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Selective Extraction and Recovery of Cytochrome c by Liquid-Liquid Extraction Using a Calix[6]arene Carboxylic Acid Derivative Tatsuya Oshima,*,† Hiroaki Higuchi,‡ Keisuke Ohto,‡ Katsutoshi Inoue,‡ and Masahiro Goto§ Department of Applied Chemistry, Faculty of Engineering, University of Miyazaki, 1-1, Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan, Department of Applied Chemistry, Faculty of Science and Engineering, Saga University, 1-Honjo, Saga 840-8502, Japan, and Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan Received February 9, 2005. In Final Form: May 6, 2005 Recently, we reported that a calix[6]arene carboxylic acid derivative can selectively extract the lysinerich protein cytochrome c by interacting with amino groups on the protein surface. In the present article, quantitative extraction and recovery of cytochrome c using this calix[6]arene carboxylic acid derivative are described. Both adjustment of the pH under acidic conditions and addition of an alcohol are necessary to strip the extracted protein from an organic solution to an aqueous solution. Separation of cytochrome c and lysozyme using the calix[6]arene was achieved under the optimal conditions. In the forward extraction stage, 93% of the cytochrome c was extracted, while lysozyme remained in the solution. In the subsequent stripping stage, the extracted cytochrome c was quantitatively recovered in an aqueous solution. Finally, separation of these proteins, which have similar molecular weights and isoelectric points, was accomplished.
Introduction The separation and recovery of proteins by liquid-liquid extraction using reverse micelles have been studied since the 1980s.1 A protein is transferred from an aqueous phase to the water pools of reverse micelles in an organic phase via interactions between the surfactant and the protein. In most cases, the major driving force for protein extraction is electrostatic interaction between the charged polar heads in the surfactant and the oppositely charged groups in the protein. Hydrophobic interaction may also be a dominant factor. To date, the extractions of various proteins using reverse micelle techniques have been published. One of the potential problems of the system is the low separation factor, since protein transfer into reverse micelles via simple electrostatic interaction alone generally makes the separation of proteins with similar molecular weights and isoelectric points (pIs) from a mixed solution difficult. Some researchers have reported selective extraction of proteins in reverse micelle systems by incorporating an affinity ligand that discriminates the target protein from contaminating proteins. Concanavalin A is selectively extracted over other proteins using an affinity reverse micelle extraction system incorporating alkylglucoside as a ligand, on the basis of their biospecific interaction.2 Concanavalin A is also utilized as a ligand in affinitybased reverse micelle extraction and separation to purify * Corresponding author. cc.miyazaki-u.ac.jp. † University of Miyazaki. ‡ Saga University. § Kyushu University.
E-mail
address:
oshimat@
(1) Pires, M. J.; Aires-Barros, M. R.; Cabral, J. M. S. Biotechnol. Prog. 1996, 12, 290. (2) (a) Woll, J. M.; Hatton, T. A.; Yarmush, M. L. Biotechnol. Prog. 1989, 5, 57. (b) Coughlin, R. W.; Baclaski, J. B. Biotechnol. Prog. 1990, 6, 307. (c) Chen, J.-P.; Jen, J.-T. Sep. Sci. Technol. 1994, 29, 1115. (d) Adachi, M.; Harada, M.; Shioi, A.; Takahashi, H.; Katoh, S. J. Chem. Eng. Jpn. 1996, 29, 983.
peroxidase and the medically important R1-acid glycoprotein.3 The extraction of lysozyme (Lyso) and bovine serum albumin (BSA) into reverse micelles is affected by addition of the triazine dye Cibacron Blue.4 Trypsin is separated from a mixture of proteins by reverse micelles containing an alkylated trypsin inhibitor through their protein-protein affinity interaction.5 Selective extraction of chymotrypsinogen with anti-chymotrypsinogen antibodies immobilized in reverse micelles has also been achieved, but the overall extraction efficiency of chymotrypsinogen is low.6 As shown by these precedents, the specificity of the ligand for a target protein dominates the selectivity in protein extraction. Recently, we demonstrated that a macrocyclic molecule calix[6]arene carboxylic acid derivative (tOct[6]CH2COOH; Figure 1) shows high extractability for the cationic protein cytochrome c (Cyt-c), representing the first report of protein extraction using a calixarene.7 The molecular structure of tOct[6]CH2COOH is ideal for binding to a protonated amino group, since it contains a well-fitted pseudocavity, C3 symmetry, and six preorganized carboxylic acid groups to include the protonated amino group.8 On the basis of these factors, several tOct[6]CH2COOH molecules strongly interact with protonated amino groups on the surface of Cyt-c to form a complex between the tOct[6]CH COOH molecules and the RNH + of the lysine 2 3 (3) (a) Paradkar, V. M.; Dordick, J. S. Biotechnol. Prog. 1993, 9, 199. (b) Choe, J.; Zhang, F.; Wolff, M. W.; Murhammer, D. W.; Linhardt, R. J.; Dordick, J. S. Biotechnol. Bioeng. 2000, 70, 484. (4) (a) Sun, Y.; Ichikawa, S.; Sugiura, S.; Furusaki, S. Biotechnol. Bioeng. 1998, 58, 58. (b) Zhang, T.; Liu, H.; Chen, J. Biochem. Eng. J. 1999, 4, 17. (5) (a) Adachi, M.; Yamazaki, M.; Harada, M.; Shioi, A.; Katoh, S. Biotechnol. Bioeng. 1997, 53, 406. (b) Adachi, M.; Shibata, K.; Shioi, A.; Harada, M.; Katoh, S. Biotechnol. Bioeng. 1998, 58, 649. (6) Adachi, M.; Harada, M.; Katoh, S. Biochem. Eng. J. 2000, 4, 149. (7) Oshima, T.; Goto, M.; Furusaki, S. Biomacromolecules 2002, 3, 438. (8) (a) Oshima, T.; Goto, M.; Furusaki, S. J. Inclusion Phenom. 2002, 43, 77. (b) Oshima, T.; Inoue, K.; Uezu, K.; Goto, M. Anal. Chim. Acta 2004, 509, 137.
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in methanol via complexation with a crown ether.13 The supermolecular complex promotes asymmetric oxidation of organic sulfoxide in methanol at lower temperatures. The present study is also related to these achievements from the viewpoint of protein surface recognition with macrocycles.14 Experimental Section
Figure 1. Molecular structure of tOct[6]CH2COOH.
residues. The complexation offsets the ionic properties of the surface of Cyt-c and affords sufficient hydrophobicity for its transfer into the organic phase, resulting in its extraction. Since the extraction is based on complexation between the calix[6]arene and a protonated amino group, t Oct[6]CH2COOH displays selectivity for lysine-rich proteins. In the present study, we propose a separation technique for cationic proteins using this calix[6]arene. The extraction system with tOct[6]CH2COOH can discriminate between cationic proteins that have similar molecular weights and pIs, on the basis of the number of amino groups on the surface of the protein molecule. In other words, a lysine-rich protein should be extracted with tOct[6]CH2COOH, while a lysine-poor protein would remain in the aqueous feed solution. Separation of the cationic proteins Cyt-c and Lyso was the goal of this study. After the conditions for selective extraction and stripping of Cyt-c were optimized, a separation test of Cyt-c and Lyso was performed practically under the optimal conditions. Recognition or modification of a protein with synthetic receptors on the basis of macrocycles is one of the latest topics in macrocyclic chemistry. Hamilton et al. have established a library of synthetic protein binding agents containing four peptide loops to a calix[4]arene core.9 The receptors function as inhibitors for Cyt-c and R-chymotrypsin by interacting with the protein surfaces. These authors further reported that one of the receptors binds strongly to the surface of platelet-derived growth factor (PDGF) and blocks binding to a native receptor. Tetraphenylporphyrin receptors have also been developed as protein “surface” receptors.10 These receptors function as protein denaturants10a or fluorescent receptors10b for proteins. Crown ethers are known as complexation agents for proteins.11 Reinhoudt et al. reported that crown ethers induce enzyme activation in organic media, probably because of interactions between the crown ethers and the RNH3+ groups of lysine residues in the enzymes.12 Tsukube et al. recently demonstrated chemical activation of Cyt-c (9) (a) Hamuro, Y.; Celama, M. C.; Park, H. S.; Hamilton, A. D. Angew. Chem., Int. Ed. Engl. 1997, 36, 2680. (b) Park, H. S.; Lin, Q.; Hamilton, A. D. J. Am. Chem. Soc. 1999, 121, 8. (c) Wei, Y.; McLendon, G. L.; Hamilton, A. D.; Case, M. A.; Purring, C. B.; Lin, Q.; Park, H. S.; Lee, C.-S.; Yu, T. Chem. Commun. 2001, 1580. (10) (a) Jain, R. K.; Hamilton, A. D. Angew. Chem., Int. Ed. 2002, 41, 641. (b) Wilson, A. J.; Groves, K.; Jain, R. K.; Park, H. S.; Hamilton, A. D. J. Am. Chem. Soc. 2003, 125, 4420. (c) Baldini, L.; Wilson, A. J.; Hong, J.; Hamilton, A. D. J. Am. Chem. Soc. 2004, 126, 5656. (11) Odell, B.; Earlam, G. J. Chem. Soc., Chem. Commun. 1985, 589. (12) (a) Reinhoudt, D. N.; Eendebak, A. M.; Nijenhuis, W. F.; Verboom, W.; Kloosterman, M.; Schoemaker, H. E. J. Chem. Soc., Chem. Commun. 1989, 399. (b) Engbersen, J. F. J.; Broos, J.; Verboom, W.; Reinhoudt, D. N. Pure Appl. Chem. 1996, 11, 2171. (c) Unen, D.-J.; Engbersen, J. F. J.; Reinhoudt, D. N. Biotechnol. Bioeng. 1998, 59, 553. (d) Unen, D.-J.; Engbersen, J. F. J.; Reinhoudt, D. N. J. Mol. Catal. B: Enzym. 2001, 11, 877. (e) Unen, D.-J.; Engbersen, J. F. J.; Reinhoudt, D. N. Biotechnol. Bioeng. 2002, 77, 248.
Reagents. The calix[6]arene hexacarboxylic acid derivative [37,38,39,40,41,42-hexakis(carboxymethoxy)-5,11,17,23,29,35hexakis (1,1,3,3,-tetramethylbutyl) calix[6]arene] (tOct[6]CH2COOH; Figure 1) was synthesized according to a previously described procedure.15 The final product was purified by recrystallization and was identified by means of FT-IR, 1H NMR, and elemental analysis. Protein reagents for the solvent extraction experiments were purchased and employed without further purification, that is, Cyt-c from horse heart (Sigma-Aldrich Co., St. Louis, MO) and Lyso from egg white (Nacalai Tesque Inc., Kyoto, Japan). All other reagents were of reagent grade and were used as received. Extraction of Cytochrome c with Calixarenes in Organic Solvents. Each aqueous protein solution was prepared by dissolving 1.0 × 10-5 mol/dm3 of the protein. The pH of the aqueous solution was adjusted with 1.0 × 10-2 mol/dm3 glycine and a small amount of hydrochloric acid. The organic solution was prepared by dissolving tOct[6]CH2COOH (3.0 × 10-3 mol/ dm3) in chloroform, toluene, or isooctane containing 10 vol % of 1-octanol.16 The two phases (5 cm3/5 cm3) were mixed in a stoppered test tube and were gently shaken for 24 h to reach the equilibrium state at 30 °C. After phase separation, the concentration of the protein in the aqueous phase was measured using an HPLC (Waters 515-2487 isocratic system) at 280 nm or a UV-vis spectrometer (PerkinElmer LAMBDA 190) by the absorbance of the Soret band peak to determine the degree of extraction (E )1 - [protein]aq,eq/[protein]aq,ini). Stripping of the Extracted Cyt-c. After operation of a forward extraction (50 cm3/50 cm3) in a manner similar to that described in the above section, an organic solution containing 9 × 10-6 mol/dm3 of Cyt-c was obtained. An aliquot of the organic solution (5 cm3) was placed in contact with a fresh aqueous solution (5 cm3) for 24 h. In some cases, a small amount of alcohol was added to the aqueous stripping solution. 1-Pentanol or 1-hexanol, which are difficult to dissolve in aqueous solution, was added to the organic solution. The phases were separated and the pH of the stripping solution was neutralized with 2.0 × 10-1 mol/dm3 of Na2HPO4 buffer for accurate measurement of the UV spectrum. The concentration of Cyt-c in the stripping solution was quantified to determine the degree of back-extraction ()[protein]aq,eq/[protein]org,ini). The activity of Cyt-c was evaluated by reduction of heme groups after the addition of ascorbic acid as follows: 2.0 × 10-4 mol/dm3 of ascorbic acid and 4.0 × 10-6 mol/dm3 of Cyt-c were mixed in a quartz cell to measure the UV spectrum. The initial reduction rate coefficient, k, was determined by the following equation:17
ln{(Amax - A)/(Amax - Amin)} ) -kt where A is the absorbance at 550 nm, and Amax and Amin are the maximum and minimum absorbances at 550 nm during the assay, respectively. (13) (a) Itoh, T.; Takagi, Y.; Murakami, T.; Hiyama, Y.; Tsukube, H. J. Org. Chem. 1996, 61, 2158. (b) Yamada, T.; Shinoda, S.; Kikawa, K.; Ichimura, A.; Teraoka, J.; Takui, T.; Tsukube, H. Inorg. Chem. 2000, 39, 3049. (c) Paul, D.; Suzumura, A.; Sugimoto, H.; Teraoka, J.; Shinoda, S.; Tsukube, H. J. Am. Chem. Soc. 2003, 125, 11478. (14) (a) Peczuh, M. W.; Hamilton, A. D. Chem. Rev. 2000, 100, 2479. (b) Itoh, T.; Takagi, Y.; Tsukube, H. J. Mol. Catal. B: Enzym. 1997, 3, 259. (15) Ohto, K.; Yano, M.; Inoue, K.; Yamamoto, T.; Goto, M.; Nakashio, F.; Shinkai, S.; Nagasaki, T Anal. Sci. 1995, 11, 893. (16) (a) Nakashima, K.; Oshima, T.; Goto, M. Solvent Extr. Res. Dev. Jpn. 2002, 9, 69. (b) Shimojo, K.; Watanabe, J.; Oshima, T.; Goto, M. Solvent Extr. Res. Dev. Jpn. 2003, 10, 185. (17) Ono, T.; Miyazaki, T.; Goto, M.; Nakashio, F. Solvent Extr. Res. Dev. Jpn. 1996, 3, 1.
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Figure 2. Conceptual illustration of protein separation using t Oct[6]CH2COOH. Table 1. Molecular Information for the Proteins Used in the Study protein
molecular weight
pI
Cyt-c Lyso
12 400 14 300
10.1 11.1
number of cationic residues Lys Arg His 19 6
2 11
3 1
Results and Discussion Recovery of Cytochrome c Extracted with Calixarene. A schematic illustration of the protein separation with calixarene used in this study is shown in Figure 2. The separation process consists of (1) selective extraction of a Lys-rich protein with calixarene and (2) recovery of the extracted protein. In the first step, a Lys-rich protein should be selectively extracted from an aqueous solution containing a protein mixture. Cyt-c and Lyso have similar molecular weights and pIs but differ in their numbers of Lys residues (Table 1), since Cyt-c has 19 lysine residues while Lyso only has 6. As reported in a previous paper, t Oct[6]CH2COOH quantitatively extracts the Lys-rich Cyt-c on the basis of recognition of the -amino groups in the Lys residues. On the other hand, Lyso is hard to extract and tends to aggregate with the extractant, resulting in the formation of a precipitate at the oil-water interface. Selective extraction of Cyt-c with tOct[6]CH2COOH from the mixture of Cyt-c and Lyso is demonstrated in the next section. The second step is quantitative recovery of the Cyt-c extracted with tOct[6]CH2COOH into a fresh aqueous solution. Since the driving force of the extraction is electrostatic interaction, the guest cation extracted with t Oct[6]CH2COOH is usually recovered by contact with an aqueous acidic solution. In a preliminary stripping test, however, the Cyt-c extracted with tOct[6]CH2COOH was not recovered from the chloroform solution by contact with an aqueous acidic solution alone. It therefore appears that transfer of Cyt-c into the organic phase is an irreversible process. Recovery of an extracted protein from an organic solution is also generally difficult in reverse micelle systems. Therefore, various techniques have been reported for protein release into a fresh aqueous solution. Generally, a protein in reverse micelles can be recovered by changing the pH to create electrostatic repulsion or by increasing the salt concentration for a size-exclusion effect in reverse micelles. In cases where a protein is not released from reverse micelles, even under conditions that would not ordinarily allow uptake, addition of an alcohol to the biphasic system promotes back-extraction.18 On the other hand, organic solvents show significantly different behavior for protein extraction and recovery with reverse micelles.17,19 According to these precedents, the extraction and stripping of Cyt-c with tOct[6]CH2COOH were in(18) (a) Aires-Barros, M. R.; Cabral, J. M. S. Biotechnol. Bioeng. 1991, 38, 1302. (b) Carlson, A.; Nagarajan, R. Biotechnol. Prog. 1992, 8, 85. (c) Pires, K. J.; Cabral, J. M. S. Biotechnol. Prog. 1993, 9, 647. (19) (a) Huang, S. Y.; Lee, Y. C. Bioseparation 1994, 4, 1. (b) Chamg, Q. L.; Lee, Y. C. Biotechnol. Bioeng. 1995, 46, 172.
Figure 3. Extraction profiles of Cyt-c with tOct[6]CH2COOH. Conditions: [Cyt-c]ini,aq ) 1.0 × 10-5 mol/dm3, [glycine]ini,aq ) 1.0 × 10-2 mol/dm3, [tOct[6]CH2COOH]org ) 3.0 × 10-3 mol/ dm3.
vestigated using chloroform, toluene, and isooctane. The effect of adding an alcohol for stripping the extracted Cyt-c was also examined. In the case of the isooctane system, 10 vol % of 1-octanol was added to dissolve the tOct[6]CH2COOH.16 Figure 3 shows the extraction profiles of Cyt-c with tOct[6]CH2COOH in chloroform, toluene, and isooctane as a function of pH. The extraction increases in proportion to the increase in the aqueous pH value. Furthermore, the slopes of the extractions are similar among the organic solvents, suggesting that the extraction mechanisms in these three solvents are the same. The slope of the relationship between the logarithm of the distribution (log D) and the pH from the results in Figure 3 is almost unity (data not shown).8 Therefore, Cyt-c is extracted through a proton-exchange reaction as follows:
nH6R + (NH3+)n-protein ) (H5R‚NH3+)n-protein + nH+ where H6R and (NH3+)n-protein denote tOct[6]CH2COOH and Cyt-c, respectively. In other words, tOct[6]CH2COOH molecules offset the positive charges on the surface of Cyt-c. Since Cyt-c is effectively extracted in chloroform, the complex between Cyt-c and tOct[6]CH2COOH appears to be stable in relatively polar organic solvents. Figure 4 shows the back-extraction profiles of Cyt-c extracted with tOct[6]CH2COOH after contact with (a) a fresh acidic solution alone or (b) an acidic solution containing 15 vol % ethanol. Under the ethanol-free condition, the extracted Cyt-c is difficult to recover from any of the organic solvents. As shown in Figure 4b, the back-extraction proceeds effectively in the presence of ethanol. The extracted Cyt-c is quantitatively recovered from the isooctane solution using an aqueous solution containing ethanol at a pH of less than 2. The effects of the organic solvents on the extraction and back-extraction of Cyt-c are similar to those in a reverse micelle system.17 Extraction of a protein into an organic solution is dominated by the hydrophilic/lipophilic balance of the protein-extractant complex. The relatively polar solvent chloroform dissolves the complex more effectively than toluene and isooctane. In the stripping stage, the extracted Cyt-c is efficiently recovered from the aliphatic solvent isooctane, but not from chloroform, because of the high solubility of the complex. Screening tests for the type and concentration of the added alcohol for stripping of Cyt-c were performed to clarify the role of the alcohol. The effects of the types of alcohol as well as acetone on stripping of the extracted Cyt-c are plotted in Figure 5 against the log P value of the added alcohol. Here, P is the partition coefficient of the
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Figure 6. Effects of the alcohol concentration on the stripping of Cyt-c extracted with tOct[6]CH2COOH. Circles: 1-propanol; triangles: ethanol; diamonds: methanol. Conditions: pH 2.0, isooctane media, [Cyt-c]ini,org ) 9 × 10-6 mol/dm3, [tOct[6]CH2COOH]org ) 3.0 × 10-3 mol/dm3.
Figure 4. Back-extraction profiles of Cyt-c extracted with tOct[6]CH2COOH into (a) an aqueous acidic solution (HCl) or (b) an acidic solution (HCl) containing 15 vol % of ethanol. Conditions: [Cyt-c]ini,org ) 9 × 10-6 mol/dm3, [tOct[6]CH2COOH]org ) 3.0 × 10-3 mol/dm3.
Figure 7. CD spectra and relative activities of Cyt-c before and after the extraction.
Figure 5. Effects of different types of added alcohol on the stripping of the extracted Cyt-c as a function of the log P value of the added alcohol. Filled circles: normal alcohols; open circles: branched alcohols; filled triangle: acetone. Conditions: pH 2.3, isooctane media, [Cyt-c]ini,org ) 9 × 10-6 mol/dm3, [tOct[6]CH2COOH]org ) 3.0 × 10-3 mol/dm3, volume of added solvent ) 0.75 cm3 (15 vol %).
solvent between water and 1-octanol, as an indicator of the hydrophilicity/lipophilicity balance of the solvent. The stripping proceeds efficiently after adding an alcohol with a log P value close to unity, such as 1-propanol and 1-butanol. In other words, addition of an alcohol that distributes into both the aqueous and organic phases promotes the stripping. The effects of the alcohol concentration on stripping of the Cyt-c extracted with tOct[6]CH2COOH are shown in Figure 6. Clearly, the backextraction increases with increasing alcohol concentration. The minimum concentration values of the alcohols for quantitative recovery of Cyt-c differ: Cyt-c is quantitatively recovered by adding less than 15 vol % of 1-propanol at pH 2.0 but not by adding more than 30 vol % of methanol. From these results, a mechanism for the observation that addition of a small amount of a cosolvent that distributes into both the aqueous and organic phases is effective for the stripping was elucidated as follows. Since the extracted
Cyt-c molecule is surrounded by tOct[6]CH2COOH molecules, the complex cannot appear at the oil-water interface because of its hydrophobicity. The addition of a cosolvent makes the properties of the aqueous and organic phases more similar. As a consequence, the extracted Cyt-c can appear at the oil-water interface and can be recovered to the aqueous phase through the stripping reaction. Next, the effects of the extraction process on the tertiary structure and activity of Cyt-c were investigated. Figure 7 shows the CD spectra and relative activities of Cyt-c before and after the extraction. The tertiary structure of Cyt-c after the back-extraction is denatured because of the acidity and the presence of the alcohol in the aqueous stripping solution but recovers after neutralization. The activities of Cyt-c as an electron-transfer protein before and after the extraction process were evaluated by the initial reduction rate coefficient, k. Relative to the value before the extraction process, 67% of the activity was retained through the forward extraction and backextraction after neutralization. The decrease in activity appears to be caused by alcohol contamination of the aqueous solution. Separation of Cationic Proteins with Calixarene. A problem for selective extraction of Cyt-c from a mixture of Cyt-c and Lyso is the aggregation of Lyso by complexation with tOct[6]CH2COOH. Since Lyso only has six Lys residues and interacts with a small number of tOct[6]CH2COOH molecules, the hydrophobicity of the complex between Lyso and tOct[6]CH2COOH molecules is too small for transfer into the organic phase and the complex aggregates at the oil-water interface. Sodium chloride was added to the aqueous phase to suppress the aggrega-
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Oshima et al. Table 2. Separation between Cyt-c and Lyso Using tOct[6]CH COOH under Optimal Conditionsa 2 solution type feed solution after forward extraction stripping solution
concentrations of cationic proteins Cyt-c: 0.7 × 10-6 mol/dm3 (%extraction ) 93%) Lyso: 1.0 × 10-5 mol/dm3 (%extraction ) 0)
Cyt-c: 9.0 × 10-6 mol/dm3 (%back-extraction ) 97%) after back-extraction Lyso: 0
Figure 8. Effects of NaCl concentration on the selective extraction of Cyt-c with tOct[6]CH2COOH over Lyso. Conditions: organic phase: isooctane media, [tOct[6]CH2COOH]org ) 2.0 × 10-3 mol/dm3; aqueous phase: pHini 6.0, [Cyt-c]aq.ini ) 1.0 × 10-5 mol/dm3, [Lyso]aq.ini ) 1.0 × 10-5 mol/dm3, [NaCl]aq.ini ) 0-200 × 10-3 mol/dm3.
Figure 9. Effects of alkali ions on the extraction of Cyt-c with t Oct[6]CH2COOH. Conditions: organic phase: isooctane media, [tOct[6]CH2COOH]org ) 2.0 × 10-3 mol/dm3; aqueous phase: pHini 6.2, [Cyt-c]aq.ini ) 1.0 × 10-5 mol/dm3, [MCl]aq.ini ) 0-200 × 10-3 mol/dm3 (M ) Na, K, or Cs).
tion, on the basis of an increase in the interfacial tension. The photograph in Figure 8 shows the extraction profiles of Cyt-c and Lyso with tOct[6]CH2COOH as a function of the NaCl concentration. Addition of NaCl suppresses precipitation of the calixarene-Lyso complex, and no precipitate is generated after the addition of more than 2.0 × 10-2 mol/dm3 of NaCl. However, the extraction of Cyt-c is decreased in the presence of excess sodium chloride. The effects of added alkali ions on the extraction of Cyt-c with tOct[6]CH2COOH were investigated to optimize the type and concentration of the alkali ions (Figure 9). Extraction of Cyt-c is suppressed after addition of small amounts of cesium ions, which have the largest ionic radius, whereas addition of 5.0 × 10-2 mol/dm3 of sodium ions (5 × 103-fold equivalent to Cyt-c) does not inhibit the protein extraction. There is competition between an alkali ion and the Cyt-c molecule for complexation with tOct[6]CH2COOH. The t Oct[6]CH2COOH molecule displays a higher extractability for cesium ions compared to smaller alkali ions, because the cavity size of the calix[6]arene fits a larger alkali ion.20 Therefore, complexation between Cyt-c and tOct[6]CH2COOH is inhibited by adding small amounts of cesium ions. On the other hand, Cyt-c extraction with tOct[6]CH2COOH is hardly affected by the addition of sodium ions, which are too small for the cavity of the calix[6]arene. As a result, NaCl is better for adjusting the salt (20) Kakoi, T.; Toh, T.; Kubota, F.; Goto, M.; Shinkai, S.; Nakashio, F. Anal. Sci. 1998, 14, 501.
a Experimental conditions are as follows. Forward extraction stage: organic phase, volume ) 5.0 cm3, [tOct[6]CH2COOH]org ) 2.0 × 10-3 mol/dm3, solvent: isooctane + 10 vol % 1-octanol. Aqueous phase: volume ) 5.0 cm3; [Cyt-c]aq.ini 1.0 × 10-5 mol/dm3, [Lyso]aq.ini ) 1.0 × 10-5 mol/dm3, [NaCl]aq.ini ) 5.0 × 10-2 mol/dm3, pHini 6.2 (glycine 1.0 × 10-2 mol/dm3). Back-extraction stage: organic phase; volume: 5.0 cm3, organic solution after the forward extraction. Aqueous phase: volume ) 5.0 cm3, pH 1.5 (HClaq) + 15 vol % ethanol.
concentration for selective extraction of Cyt-c than larger alkali ions. On the basis of the obtained results, separation of Cyt-c and Lyso by solvent extraction with tOct[6]CH2COOH was demonstrated under the optimal conditions as shown in Table 2, namely, selective extraction of Cyt-c at pH 6.2 in the presence of 5.0 × 10-2 mol/dm3 of sodium ions, followed by back-extraction to an aqueous solution at pH 1.5 containing 15 vol % of ethanol. Aqueous solutions were analyzed using an HPLC equipped with Waters µ bondasphare 5µ C18 as a column to determine the concentrations of the proteins. The results of the separation test are summarized in Table 2. In the forward extraction stage, an aqueous solution containing Cyt-c and Lyso was placed in contact with an organic solution containing the calix[6]arene. After phase separation, 93% of the Cyt-c in the aqueous solution was extracted by tOct[6]CH2COOH, while Lyso remained in the solution. In the subsequent stripping stage, 97% of the extracted Cyt-c was recovered in the aqueous solution, whereas Lyso was not detected. Finally, separation of cationic proteins with a calixarene derivative has been accomplished by discrimination of the numbers of Lys residues on the surfaces of the protein molecules. Conclusions The calix[6]arene carboxylic acid derivative used shows specific extractability for the lysine-rich protein cytochrome c. Under the optimal conditions, separation of cytochrome c and lysozyme was accomplished through the selective extraction and quantitative recovery of cytochrome c. The selectivity is based on the number of calix[6]arene molecules that complex with the amino groups on the surface of the protein. This report describes a novel separation technique via recognition of amino acid residues on the protein surface by macrocyclic molecules. On the other hand, we are currently preparing a manuscript on enzymatic oxidation of aromatics in organic media using cytochrome c complexed with the calix[6]arene. Recognition or modification of biomacromolecules with synthetic receptors on the basis of macrocycles should be recognized because of their recognition properties. Information regarding host-guest chemistry that has been obtained in previous years will be useful for identifying synthetic receptors for various biomacromolecules. Acknowledgment. The authors gratefully acknowledge the financial support of a Grant-in-Aid for Scientific Research (Fundamental Research B, No. 14350419) from the Ministry of Education, Science, Sports, and Culture of Japan. LA050364A
