. IMAI* and M. SHIMIZU* Department of Chemical Engineering, Nara National College of Technology, Nara, Japan, *Department of Chemical Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan

T

he minimal AOT concentration of a binary protein mixture containing cytochrome c and lysozyme was investigated, and was found to be smaller than the sum of the minimal AOT concentration of each protein obtained in single-protein systems, whatever the lysozyme molar fraction in the mixture. The water content, W0 (= the water/amphiphile molar ratio), of the reverse micellar organic phase under the binary minimal AOT concentration became maximum at a lysozyme molar fraction of 0.5. The apparent interfacial area per single head group of AOT in the binary protein system, estimated from viscosity data, was greater than that of a protein-free system, and became maximum at a lysozyme molar fraction of 0.5. The interfacial tension between the protein mixture aqueous solution and AOT/isooctane solution was lower than that for each single-protein aqueous solution. The binary protein mixture containing cytochrome c and lysozyme thus exhibited higher amphiphilic characteristics. These results indicated that the proteins behaved like amphiphilic molecules, especially when both of the proteins coexisted in the reverse micellar phase. Keywords: extraction; reverse micelle; cytochrome c; lysozyme; AOT; minimal AOT concentration

INTRODUCTION

In particular for low-molecular-weight protein, the electrostatic interaction is a dominant factor in the protein solubilization behaviour. Low-molecular-weight proteins similar in the electrostatic properties of their surface can be simultaneously solubilized into reverse micelles under a favourable aqueous phase condition of pH, ionic strength, and salt types. In the present study, the minimal AOT concentration of a binary protein mixture containing lysozyme and cytochrome c in various proportions, which is intended to serve as a model protein mixture is investigated. The solubilization state of the binary protein components in the reverse micelles is discussed with respect to water content, viscosity of the reverse micellar phase, and interfacial tension.

In practical protein separation in the bioindustry, the target protein is generally puri® ed from a complex feed system such as a fermentation broth or microorganism culture. To develop protein separation techniques further, it is important to determine the most suitable operating conditions for separation from complex feed systems. In reverse micellar extraction processes, several operating parameters, including the pH, ionic strength, amphiphile concentration, and types of the organic solvent, have an effect on the behaviour of protein solubilization into the reverse micellar organic phase4,7,9,10. In particular, the amphiphile concentration in the micellar organic phase plays an important role in forming reverse micelles that solubilize the host protein. In an aerosol-OT (AOT)/ isooctane reverse micellar system, the minimal AOT concentrations needed for the forward extraction of cytochrome c and lysozyme were separately determined by Ichikawa et al.7 and Naoe et al.10 , respectively. The minimal AOT concentration means the minimum amount of AOT required for the complete forward extraction of the protein. Natsume et al. reported that the minimal AOT concentration depends on the protein species11 . Since, under minimal AOT conditions, a linear correlation has been observed between the solubilizing water and the extracted protein7 , the minimal AOT concentration is important not only for the stoichiometry in the practical design of reverse micellar extraction processes, but also for investigating the solubilization behaviour of proteins in reverse micelles. Knowing the minimal AOT concentration in a multiprotein component system is necessary for practical extraction from feed solutions containing various proteins.

EXPERIMENTAL An AOT/isooctane reverse micellar system was used. Aerosol-OT (AOT) (di-2-ethylhexyl sodium sulphosuccinate; C20 H37 O7 Na) was obtained from Nacalai Tesque, Inc (Kyoto, Japan), and isooctane (2,2,4-trimethylpentane) from Wako Pure Chemical Industries Ltd (Osaka, Japan). They were used without further puri® cation. Lysozyme and cytochrome c were used as model proteins. Lysozyme from chicken egg white (MW = 14300, pI = 11.2) and cytochrome c from horse heart (MW = 12400, pI = 10.1) were obtained from Sigma Chemical Co. (St Louis, MO). The total protein concentration of each feed protein mixture (lysozyme and cytochrome c) was ® xed at 2 ´ 10- 4 M, in which the molar fraction of lysozyme was zero, 0.25, 0.50, 0.75, or 1.0. The forward extraction step 143

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was carried out by mixing an equal volume of reverse micellar solution with 0.1 M KCl aqueous solution containing the protein at pH 8.5. The concentration of cytochrome c was determined by UV absorbance at 409.5 nm in the aqueous solution and at 410 nm in the reverse micellar organic solution. Quantitative analysis of each protein component in the aqueous solution was performed by HPLC. A Shimadzu Shim-pack HPC-C2 column was employed on a Shimadzu A-6 chromatography system. The UV detection was done at 280 nm. A solution of 1.60 M (NH4 )2 SO4 in 0.1 M phosphate buffer (pH 7.0) was used as a carrier. The ¯ ow rate was maintained at 0.5 ml/min. The minimal AOT concentration of the binary protein test mixture was de® ned in the following way. The concentration of cytochrome c in the reverse micellar organic phase was measured from the absorbance at 410 nm and the raf® nate aqueous phase was analysed by HPLC. The minimal AOT concentration for the binary protein mixture was de® ned as the AOT concentration when complete forward extraction of cytochrome c was achieved without sediment at the W/O interface, and no HPLC signal for lysozyme was observed in the raf® nate aqueous phase. The water content of the reverse micellar organic phase was measured by Karl-Fisher titration. The viscosity and density of reverse micellar solution were determined with an Ubbelohde viscometer and a pychnometer, respectively. Viscosity data were analysed by the method of Day et al.3 , in which it is assumed (1) reverse micelles are spherical in shape, (2) AOT molecules in reverse micelles form a monolayer (the length of the hydrophobic part of AOT = 0.9 nm), and (3) the density of the solubilizing water in the reverse micelle is the same as that of the bulk water. According to Day et al.3 , the volume fraction of the solvent taken up by reverse micelle is shown,

w = (N[AOT ]Vm )/ (1000n) (1) where V m is the volume of a reverse micelle, n is the average aggregation number (number of AOT molecules per micelle) and N is Avogadro’ s number. Total micellar volume, Vm , is made up of a contribution from the water pool (VW ) and the AOT coat (V S ), V m = VW + VS and VW = SvH2 O nW0 (2)

RESULTS AND DISCUSSION Minimal AOT Concentration of Binary Protein Mixture The minimal AOT concentrations in single-protein component systems with cytochrome c and lysozyme were previously determined by Ichikawa et al.7 and Naoe et al.10, respectively. The calculated minimal AOT concentration of each corresponding protein fraction in the present work could thus be obtained from the correlative relationship determined for the two proteins. The calculated binary minimal AOT concentration is the sum of the minimal concentration of each single protein component system. Figure 1 shows the experimental and calculated binary minimal AOT concentrations at each lysozyme molar fraction in the protein mixture. The binary minimal AOT concentrations obtained experimentally were clearly smaller than the calculated values at all lysozyme molar fractions (see Figure 1). In particular, at a lysozyme molar fraction of 0.5, the experimental value was only about half that of the calculated one, indicating that in practice, a protein mixture could be extracted at a considerably smaller

Figure 1. Binary minimal AOT concentrations of a binary protein mixture containing cytochrome c and lysozyme. Total protein concentration = 2 ´ 10- 4 M. CKCl = 0.1, pH 8.5. The calculated data are the sums of the minimal AOT concentrations for the individual proteins obtained by Ichikawa et al.7 and Naoe et al.10 .

where SvH2 O is the speci® c volume of a water molecule in the micelle and W0 is the molar ratio of water to AOT. The outer micellar radius, rm , and the radius of the water pool, rW ; are represented as follows, rm

= rW + l and rW = (3VW / 4p

Vm

= (4p

)1/ 3

(3)

where l is the thickness of AOT coat (l = 0.9 nm). Hence, / 3)([3SvH2 OnW0 / 4p ]1/ 3 + 1)3

(4)

Using equations (1) and (4), the estimation of n was computed, and the apparent interfacial area per single head group of AOT, SAOT , was obtained using the following equation: SAOT

= (4p

2 rW )/ n

(5)

The interfacial tension between the protein aqueous solution and the AOT/isooctane organic solution was measured by the method of Harkins and Brown5 . All experiments were carried out at 298 K.

Figure 2. W0 values of reverse micellar organic phase containing binary protein mixture (cytochrome c/lysozyme) at binary minimal AOT concentrations. The binary minimal AOT concentration at each lysozyme molar fraction are summarized in Table 1.

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Table 1. Physical properties of AOT reverse micellar organic phase in protein-free system. Measured point a,b A AOT conc.b [mM] W 0 [- ] g sp [- ] SAOT [nm2 ]

25.9 12.0 0.0466 0.515

B

C

18.0 12.0 10.4 9.75 0.0285 0.0164 0.490 0.431

D

E

15.0 10.3 0.0238 0.496

20.1 11.4 0.0249 0.490

a

The marked plots in Figures 2, 5, and 6. They are prepared as the same AOT concentrations as the binary minimal AOT concentration at the marked measured points A±E. Forward extraction conditions: pH 8.5; KC1 conc. = 0.1 M. b

AOT concentration than that calculated on the basis of the minimal AOT concentrations of single-protein component systems. Water Content of Reverse Micellar Organic Phase Containing a Binary Protein Mixture Figure 2 shows the W0 value of the reverse micellar organic phase under the binary minimal AOT condition at each protein composition. The W0 value is the molar ratio of water to amphiphile. The W0 values experimentally obtained in the protein-free system are shown for a comparison in Table 1. The term `protein-free’ means the blank extraction experiment without containing protein mixture at the same concentration corresponding to the minimal AOT concentrations for each lysozyme molar fraction. The W0 value in protein-mixture-containing system is larger than that of protein-free system. The W0 value reached a maximum at a lysozyme molar fraction of 0.5. On the other hand, in the protein-free system, W0 value was almost constant with respect to the molar fraction of lysozyme changed. From small angle X-ray scattering (SAXS) measurements, the characteristic radius (rW ) of the water pool of reverse micelles in an AOT/isooctane/water system has been reported to be proportional to W0 , represented as rW = 0.15 W 0 13 . Thus, as the size of the reverse micelles increases, the concentration of discrete micelles decreases,

Figure 3. Water accompanying extracted protein mixture into the reverse micellar organic phase under the minimal AOT concentration at each lysozyme molar fraction. The binary minimal AOT concentration at each lysozyme molar fraction are summarized in Table 1.

Trans IChemE, Vol 75, Part C, June 1997

Figure 4. Water concentration of reverse micellar organic phase under the minimal AOT concentration at each lysozyme molar fraction. The binary minimal AOT concentration at each lysozyme molar fraction are summarized in Table 1.

and the W0 value becomes higher. Our results therefore indicate that solubilization of a binary protein mixture is especially inducible of an increase in micellar size. Zampieri et al.14 investigated the size of reverse micelles containing single-component proteins by ultracentrifugation, and found that the micellar solution containing protein consisted of larger protein-containing micelles and smaller un® lled micelles. On the other hand, Kuboi et al.8 , using dynamic light-scattering measurements, reported that the size of reverse micelles containing a -chymotrypsin was the same as that of protein-free micelles, although they attributed this result to the small fraction of protein-® lled micelles (<10%). Since, under the minimal AOT condition, protein-® lled micelles comprise the largest fraction of reverse micelles, the characteristics of the reverse micellar organic phase at minimal AOT concentration will mainly re¯ ect the behaviour of protein-® lled micelles. Figure 3 shows the accompanying water of the mixed protein system under the binary minimal AOT condition. The accompanying water is the number of water molecules accompanying one protein molecule solubilized in the reverse micellar phase, (CWP -CW )/ CPO , where (CWP -CW ) is the difference in the concentration of solubilizing water between the protein-containing and the protein-free systems7 . The number of accompanying water molecules in the binary system was larger than that in the single protein systems11. The maximum value of accompanying water occurred at a lysozyme molar fraction of 0.5. The simple addition of the accompanying water values for the separate lysozyme and the cytochrome c systems was not valid. On the other hand, as shown in Figure 4, no signi® cant change in the water concentration of the reverse micellar phase was observed over the whole lysozyme molar fraction range even though each binary minimal AOT concentration was different. These results regarding water content imply that both the protein components are located on the inner side of the interface of reverse micelles, especially when the molar ratio of lysozyme to cytochrome c is unity. Pileni and co-workers, who investigated the location of proteins in reverse micelles by pulse radiolysis12 and SAXS2,12,13, reported cytochrome c to be located on the inner side of the interface of reverse micelles. Natsume et al.11 investigated the accompanying water in several protein extractions, and

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NAOE et al. Table 2. Relative interfacial tensions of protein-containing system to protein-free in AOT/isooctane solution. Relative interfacial tension r / r AOT conc. [M] 0 1.0 ´ 10- 5

0

[- ]

Lysozyme

Cytochrome c

Mixture (lyso:cyto = 1:1)

0.93 0.93

0.86 0.87

0.86 0.83

Protein conc. in aqueous phase = 1.0 ´ 10- 5 M. r 0 = interfacial tension between water and AOT/isooctane solution in protein-free system.

Figure 5. Speci® c viscosity of reverse micellar organic phase under the binary minimal AOT concentration at each lysozyme molar fraction. The binary minimal AOT concentration at each lysozyme molar fraction are summarized in Table 1.

concluded that the accompanying water was induced by the location of the protein at the micellar interface. The above ® ndings thus support the implication of this study’ s results that the binary protein mixture is located on the inner side of the interface of reverse micelles. Viscosity of Reverse Micellar Organic Phase Containing a Binary Protein Mixture Viscosity measurement can deepen our understanding of micellar systems. The volume of the water pool of reverse micelles, as well as the length of the surfactant tail, can be determined using viscometry, which yields the apparent interfacial area per single head group of amphiphile in a reverse micelle3 . The speci® c viscosity, g sp , of the reverse micellar phase containing the binary protein components is shown in Figure 5. The binary protein components were solubilized under the binary minimal AOT conditions. There were no obvious changes in the speci® c viscosity at any of the lysozyme molar fractions. The speci® c viscosity in the protein-free system was shown in Table 1. The speci® c viscosity in the protein-mixture-containing system was larger than that of the protein-free system in Table 1.

Figure 6. Apparent interfacial area per single head group of the AOT molecule under the binary minimal AOT concentration at each lysozyme molar fraction. The binary minimal AOT concentration at each lysozyme molar fraction are summarized in Table 1.

According to Day et al.3 , the apparent interfacial area per single polar head group of AOT can be estimated using viscosity data. Figure 6 shows the apparent interfacial area per single head group of AOT at each lysozyme molar fraction. The apparent interfacial area was increased by the solubilization of the protein, and became maximum at a lysozyme molar fraction of 0.5. In the protein-free system, no signi® cant change in the apparent interfacial area was observed (see Table 1). It is considered that the W/O interface of the reverse micelles was occupied by an amphiphilic component(s) other than the AOT molecule, e.g., the protein mixture, because the actual interfacial area per single head group of AOT is constant. This suggests that solubilization of the protein mixture induced an increase in the interfacial area of the reverse micelles, especially when both proteins coexisted in the reverse micellar phase. Interfacial Tension Between Protein Mixture and AOT/Isooctane Solutions The interfacial tensions between protein aqueous solutions and AOT/isooctane solution were measured. Protein components have an amphiphilic characteristic that decreases interfacial tension. As shown in Table 2, the protein solutions had lower interfacial tensions than that of a protein-free solution. In the presence of AOT, the amphiphilic character of protein mixture was larger than that of each single protein, and in the single protein system, the amphiphilic character of cytochrome c was larger than that of lysozyme. The binary protein mixture with AOT had the most amphiphilic character among the fractions examined, especially at lysozyme:cytochrome c = 1:1. On the other hand, in the absence of AOT, the protein mixture showed the same amphiphilic character as that of cytochrome c, and cytochrome c was more amphiphilic than lysozyme. These results indicate that cytochrome c has a high amphiphilic character to behave like a cosurfactant, and that the decrease in the interfacial tension might be caused synergistically by the intermediate complex between the binary protein and AOT at the oil-water interfaces not the binary protein alone. The location of solubilized proteins in AOT reverse micelles has been investigated by SAXS1,2,12,13 and pulse radiolysis12 , from which it was concluded that cytochrome c is solubilized on the inner side of the interface of reverse micelles. Judging from the larger water uptake by protein extraction in the reverse micellar organic phase, similar behaviour can be considered in the case of lysozyme11 . This study’ s results on interfacial tension agree well with previous reports1,2,12,13 . Trans IChemE, Vol 75, Part C, June 1997

MINIMAL AOT CONCENTRATION OF BINARY PROTEIN COMPONENT CONCLUSIO N The minimal AOT concentration in a binary protein (lysozyme/cytochrome c) component system was investigated. The binary minimal AOT concentration was smaller than the sum of the minimal AOT concentrations obtained for the two proteins in single-protein systems; i.e., the protein mixture was extracted at a smaller AOT concentration than would be expected from the AOT concentrations in single protein extraction systems. The W0 value and the accompanying water both became a maximum at a lysozyme molar fraction of 0.5. The apparent interfacial area per single head group of AOT in the system containing the protein mixture was greater than that in a protein-free system, and became maximum at a lysozyme molar fraction of 0.5. A protein mixture containing equal fractions of the two protein components (lysozyme:cytochrome c = 1:1) exhibited the lowest interfacial tension between the protein aqueous and the AOT/isooctane solutions. These results indicate that each protein tends to be located synergistically on the inner side of the interface of reverse micelles, especially when the two proteins coexist in the AOT reverse micellar phase. NOMENCLATUR E CAOT * CAOT * * CAOT CKCl CPO CW CWP l n N rm rW SAOT SvH2 O Vm Vs VW W0 g

w r

sp

concentration of AOT in reverse micellar organic phase, M minimal AOT concentration of single protein component, mM minimal AOT concentration of binary protein component, mM concentration of potassium chloride, M concentration of protein in reverse micellar organic phase, M concentration of water in protein-free reverse micellar organic phase, M concentration of water in protein-containing reverse micellar organic phase, M thickness of AOT coat = 0.9, nm from ref. 3 average aggregation number of AOT, mol-AOT/mol-reverse micelle Avogadro’s number outer radius of reverse micelle, nm radius of water pool of reverse micelle, nm apparent interfacial area per single head group of surfactant molecule, nm2 speci® c volume of water molecule, nm3 /H2 O-molecule total volume of a reverse micelle, nm3 volume of AOT coat of reverse micelle, nm3 volume of water pool of reverse micelle, nm3 molar ratio of water to AOT in the protein-containing reverse micellar organic phase de® ned as CW / CAOT , mol-H2 O/mol-AOT volume fraction of the solvent taken up by reverse micelle, speci® c viscosity of reverse micellar organic phase; ratio of viscosity of micellar organic phase to that of the solvent, interfacial tension between protein aqueous solution and AOT/ isooctane solution, mNm- 1

Trans IChemE, Vol 75, Part C, June 1997

r

0

147

interfacial tension between water and AOT/isooctane solution in protein-free system, mNm- 1

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ADDRESS Correspondence concerning this paper shoulld be addresseed to Dr K. Naoe, Department of Chemical Engineering, Nara National College of Technology, 22 Yata, Yamato-Koriyama, Nara 639-11, Japan. The manuscript was communicated via our International Editor for Japan, Professor S. Furusaki. It was received 26 July 1996 amd accepted for publication after revision 21 February 1997.