Bioconjugate Chem. 2001, 12, 3−6
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Hemin (Fe3+)- and Heme (Fe2+)-Smectite Conjugates as a Model of Hemoprotein Based on Spectrophotometry Tetsuji Itoh, Takuya Yamada, Yoh Kodera, Ayako Matsushima, Misao Hiroto, Katsukiyo Sakurai, 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, Japan. Received May 29, 2000
Hemin (Fe3+) was adsorbed onto synthetic smectite (clay mineral) in acetone to form a hemin-smectite conjugate. The hemin-smectite conjugate became soluble in water to form a transparent colloidal solution with a dark brown color. Its absorption spectrum in water showed a sharp Soret band at 398 nm with the molar extinction coefficient as 398nm ) 11.6 × 104 M-1 cm-1, which is in good agreement with 398nm ) (12.2 ( 3) × 104 M-1 cm-1 of monomeric hematin (1). Hemin (Fe3+)-smectite conjugate had a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and guaiacol (a hydrogen donor) in aqueous solution and its activity was higher than that of hematin. Hemin (Fe3+)smectite conjugate in water was reduced by adding sodium dithionite to form a heme (Fe2+)-smectite conjugate which is also a transparent colloidal solution in water. Its absorption spectrum in aqueous solution was surprisingly in close agreement with that of oxyhemoglobin. Its peak positions of R, β, and Soret bands were located in only a 9-3 nm shift to shorter wavelengths in comparison with those of oxyhemoglobin. Therefore, heme (Fe2+)-smectite conjugate was bound to O2 to form O2-heme (Fe2+)-smectite conjugate. The addition of carbon monoxide, CO, to O2-heme (Fe2+)-smectite conjugate caused the formation of CO-heme (Fe2+)-smectite conjugate with a similar absorption spectrum of carboxyhemoglobin (HbCO) accompanied by shifting 8-10 nm to shorter wavelength. Therefore, the transformation of O2-heme (Fe2+)-smectite conjugate to CO-heme (Fe2+)-smectite conjugate was accompanied by shifting of 7, 4, and 3 nm to shorter wavelengths in the R, β, and Soret bands respectively, which are similar to the spectral change from oxyhemoglobin to carboxyhemoglobin. Also the ratio (1:1.6) of the molar extinction coefficient of Soret band of O2-heme (Fe2+)-smectite conjugate and CO-heme (Fe2+)-smectite conjugate was surprisingly agreement with ratio (1:1.5) of oxyhemoglobin and carboxyhemoglobin. The phenomenon shown above was unexpectedly found during the course of study of bioconjugate of a bioactive substance, hemin (Fe3+) or heme (Fe2+), and a clay mineral, smectite, in place of the protein of globin in hemoglobin.
Human hemoglobin with the molecular weight of 64 500 in red blood cells consists of 6% heme and 94% globin and plays, in vivo, an important role in transport of oxygen. In its oxygenation form it is called oxyhemoglobin, HbO2,1 and when the oxygen is displaced by carbon monoxide, it is called carboxyhemoglobin, HbCO (3). This splendid function depends on the cooperative action of heme and globin protein (R2 β2) having a specific conformation composed of primary (R chain, 141 amino acids; β chain, 146 amino acids), secondary (helix content, 70%), tertiary (the steric conformation), and quaternary structures (four subunits) (4, 5). The hemoglobin molecule has a compact spheroidal structure of 6.4 by 5.5 by 5.0 nm in dimension (6). When one of amino acids in hemoglobin molecule is deleted or substituted by another amino acid by mutation, the normal function of hemoglobin can be harmed to form what is called abnormal hemoglobin (7). On the other hand, heme (Fe2+) is a prosthetic group of hemoglobin and is obtained by reducing hematin (Fe3+) in alkali solution by sodium dithionite (Figure 1a) (2). The absorption spectrum of monomeric * To whom correspondence should be addressed. Phone: +8145-974-5060. Fax: +81-45-972-5972. 1 Abbreviations: HbO , oxyhemoglobin; HbCO, carboxyhe2 moglobin; SME, smectite.
hematin in aqueous solution could be measured at pH 6.8 and found to have a sharp Soret band with a molar extinction coefficient as 398nm ) (12.2 ( 3) × 104 M-1 cm-1, as reported by Inada and Shibata (1). 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 complexes (8, 9). 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 as shown in Figure 1b. 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. The properties of smectite prepared by Torii and Iwasaki (810) are as follows: elemental composition Si 8.00, Mg 5.65, Li 0.70, and Na 1.05; transmittance 95% in 1% aqueous solution at 500 nm; and methylene blue absorption 101 milliequiv/100 g which is obtained from Co-op Chemical Co., Ltd (Tokyo, Japan). Chlorohemin is soluble in dilute potassium hydroxide solution to form hematin. The monomeric hematin molecule is unstable and has a marked tendency to take a polymerized form. The hemin-smectite conjugate was prepared by adding 300 mg of smectite powder to 33 mL
10.1021/bc000055q CCC: $20.00 © 2001 American Chemical Society Published on Web 01/17/2001
4 Bioconjugate Chem., Vol. 12, No. 1, 2001
Itoh et al.
Figure 1. (a) Heme or hemin consists of protoporphyrin being made up of four pyrrole groups and an iron ion. The iron atom in heme or hemin binds to the four nitrogens in the center of the protoporphyrin ring. The iron can form two additional bounds. Their bonding sites are termed the fifth and sixth coordination positions. The iron atoms in the heme can be in the ferrous (Fe2+) or the ferric (Fe3+) oxidation state. (b) The chemical structure of smectite in a crystal state consists of two sheets of linked SiO4 tetrahedra sandwiching 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/100 g.
of acetone solution containing chorohemin (0.85 mg). The suspension was then shaken for 1 h at 25 °C to establish the adsorption equilibrium between hemin and smectite. The hemin (Fe3+)-smectite conjugate thus formed was collected by centrifugation and was dried under reduced pressure. The amount of hemin adsorbed onto smectite was spectrophotometrically determined by measuring the absorbance of the supernatant liquid obtained by centrifugation of the sample suspension using the molar extinction coefficient of hematin. The hemin-smectite conjugate, in which amount of hemin (0.2 mg) was adsorbed onto 100 mg of smectite, was dissolved in water to from transparent solution with dark brown color. Absorption spectrum of hemin (Fe3+)-smectite conjugate (1.14% in water) shows a sharp Soret band at 398 nm and two peaks at 490 and 610 nm as shown in Figure 2a. And its molar extinction coefficient at 398 nm as 398nm ) 11.6 × 104 M-1 cm-1 was in good agreement with that of monomeric hematin (1). The absorption spectrum of hematin, dashed line in Figure 2a, shows a polymerized form of hematin with a broad Soret band at approximate 360 nm. Figure 2b shows the peroxidase activities of hemin (Fe3+)-smectite conjugate (closed circle) and hematin (open circle) against incubation time using hydrogen peroxide (a hydrogen acceptor) and guaiacol (a hydrogen donor) in aqueous solution. The activity of the hemin (Fe3+)-smectite conjugate was increased linearly with time, while the activity of hematin was lowered with time. The peroxidase-like activity of the conjugate was well retained at high concentration of H2O2 and in high temperature at more than 60 °C in the reaction system in comparison with that of natural peroxidase composed of protein and hemin. Hemin (Fe3+)-smectite conjugate in the thunberg tube was deaerated by successive freeze-pump-thaw cycles and was reduced by the addition of sodium dithionite to form heme (Fe2+)-smectite conjugate. Absorption spectrum of heme (Fe2+)-smectite conjugate in water is shown by solid line in Figure 3a, which is quite different from that of hemin (Fe3+)-smectite conjugate shown by dashed line in Figure 3a. The peak positions of R, β, and Soret bands in heme (Fe2+)-smectite conjugate were located at 568, 534, and 411 nm, respectively, which represent an only approximately 9-3 nm shift to shorter wavelengths compared with those of oxyhemoglobin
Figure 2. Absorption spectra of hemin (Fe3+)-smectite conjugate and hematin (Fe3+) in water (a) and peroxidase activity of hemin-smectite conjugate in the presence of hydrogen peroxide and guaiacol in aqueous solution (b). (a) The spectrum of hemin (Fe3+)-smectite conjugate (17 µM) is shown by solid line with a sharp Soret band at 398 nm. The absorption spectrum of hematin at the same concentration as the hemin (Fe3+)-smectite conjugate is shown by dashed line with a broad band at about 360 nm as a Soret band which is a polymerized form of hematin. (b) The peroxidase activity was spectrophotometrically determined by measuring the absorbance at 460 nm caused by the oxidation of guaiacol (0.3 mM) with hydrogen peroxide (3.2 mM) in 3.1 mL of 0.1 M phosphate buffer (pH 7.0) in the presence of hemin-smectite conjugate (8.6 µM) or hematin (8.2 µM). The absorbance at 460 nm was plotted against incubation time (min) at 25 °C.
(Table 1). To confirm the binding of O2 to heme (Fe2+)smectite conjugate to form O2-heme (Fe2+)-smectite conjugate, states of heme (Fe2+) and hemin (Fe3+) conjugated with smectite were studied by measuring the ESR spectrum with a Japan Electron Optics Laboratory spectrometer model JES-FA 200 operating at 100 kHz. The ESR spectrum of hemin (Fe3+)-smectite conjugate in water showed the concomitant formation of the two high-spin states of (g ) 6.160) and (g ) 5.865). On the other hand, heme (Fe2+)-smectite conjugate obtained by adding sodium dithionite to hemin (Fe3+)-smectite conjugate showed a reduced ESR signal in high-spin state (g ) 6.160). A similar phenomenon had been observed in transformation of methemoglobin (Fe3+) to oxyhemoglobin (Fe2+) in which ESR spectrum with high-spin state
Communications
Bioconjugate Chem., Vol. 12, No. 1, 2001 5
Table 1. Spectrophotomeric Characteristics of Hemin (Fe3+)- and Heme(Fe2+)-smectite Conjugates in Comparison with Those of Hemi(Fe3+) and Hemo(Fe2+) Proteinsa ligands
λmax
compound
oxidation state of Fe
fifth
sixth
R
β
Soret
(mM)
O2-heme-smectite CO-heme-smectite oxyhemoglobin(1) carboxyhemoglobin(3) hemin-smectite monomeric hematin(1) hemiglobin(3)
Fe2+ Fe2+ Fe2+ Fe2+ Fe3+ Fe3+ Fe3+
(SME (SME His His (SME OHHis
O 2) CO) O2 CO Cl-) H 2O H 2O
568 561 577 570 610 630
534 530 541 538 490 500
411 408 414 418 398 398 406
93 148 125 191 116 122
a The absorption spectra of hemin- and heme-smectite conjugates in water, a transparent colloidal solution, was measured with a Shimazu spectrophotometer MPS-2000 (Kyoto, Japan). Soret (, mM): the peak position (λmax) of Soret band with molar extinction coefficient (, mM).
Figure 3. Absorption spectra of O2-heme (Fe2+)-smectite conjugate (a) and CO-heme (Fe2+)- smectite conjugate (b) in water. (a) Spectrum of O2-heme (Fe2+)-smectite conjugate in water (17 µM) obtained by adding dithionite to hemin (Fe3+)smectite conjugate. The peak positions are located at 568, 534, and 411 nm. (b) Spectrum of CO-heme (Fe2+)-smectite conjugate (17 µM) obtained by adding carbon monoxide, CO, to O2heme (Fe2+)-smectite conjugate. The absorption bands are at 561, 530, and 408 nm which are located at shorter wavelengths compared with the spectrum of O2-heme (Fe2+)-smectite conjugate. Dashed lines shown in panels a and b indicate the spectrum of hemin (Fe3+)-smectite conjugate having the peak positions of 610 and 490 nm and the Soret band at 398 nm.
(g ≈ 6) of methemoglobin (Fe3+) is hardly detected in oxyhemoglobin (HbO2) due to diamagnetism (11). It suggests the possibility of heme (Fe2+)-smectite conjugate being bound to oxygen to form O2-heme (Fe2+)smectite conjugate. Furthermore, the addition of carbon monoxide, CO, to O2-heme (Fe2+)-smectite conjugate caused the formation of CO-heme (Fe2+)-smectite conjugate with the absorption maxima at 561, 530, and 408 nm, which are also similar to carboxyhemoglobin (HbCO) with peak positions at 570, 538, and 418 nm (Figure 3b and Table 1). It is well-known that oxyhemoglobin changes easily to carboxyhemoglobin (HbCO) in the presence of carbon monoxide, CO, with the spectral change as shown in Table 1. A similar phenomenon was observed in heme (Fe2+)-smectite conjugate, in a form of a spectral change of O2-heme (Fe2+)-smectite to CO-heme (Fe2+)-smectite from 568, 534, and 411 nm to 561, 530, and 408 nm, respectively, with a 7-3 nm shift to shorter wavelength as shown in Table 1. The ratio of the molar extinction coefficient of O2-heme (Fe2+)-smectite conjugate and CO-heme (Fe2+)-smectite conjugate was 1:1.6 which is closely agreement with that of oxyhemoglobin and carboxyhemoglobin, 1:1.5 (Table 1). From the results obtained above, it may be concluded that the heme (Fe2+)-
smectite conjugate prepared by the above method is bound to an oxygen molecule to form O2-heme (Fe2+)smectite conjugate. It was previously known in a similar line of study conducted extensively by us that conjugate of tetrapyrrole ring including magnesium or chlorophylls adsorbed onto clay minerals such as bentonite and smectite is closely associated with the development of the photochemical function of chlorophylls (10, 12-14). It is well-known that chlorophylls a and b extracted with organic solvents from fresh green leaves are quite unstable and are rapidly bleached by light-illumination, although chlorophylls bound to protein in chloroplast are quite stable in vivo. In expectation of making chlorophylls much more stable against light, chlorophylls were adsorbed onto clay minerals such as bentonite or smectite in place of protein. Chlorophyll a-smectite conjugate was quite photostable, and the absorption maximum of the conjugate approached 678 nm, which is the absorption maximum of intact spinach leaves. And the chlorophyll-smectite conjugate becomes soluble in aqueous solution although chlorophylls are never soluble in water. The conjugate of chlorophyll a, poly(vinylpyrrolidone), and smectite had an absorption maximum at 677 nm thus becoming more photostable than chlorophyll-smectite conjugate and catalyzing hydrogen gas evolution by visible light in aqueous solution. The similar line of study was conducted by Wang (15): heme embedded in a hydrophobic matrix of polystyrene and 1-(2-phenylethyl)-imidazole has the ability of O2 and CO binding. During the course of the study mentioned above, we happened to prepare hemin (Fe3+)- and heme (Fe2+)smectite conjugates in which smectite, soluble in water, was used in place of the protein of globin in hemoglobin. It was unexpected that the heme (Fe2+)-smectite conjugate is bound to O2 and CO in a manner similar to hemoprotein. Soon, bioconjugate compounds of smectite with chlorophylls are expected to make artificial photosynthesis possible and that of smectite with heme serves as an oxygen carrier in place of hemoglobin. ACKNOWLEDGMENT
The authors are grateful to Dr. Masahiro Kohno, Research and Development Department Analytical Instruments Division, JEOL Ltd., and Dr. Youkoh Kaizu, Professor, Faculty of Science, Department of Chemistry, Tokyo Institute of Technology, for his basic and technical advice in measurement of ESR spectra. We are deeply indebted to professor Saburo Osabe, Toin University of Yokohama, for his assistance in preparation of the manuscript.
6 Bioconjugate Chem., Vol. 12, No. 1, 2001 LITERATURE CITED (1) Inada, Y., and Shibata, K. (1962) The Soret band of monomeric hematin and its changes on polymerization. Biochem. Biophys. Res. Commun. 9, 323-327. (2) Mahler, H. R., and Cordes, E. H. (1969) Biological Chemistry, pp 583-589, Harper & Row, London. (3) Susan, B. (1996) The Merck Index, pp 4667-4668, Merck & CO., Inc. (4) Perutz, M. F. (1969) The hemoglobin molecule. Proc. R. Soc. (B) 173, 113-140. (5) Perutz, M. F. (1964) The hemoglobin molecule. Sci. Am. 211 (5), 64-76. (6) Lehninger, A. L. (1975) Biochemistry, p 145, Worth Publishers Inc., New York. (7) Morimoto, H., Lehmann, H., and Perutz, M. F. (1971) Molecular pathology of human hemoglobin: stereochemical interpretation of abnormal oxygen affinities. Nature 232, 404-413. (8) Torii, K., and Iwasaki, T. (1987) Synthesis of hectorite. Clay Sci. 6, 1-10. (9) Torii, K., and Iwasaki, T. (1986) Synthesis of new trioctahedral Mg-smectite. Chem. Lett. 2021-2024.
Itoh et al. (10) Ishii, A., Itoh, T., Kageyama, H., Mizoguchi, T., Kodera, Y., Matsushima, A., Torii, K., and Inada, Y. (1995) Photostabilization of chlorophyll a adsorbed onto smectite. Dyes Pigm. 28, 77-82. (11) Jones, R. D., Summerville, D. A., and Basolo, F. (1978) Synthetic oxygen carriers related to biological systems. Chem. Rev. 79, 139-179. (12) Kodera, Y., Kageyama, H., Sekine, H., and Inada, Y. (1992) Photostable chlorophylls conjugated with montmorillonite. Biotechnol. Lett. 14, 119-122. (13) Ishii, A., Itoh, T., Kodera, Y., Matsushima, A., Hiroto, M., Nishimura, H., and Inada,Y. (1997) Photostable chlorophyll a-bentonite conjugate exhibits high photosensitive activity. Res. Chem. Intermed. 23, 683-689. (14) Itoh, T. Ishii, A., Kodera, Y., Matsushima, A., Hiroto, M., Nishimura, H., Tsuzuki, T., Kamathi, T., Okura, I., and Inada, Y. (1998) Photostable chlorophyll a with poly(vinylpyrrolidone)-smectite catalyzes photoreduction and hydrogen gas evolution by visible light. Bioconjugate Chem. 9, 409-412. (15) Wang, J. H., (1970) Synthetic biochemical models. Acc. Chem. Res. 3, 90-97.
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