Bioconjugate Chem. 2002, 13, 167−171
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ARTICLES Formation of a Bioconjugate Composed of Hemin, Smectite, and Quaternary Ammonium Chloride That Is Soluble and Active in Hydrophobic Media Masaru Kurosawa, Tetsuji Itoh, Yoh Kodera, Ayako Matsushima, Misao Hiroto, Hiroyuki Nishimura, and Yuji Inada* Toin Human Science and Technology Center, Department of Biomedical Engineering, Toin University of Yokohama, Kurogane-cho, Aoba-ku, Yokohama 225-8502. Received November 2, 2000; Revised Manuscript Received August 27, 2001
Hemin (Fe3+) was adsorbed onto synthetic smectite (clay mineral) intercalated with a quaternary alkenylammonium compound, dioleyldimethylammonium chloride (DOA), to form a hemin-smectiteDOA conjugate. The hemin-smectite-DOA conjugate was soluble in organic solvents such as benzene and toluene to form a transparent colloidal solution with a light yellow color. Its absorption spectrum in benzene showed two bands, 600 and 568 nm, in the visible region and a sharp Soret band at 400 nm with the molar extinction coefficient of 7.5 × 104 M-1 cm-1. The formation of the conjugate of smectite and DOA was confirmed by X-ray diffraction analysis: the basal spacing, d(001), of heminsmectite-DOA conjugate was 19 Å which is an expansion of the interlayer space by 5 Å based upon the basal spacing of smectite of 14 Å. Hemin-smectite-DOA conjugate catalyzed the peroxidase-like reaction in organic solvents using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The temperature-dependent peroxidase-like activity of the conjugate was compared with peroxidase activity of horseradish peroxidase. The hemin-smectite-DOA conjugate exhibited higher activity as the temperature was increased from 30 to 70 °C, while horseradish peroxidase activity was reduced as the temperature was increased.
INTRODUCTION
Hemin (Fe3+), a prosthetic group of heme-proteins and enzymes, plays an important role by itself in oxidoreductions such as oxidation of a compound with peroxide. Hemin (Fe3+) liberated from holoenzyme is not soluble in neutral aqueous solution and in organic solvents such as benzene and chloroform. However, hemin (Fe3+) itself has a catalytic potential, and several studies have been performed concerning the peroxidase activity (1) and catalase activity (2) in aqueous solution. It should be noted that hemin molecules tend to aggregate and dimerize in aqueous solutions (3), and its catalytic activity is often decreased through dimerization and polymerization (4). There are several ways of avoiding the aggregation and dimer formation of hemin (Fe3+) (5), and the utilization of detergents was found to be one of the simple but useful techniques (6). On the other hand, hemin (Fe3+) has been seldom used as a catalyst in organic media because it is not soluble in organic media. Chemical modification of proteins with poly(ethylene glycol) (PEG),1 a nontoxic, nonimmunogenic, and amphipathic polymer, has been extensively studied for the purpose of applying proteins to biomedical and biotechnological processes (7, 8). In the biotechnology field, PEG* To whom correspondence should be addressed. Phone: +8145-974-5060. Fax: +81-45-972-5972. 1 Abbreviations: DOA, dioleyldimethylammonium chloride; PEG, poly(ethylene glycol).
modified hydrolases, which are soluble and active in hydrophobic media, catalyze the reverse reactions of hydrolysis, ester synthesis, and ester exchange reactions (9, 10). Along this line of investigations, Takahashi et al. (11) prepared PEG-modified hemin (Fe3+) with peroxidase activity in hydrophobic media. Recently, we have found a clay mineral, smectite, having a catalytic function as seen in a living body, by binding it with bioactive substances. The heme (Fe2+)smectite conjugate, formed by combining smectite with heme (Fe2+), has similar properties to those of hemoglobin or myoglobin in terms of adsorption and desorption of oxygen and carbon monoxide (12). Furthermore, chlorophyll a-smectite conjugate, smectite conjugated with chlorophyll a in place of heme (Fe2+), has proved to be stable in sunlight and to produce hydrogen gas (13). The present study deals with a conjugate of hemin (Fe3+), smectite, and quaternary ammonium compound, dioleoyldimethylammonium chloride (DOA). The layer of smectite was expanded by conjugating with DOA, and consequently the hemin-smectite-DOA conjugate became soluble in organic solvents such as benzene and toluene to form a transparent colloidal solution. Furthermore, the conjugate had a peroxidase-like activity in hydrophobic media. EXPERIMENTAL PROCEDURES
General. Hemin (crystalline) and horseradish peroxidase (116 purpurogallin units/mg solid) was purchased
10.1021/bc000133+ CCC: $22.00 © 2002 American Chemical Society Published on Web 02/21/2002
168 Bioconjugate Chem., Vol. 13, No. 2, 2002
Figure 1. (a) Structure of dioleyldimethylammonium chloride (DOA). (b) The chemical structure of smectite in a crystal state consists of two sheets of linked SiO4 tetrahedra sandwiching an octahedral cation (Mg) between them, and the interlayer. Elemental composition Si 8.00, Mg 5.65, Li 0.70, Na 1.05; transmittance 95% in 1% aqueous solution at 500 nm; and methylene blue absorption 101 milliequiv per 100 g.
from Sigma Chemical Co. (St. Louis, MO). Dioleoyldimethylammonium chloride (DOA) was kindly provided by Nippon Oil and Fats Co., Ltd. (Tokyo, Japan). Smectite powder (hectorite), which was hydrothermally synthesized by Torii et al. (14), was obtained from Co-op Chemical Co., Ltd. (Tokyo, Japan). The chemical structure and property of DOA and smectite were shown in Figure 1. Leucocrystal violet was obtained from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Other reagents used were of analytical grade. Preparation of Hemin-Smectite-DOA Conjugate. The hemin (Fe3+)-smectite conjugate was prepared from hemin and smectite by the method of Itoh et al. (12). To 36 mL of acetone solution containing hemin (0.9 mg) was added 300 mg of smectite powder. The suspension was shaken for 1 h at 25 °C to establish the adsorption equilibrium between hemin (Fe3+) and smectite. The hemin (Fe3+)-smectite conjugate thus formed was collected by centrifugation and was dried under reduced pressure. The amount of hemin (Fe3+) adsorbed onto smectite was spectrophotometrically determined by measuring the absorbance of the supernatant liquid obtained by centrifugation of the sample suspension. Since smectite2 has cation-exchange properties of 101 mequiv/100 g of smectite, hemin-smectite conjugate must be bound with quaternary alkenylammonium compound, dioleyldimethylammonium chloride (DOA) (Figure 1a), with ion-exchange reaction. To 20 mL of heminsmectite conjugate dissolved in water (15 mg/mL) was added 0.68 mL of DOA colloidal solution (70%), and the suspension was then stirred for 7 h at 25 °C to form hemin-smectite-DOA conjugate. The conjugate was collected by centrifugation and was dried under reduced pressure. The absorption spectra of hemin-smectite in
Kurosawa et al.
water and hemin-smectite-DOA conjugates in organic solvents were measured with a Shimadzu MPS-2000 multipurpose spectrophotometer (Kyoto, Japan). One gram of hemin-smectite-DOA conjugate consists of 2 mg of hemin, 633 mg of smectite, and 365 mg of DOA. X-ray powder diffraction (XRD) analyses were carried out with a Rigaku diffractometer (RTP-300) using monochromatized Cu KR radiation. Measurement of Peroxidase Activity of HeminSmectite-DOA Conjugate in Organic Solvents. The peroxidase activity of hemin-smectite-DOA conjugate was tested by the modified method of Ajima et al. (16). The first series of experiments was conducted by measuring the peroxidase activity of hemin-smectite-DOA conjugate (0-0.32 µM) in an organic solvent, benzene. The reaction system consists of benzoyl peroxide (0-0.66 mM) as a hydrogen acceptor and leucocrystal violet (16.7 mM) as a hydrogen donor. The reaction was carried out for 5 min at 25 °C in the dark. The peroxidase activity of hemin-smectite-DOA conjugate in benzene was spectrophotometrically determined by measuring the amount of oxidized crystal violet using the molar extinction coefficient of 604 nm ) 1.2 × 105 M-1 cm-1 (17). The second series of experiments was conducted by measuring temperature-dependent peroxidase activity of heminsmectite-DOA conjugate using benzoyl peroxide and leucocrystal violet as well as horseradish peroxidase using hydrogen peroxide and guaiacol. The peroxidase activity of the conjugate in toluene was spectrophotometrically determined by measuring the absorbance change of oxidized crystal violet at 604 nm/min at temperatures from 30 to 70 °C. As a control experiment, the peroxidase activity of the horseradish peroxidase, instead of hemin-smectite-DOA conjugate, in 0.1 M phosphate buffer (pH 7.4) was determined as follows: the reaction system consisted of horseradish peroxidase in 0.1 M phosphate buffer (pH 7.4), hydrogen peroxide (0.13 mM), and guaiacol (0.28 mM), and its activity was spectrophotometrically determined by measuring the absorbance change of oxidized guaiacol at 460 nm/min at temperatures from 30 to 70 °C. RESULTS AND DISCUSSION
Absorption Spectra of Hemin-Smectite Conjugate in Water and Hemin-Smectite-DOA Conjugate in Benzene. Hemin, or ferriprotoporphyrin, is practically insoluble in water and in organic solvents except for strong organic bases such as trimethylamine and dimethylaniline. Hemin conjugated with smectite, hemin-smectite conjugate, was soluble in water to form a brownish, transparent colloidal solution. Addition of DOA, quaternary alkenylammonium compound, to hemin-smectite conjugate in water caused the precipitation of hemin-smectite-DOA in which DOA was bound to smectite by a cation-exchange reaction in the interlayer space of hemin-smectite. Then hemin-smectiteDOA conjugate was dissolved in hydrophobic media such as benzene and toluene. 2 The smectite (hectorite), which was hydrothermally synthesized, had unique physical and chemical properties such as high transparency in water, cation exchange, and the ability to form organic and inorganic interlayer complex (14). Expandable phyllosilicates have a structure composed of alternating 2:1 layer, which consists of two sheets of linked SiO4 tetrahedra sandwiching octahedral cations, Mg, between them (Figure 1 b). The 2:1 layers, which have a net negative charge (layer charge), are loosely tied together by interlayer cations. Water also is present between the layers.
Hemin−Smectite−Quaternary Ammonium Chloride Bioconjugate
Bioconjugate Chem., Vol. 13, No. 2, 2002 169 Table 1. Physicochemical and Spectrophotometric Properties of Hemin-Smectite-DOA Conjugate Together with Its Compositions Soret solubilitya d (001), band, nm conjugate Å (mM) water benzene DOAb hematin smectite hemin-smectite hemin-smectite-DOA
14 14 19
398 (122)c 398 (116) 400 (75)
( ( + + -
+ +
a +, -, (: transparent colloidal solution, insoluble, and sparingly soluble, respectively. bDioleyldimethylammonium chloride. c Monomeric hematin (15).
Figure 2. Absorption spectra of hemin-smectite-DOA conjugate and hemin-smectite conjugate. The spectrum of hemin (Fe3+)-smectite-DOA conjugate (13 µM) in benzene is shown by a solid line with a sharp Soret band at 400 nm. The spectrum of hemin (Fe3+)-smectite conjugate (10 µM) in water is shown by a dotted line with a sharp Soret band at 398 nm.
Figure 2 presents the absorption spectra of heminsmectite-DOA conjugate in benzene (solid line) and hemin-smectite conjugate in water (dotted line). The absorption spectrum of hemin-smectite-DOA conjugate in benzene has the absorption maxima at 568 nm with a shoulder at 600 nm and Soret band at 400 nm in visible region. The molar extinction coefficient at the Soret band was calculated to be 400 nm ) 7.5 × 104 M-1cm-1 in benzene (Table 1). On the other hand, the spectrum of hemin-smectite conjugate in water shows two peaks at 610 and 490 nm and the sharp Soret band at 398 nm with 398 nm ) 11.6 × 104 M-1 cm-1 in water which is closely in agreement with the molar extinction coefficient of monomeric hematin in water, 12.2 × 104 M-1 cm-1 (15). Table 1 summarizes the physicochemical and spectrophotometric properties of hemin-smectite-DOA conjugate together with its compositions. As is clear from the table, the basal spacing of smectite was determined to be 14 Å and that of smectite conjugated with DOA was determined to be 19 Å, indicating an expansion of the
interlayer space by 5 Å. Furthermore, the layer of smectite conjugated with hemin was hardly expanded as described above. Therefore, it can be reasonably assumed that hemin is adsorbed onto surface of smectite, and DOA is intercalated in the basal spacing of smectite. Although hemin-smectite conjugate became easily soluble in water to form a transparent colloidal solution with a dark brown color, the conjugate is never soluble in benzene. However, hemin-smectite-DOA conjugate was freely soluble in organic solvents such as benzene and toluene to form a transparent colloidal solution with light yellow color (Figure 2). The solubility of hemin-smectite-DOA conjugate together with its components are shown in Table 1. Peroxidase Activity of Hemin-Smectite-DOA Conjugate in Hydrophobic Media. The finding shown above leads to the study on the peroxidase activity induced by hemin-smectite-DOA conjugate in organic solvents. The first series of experiments was conducted with oxidation of leucocrystal violet (AH) by benzoyl peroxide in the presence of hemin-smectite-DOA conjugate in benzene as shown below (Scheme 1).
Scheme 1 Peroxide + in benzene
8 Reduced peroxide + 2A 2AH 9 hemin-smectite-DOA conjugate Figure 3a shows the oxidation of leucocrystal violet (AH) with benzoyl peroxide by the catalytic action of hemin-smectite-DOA conjugate with various concentrations (0-0.32 µM) in benzene. The oxidation of leuco-
Figure 3. (a) Oxidation of leucocrystal violet (16.7 mM) with benzoyl peroxide (0.66 mM) in benzene by the catalytic action of hemin-smectite-DOA conjugate with various concentrations (0-0.32 µM). (b) Oxidation of leucocrystal violet (16.7 mM) with benzoyl peroxide (0-0.66 mM) in benzene by the catalytic action of hemin-smectite-DOA conjugate (0.32 µM) at 25 °C.
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photostable and catalyzed hydrogen gas evolution by the illumination of visible light in aqueous solution. Along this line of investigations, the organic insolubility of hemin becomes soluble by conjugating with smectite and DOA, and the conjugate exerts peroxidase-like activity in organic media as well. ACKNOWLEDGMENT
The authors are grateful to Dr. Toshi Tsuzuki and Dr. Hiroaki Kaji, and to Showa Denko K.K. for his basic and technical advice in the X-ray powder diffraction (XRD) analyses. LITERATURE CITED
Figure 4. Temperature-dependent peroxidase activity of heminsmectite-DOA conjugate in hydrophobic media (toluene) and of horseradish peroxidase in hydrophilic media [0.1 M phosphate buffer (pH 7.4)]. Curve a: Oxidation of leucocrystal violet (16.7 mM) with benzoyl peroxide (0.66 mM) by hemin-smectite-DOA conjugate (0.32 µM). Curve b: Oxidation of guaiacol (0.28 mM) with hydrogen peroxide (0.13 mM) by horseradish peroxidase (0.26 purpurogallin units).
crystal violet was linearly enhanced by increasing the amount of the conjugate in the reaction system. No reaction took place in the absence of the conjugate. The similar line of study was conducted by changing the benzoyl peroxide concentration. Figure 3b shows the oxidation of leucocrystal violet by the catalytic action of hemin-smectite-DOA conjugate (0.32 µM) in the presence of benzoyl peroxide (0-0.66 mM) in benzene. Crystal violet in the reduced form was linearly oxidized by the enhancement of benzoyl peroxide concentration in benzene. From the results obtained above, it can be concluded that hemin-smectite-DOA conjugate has peroxidase activity in an organic solvent. Heat stability of peroxidase activity was tested for hemin-smectite-DOA conjugate and horseradish peroxidase at the temperature range from 30 to 70 °C. Although the absolute activity of horseradish peroxidase is much higher than that of the conjugate at 30 °C, these activities at 30 °C are regarded as 100%. Figure 4 shows the temperature-dependent activity curves of the conjugate and horseradish peroxidase. The peroxidase-like activity of the conjugate is enhanced by increasing the temperature (curve a). On the other hand, the enzymic activity of horseradish peroxidase is lowered by increasing temperature and is completely lost at 70 °C, due to the protein denaturation (curve b). From the result obtained above, the hemin-smectite-DOA conjugate may be available to be catalyst in hydrophobic media at high temperature. A similar line of studies has been successfully carried out with the conjugate of chlorophyll adsorbed onto clay minerals such as bentonite and smectite which are closely associated with the development of the photochemical function of chlorophylls (13, 18-20). Chlorophyll-smectite conjugate becomes soluble in aqueous solution, although chlorophylls are never soluble in water. chlorophyll-poly(vinylpyrrolidone)-smectite conjugate with the absorption maximum at 677 nm was extensively
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