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Peroxidase activity and stability of surfactant-heme complex in nonaqueous media N. Kamiya, S. Furusaki and M. Goto* Department of Chemical Science & Technology, Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812–81, Japan A surfactant-heme complex was prepared from hemin using a water-in-oil emulsion with a synthetic nonionic surfactant. The heme complex was soluble in anhydrous benzene with peroxidase activity for the oxidation of o-phenylenediamine using tert-butyl hydroperoxide as an oxidant. An absorption spectrum of the heme complex in benzene was distinct from that of free heme in an aqueous buffer solution owing to the different aggregation states in the respective solution. Moreover, the heme complex could not be decomposed in benzene even in excess of the hydroperoxide due to enhanced stability.
Introduction Protoferriheme plays a crucial role in the catalytic function of heme enzymes such as catalase and horseradish peroxidase as the prosthetic group in their active sites. It can be extracted from the apoprotein while hemin (protoferriheme chloride) is a commercially available compound. Protohemin and other simple hemins have been shown to possess peroxidase-like activities in aqueous solution (Portsmouth and Beal, 1971). Further, the catalytic activity of hemin in aqueous media is often decreased by the dimerization of heme molecules due to the low reactivity of dimeric ferriheme toward H2O2 in comparison with monomeric form (Jones et al., 1983). Moreover, the poor solubility of hemin in neutral aqueous solution and organic solvents such as alcohols, ether, benzene and chloroform limits the application of hemin as an effective oxidation catalyst in a homogeneous nonaqueous medium. In order to overcome this defect, Takahashi et al. (1986) prepared the polyethylene glycol-modified hemin which was soluble not only in neutral aqueous solution but in some organic solvents. However, more often than not, complicated steps are required to chemically modify a biocatalyst. Enzymes though can be easily modified by using surfactants to yield complexes with superior activities than their native counterparts in homogeneous organic solvents (Goto et al., 1994, Kamiya et al., 1995). Our recent works have focused on the new and easier preparation method for the surfactant-enzyme complexes using water-in-oil (W/O) emulsions (Okazaki et al., 1997a). The proposed method enables us to convert all enzymes, which are soluble in water, into © 1997 Chapman & Hall
surfactant-enzyme complexes without loss of enzymes. To assess the feasibility of the novel preparation method, the procedure was applied to proteases (Okazaki et al., 1997b) and horseradish peroxidase (Kamiya et al., 1997). The complexes have a high activity in a wide range of organic solvents. Here, the question has been raised whether a biocatalyst, except for an enzyme, can be coated with surfactant molecules. In the present work, we have investigated the modification of a coenzyme, a prosthetic group of heme enzymes, protoferriheme. The surfactant-heme complex was successfully prepared by the novel method with a synthetic nonionic surfactant. This is the first report demonstrating the peroxidase activity of the surfactant-heme complex in anhydrous, homogeneous benzene. The stability of heme in an environment of excess hydroperoxide in an aqueous and organic medium is also discussed. Experimental methods Materials Hemin was purchased from Tokyo Chemical Industry Co., Ltd. and used as received. The substrates and a solvent employed in this work were obtained commercially having the following analytical grades: o-phenylenediamine (o-PDA, > 98% purity, Tokyo Chemical Industry Co., Ltd.), approximately 5.5 M anhydrous decane solution of tert-butyl hydroperoxide (t-BuOOH, Aldrich Chemical Co., Inc.), 70% (v/v) aqueous solution of tert-butyl hydroperoxide (Sigma Chemical Co.), 31% H2O2 (Mitsubishi Gas Chemical Co., Ltd.) and benzene (Kishida Chemical Co., Ltd.). Benzene was dried with 3A-molecular sieves prior to use (the water content was 35 ppm as measured by Karl Biotechnology Letters · Vol 19 · No 10 · 1997
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Fischer potentiometric titration using a Mitsubishi moisturemeter model CA-05). The nonionic surfactant dioleyl glutamate ribitol amide (2C18∆9GE) was synthesized as described in the previous paper (Goto et al., 1987). Preparation method for surfactant-heme complex The preparation method for the surfactant-heme complex utilizing a W/O emulsion is as follows (Okazaki et al., 1997a): hemin was dissolved in 0.1 M KOH solution and the solution was diluted with 20 mM KH2PO4/KOH buffer. The 10 mL aqueous solution of heme (0.1 mM, pH 7.0) and 30 mL toluene containing the surfactant (10 mM) were mixed with a homogenizer (Ultra-Turrax T25, Junkel & Kunkel) at 13500 rpm for 3 minutes. A stable W/O emulsion was formed. The emulsion was poured into a round-bottom flask (100 mL), followed by rapid freezing in liquid N2 and lyophilized in a freeze-drying machine (FD-5N, EYELA) for 48 h. A light green solid was obtained. The sample was employed as the surfactant-heme complex. The yield of the complex was theoretically 100% (because all heme molecules are entrapped in the water phase of emulsions and recovered as a solid after freezedrying) and the heme content in the complex was 0.27 wt%. UV-vis spectra of free heme in aqueous buffer solution (pH 7.0) and the complex in benzene solution were recorded spectrophotometrically. Measurement of catalytic activity and stability of heme Before the oxidation of o-PDA was conducted with t-BuOOH, 2.5 mL 50 mM o-PDA in benzene and 20 mL 5.5 M t-BuOOH in decane were mixed in a quartz cell for UV measurement. The oxidation reaction was initiated by addition of 200 mL surfactant-heme complex in benzene (10 mM) at room temperature. Final experimental conditions are as follows; [o-PDA] = 45.7 mM, [t-BuOOH] = 40.4 mM and [heme] = 0.735 mM. The initial oxidation reaction rates of o-PDA were determined spectroscopically by measuring the absorbance at 470 nm (Takahashi et al., 1986). The stability of the heme complex in the presence of hydroperoxide was evaluated by the oxidative breakdown of the porphyrin moiety. The decomposition of heme was monitored by measuring the decrease in the Soret band at 405 nm in benzene and at 388 nm in aqueous solution. The reaction was started with the addition of 20 mL of an appropriate concentration of t-BuOOH solution to 10 mM heme containing solution.
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Figure 1 Absorption spectra of surfactant-heme complex prepared from pH 7.0 aqueous buffer solution in benzene (–) and free heme in pH 7.0 aqueous buffer solution (– – –). [heme] = 10 [mM].
Results and discussion Comparison of absorption spectra between free heme and surfactant-heme complex Figure 1 shows the UV-vis spectra of free heme in aqueous buffer solution (pH 7.0) and the surfactantheme complex in anhydrous benzene when the concentration of heme was adjusted to 10 mM in both cases. In the aqueous solution, a broad Soret band was observed around 390 nm due to the m-oxo dimer species and their aggregation (Brown et al., 1970). To our surprise, the heme complex exhibited a sharp Soret band at 405 nm in benzene; notwithstanding the fact that the complex was prepared from the same buffer solution. The result suggests that the dimerization of heme molecules could be suppressed by forming the complex with surfactant molecules and solubilizing it in benzene. However, it seemed that at the Soret absorption of 370 nm dimerization residue was evident even in the surfactant-heme complex because a similar spectra profile is obtainable in the unmodified heme shown for the aqueous system at the same wavelength. The absorption at 370 nm probably reflect the existence of residue m-oxo dimers. The absorption spectra of the free heme in the visible region (494, 613 nm) may be attributed to the hematin. It can be concluded that the chelation of an iron ion with porphyrin ring is not disrupted by the modification with surfactant molecules because two peaks in the visible region are also observed in the hemecomplex (482, 605 nm).
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Catalytic activity of surfactant-heme complex in benzene Ferrihemes are known to display peroxidase-like activity in aqueous solution. Can the surfactant-heme complex have a similar catalytic property in organic media? The catalytic activity of the heme complex was investigated by the oxidation of o-PDA with t-BuOOH in benzene. As shown in Figure 2, no reaction occurred until the heme complex was added to the substrate solution with t-BuOOH. It is obvious from Figure 3 that the initial velocity of the reaction was proportional to the heme concentration. The result clearly demonstrates that the reaction proceeded catalytically with the complex. Furthermore, the result also suggests that the dimerization of hemes due to the increase in the heme concentration did not occur in benzene because monomeric and dimeric hemes show different reactivity toward peroxides (Jones et al., 1983). The complex also performed well with hydrogen peroxide as an oxidant in benzene. However, the reaction rate was lower than in the case of using t-BuOOH due to the low concentration of hydrogen peroxide arising from poor solubility in benzene (data not shown). Stability of surfactant-heme complex in benzene The stability of the surfactant-heme complex against hydroperoxide (t-BuOOH) in benzene was investigated by measuring the changes in absorbance at the maximum wavelength. When the peroxide concentration was in excess compared to the heme concentration, the oxidation breakdown of the porphyrin occurs and hence there is complete loss of Soret and visible absorption spectra (Portsmouth and Beal, 1971). Surprisingly, the complex in benzene was completely stable in the presence of approx. 30 equivalent of t-BuOOH at a particular heme concentration, while the free heme in aqueous solution decomposed immediately at the same conditions (Figure 4). The regeneration of the original spectrum of free heme in aqueous solution could not be detected under the present experimental condition. The excess amount of the hydroperoxide (approx. 4400 equivalent) is considered to cause the decomposition of the heme complex. Assuming that the half-life period is the time required for the absorbance to be reduced to half of the initial value, the period of the complex in benzene reached approximately 30 min. In the case of the free heme in aqueous solution, half of the heme decomposed simultaneously with addition of the hydroperoxide in such a harsh condition. The results indicate that the heme possessed enhanced stability by forming the complex with surfactant molecules in benzene.
Figure 2 Time course of the oxidation of o-PDA catalyzed by surfactant-heme complex in anhydrous benzene with tBuOOH.
Figure 3 Relationship between heme concentration and the catalytic activity of surfactant-heme complex in benzene.
Conclusions The surfactant-heme complex which was prepared in the present study was readily soluble in dry benzene and showed a peroxidase activity with the hydroperoxides. Moreover, the stability of the complex toward the hydroperoxide in benzene was much higher than that Biotechnology Letters · Vol 19 · No 10 · 1997
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study of this novel biocatalyst including possible enhancement of its catalytic activity along with some practical applications is now underway. Acknowledgment This work was supported by a Grant-in-Aid for Scientific Research (No.09750841) from the Ministry of Education, Science, Sports and Culture of Japan, and by a foundation from The Association for the Progress of New Chemistry (ASPRONC). N.K. was supported by Research Fellowships of the Japan Society for the Promotion of Science (JSPS) for Young Scientists. References
Figure 4 Decomposition of heme with heme complex in benzene (A405, –) and free heme in pH 7.0 aqueous solution (A388,– – –). The concentrations of the measurement are [heme] = 9.9 [mM], (a) [t-BuOOH] = 0.31 [mM] and (b) [t-BuOOH] = 44 [mM].
of the free heme in aqueous solution. The surfactant molecules may provide a protective environment for porphyrin oxidation like an apoprotein of native heme enzymes. The surfactant-heme complex prepared from commercially available hemin is a promising biocatalyst that would work in nonaqueous media. Further
Brown, S.B., Dean, T.C. and Jones, P. (1970) Biochem.J., 117, 733–739. Goto, M., Matsumoto, M., Kondo, K. and Nakashio, F. (1987) J.Chem.Eng.Jpn., 20, 157–164. Goto, M., Kamiya, N., Miyata, M. and Nakashio, F. (1994) Biotechnol.Prog., 10, 263–268. Jones, P., Mantle, D., and Wilson, I. (1983) J.Chem.Soc.,Dalton Trans., 161–164. Kamiya, N., Goto, M. and Nakashio, F. (1995) Biotechnol.Prog., 11, 270–275. Kamiya, N., Okazaki, S. and Goto, M. (1997) Biotechnol.Tech., 11, 375–378. Okazaki, S., Kamiya, N., Abe, K., Goto, M. and Nakashio, F. (1997a) Biotechnol.Bioeng., 55, 455–460. Okazaki, S., Kamiya, N. and Goto, M. (1997b) Biotechnol.Prog., in press. Portsmouth, D. and Beal, E.A. (1971) Eur.J.Biochem., 19, 479–487. Takahashi, K., Matsushima, A., Saito, Y. and Inada, Y. (1986) Biochem.Biophys.Res.Commun., 138, 283–288.
Received 14 July 1997; Revisions requested 23 July 1997; Final Revisions received 5 August 1997; Accepted 8 August 1997
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