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Oxidative burst by acellular haemoglobin and neurotransmitters
Medical Hypotheses (2002) 59(1), 11–15 ª 2002 Elsevier Science Ltd. All rights reserved. doi: 10.1016/S0306-9877(02)00197-4, available online at http://www.idealibrary.com
Oxidative burst by acellular haemoglobin and neurotransmitters T. Kawano,1 H. Hosoya2 1
Hiroshima University, Higashi-Hiroshima, Japan; 2PRESTO, Japan Science and Technology Corporation (JST), Japan
Summary Acellular haemoglobin (Hb) has intrinsic toxicity to the tissues since harmful reactive oxygen species are readily produced during auto-oxidation of Hb. On the other hand, Hb is known to have peroxidase-like activity monovalently oxidizing various peroxidase substrates. Thus, monovalently oxidized organic free radical species may be produced. This may relay the radical reactions leading to the production of reactive oxygen species such as superoxide. Such substrates possibly generating superoxide, include aromatic monoamines such as neurotransmitters and their precursors rich in neural a tissues. Based on our knowledge on the reactivity of haemoproteins against phenolics and aromatic monoamines, we proposed a hindered danger in use of Hb as a reperfusion agent. Clinical use of recently developing Hb-based blood substitutes must be reconsidered. ª 2002 Elsevier Science Ltd. All rights reserved. INTRODUCTION
Hb–FeII þ O2 $ Hb–FeIII O2
½1
Human haemoglobin (Hb) consists of a- and b-polypeptide chains, and a non-protein component within each polypeptide chain containing a prosthetic haem group that reversibly binds one oxygen molecule. Recently Hb solution-based blood substitutes are developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock (1). However, cell-free Hb has intrinsic toxicity to tissues since it causes distortion in the vascular oxidant/antioxidant balance (1,2), and it readily produces the reactive oxygen species during its auto-oxidation from ferrous state to the ferric state (3). There are some reports concerning the generation of reactive oxygen species due to the auto-oxidation of Hb (1). It is well known that superoxide anion (O 2 ) is produced by spontaneous oxidation of the ferrous haem iron of oxyHb (FeII –O2 ) as the following:
Hb–FeII O2 ! Hb–FeII þ O 2
½2
Received 22 October 2001 Accepted 20 December 2001 Correspondence to: Dr. Tomonori Kawano, Department of Biological Science, Graduate School of Science, Hiroshima University, HigashiHiroshima 739-8526, Japan. Phone: +81 824 24 7443; Fax: +81 824 24 0734; E-mail: [email protected]
Basic problems are associated with the use of Hb outside the erythrocytes. Erythrocytes provide abundant antioxidant enzymes such as catalase and superoxide dismutase (SOD), that catalyse the breakdown of H2 O2 and O 2 , respectively, and the reductase systems that catalyse the reduction of ferric iron (FeIII ) back to the ferrous state, the only form that reversibly binds O2 (4). In addition to auto-oxidation of Hb, there may be another mechanism that produces reactive oxygen species. When Hb is circulated in the vascular system as a blood substitute, H2 O2 naturally supplied by platelets, neutrophils and macrophages may react with the deoxy or ferric forms of Hb and consequently the highly reactive ferryl Hb intermediates are readily formed (5). Formation of ferryl Hb is initially accompanied with globincentred free radicals (6). Formation of the ferryl Hb is problematic since its pseudoperoxidase activity results in peroxidation of lipids, degradation of carbohydrates, and modification of proteins despite its short half-life (7). In this paper, we propound a hindered danger of Hb’s pseudoperoxidase activity in clinical use of Hb as reperfusion agent. Based on our knowledge of the chemistry
11
12 Kawano and Hosoya
of haemoproteins, we assume that the Hb’s peroxidatic reactivity against neurotransmitters and their precursors such as b-phenylethylamine (PEA) may lead to burst of O 2 and/or monovalent oxidation of ascorbate to ascorbate free radicals (Asc ). PSEUDOPEROXIDASE CYCLE Hb has been known to possess a weak peroxidase activity since it degrades H2 O2 in peroxidase-like manner (8), but it has no catalase-like activity (7). Other haemoproteins possess the properties similar to Hb, and thus studies on various haemoproteins such as myoglobin (Mb) and horseradhish peroxidase (HRP) provide us with the general knowledge about the redox chemistry of haems. When reacted with H2 O2 , both peroxidase and catalase primarily form the oxidized intermediate Compound I (9,10). This intermediate is formed by accepting both oxidizing equivalents of H2 O2 , thus compound I contains two additional oxidizing equivalents over the native ferric form of the enzyme (9,11). The heam iron of Compound I is known to be at ferryl (FeIV ¼ O) state (12), but the absorption maxima of Compounds I of various proteins display varied features. This has been attributed to the localization site of the second oxidizing equivalent (11). For HRP (12), chloroperoxidase (13), plant ascorbate perodixdase (14), prostaglandin H synthase (15) and lignin peroxidase (11), the second oxidizing equivalent is stored as a porphyrin p-cation radical. However, for compound ESs of yeast and Pseudomonas cytochrome c peroxidase (16,17), horse myoglobin (18), leghaemoglobin (Lb) (19) and human Hb (20), the second oxidizing equivalent is on an amino acid side chain of the protein. Recently it has been shown that catalase compound I has a porphyrin p-cation radical and it is likely being converted to tyrosyl radical on protein depending on pH and temperature (21). The FeIV haemoproteins produced by reaction of metHb with H2 O2 , has highly reactive nature and may cause oxidative damages to vascular systems (1). Although there is no in vivo evidence in support of the cytotoxicity of cell-free Hb directly associated with the ferryl iron, a causative role for the redox cycling of Mb in rhabdomyolysis-induced renal failure has recently been reported (22). In human Hb (4,22) and sperm whale Mb (1), ferryl intermediates are formed in a similar manner. The oxidative power of the ferryl haemoproteins lies within its high redox potential (E0 ¼ þ1:4 V for FeIV compared with +1.5 V and +0.04 V for FeII –O2 and FeIII , respectively) (7). Thus, the transition of Hb to a redoxactive compound sets Hb aside from other known bloodderived biologics and may also explain some of the unique toxicological effects associated with its use in clinical medicine. This can be best illustrated by a recent
Medical Hypotheses (2002) 59(1), 11–15
study by Goldman and Breyer (23), in which a modified Hb prepared from cross-linking of a-chains by bis(3,5dibromosalicyl)fumarate was shown to produce both necrotic and apoptotic cell deaths in the presence of trace H2 O2 in bovine endothelial cell culture. A persistent ferryl form of this cross-linked Hb was detected in an endothelial cell model of ischemia/reperfusion without the addition of H2 O2 to the system. The long-lived ferryl intermediate of the cross-linked Hb was correlated with the increases in release of lactate dehydrogenase and in the extent of DNA fragmentation in these cells. There has been an implication that the ferryl state of Hb is involved in oxidation of aromatic amines in erythrocytes (24), supporting our hypothesis that Hb can oxidize PEA and related amines. However, the mechanism and involvement of the Hb-catalysed amine oxidation in reactive oxygen species production have not been reported. It has been intensively studied how the ferryl state of HRP (Compounds I and II) oxidizes the anilino substrates (25). We have been studying the biological significance of plant peroxidases in production of reactive oxygen species coupled to oxidation of salicylates (26– 28) or various amines including PEA (29–31), and proposed a mechanism of monoamine-dependent production of O by plant peroxidases such as HRP and 2 guaiacol-utilizing peroxidase from tobacco cell culture as follows: POX þ H2 O2 ! Compound I þ H2 O ½3 Compound I þ AH ! A þ Compound II
½4
Compound II þ AH ! A þ POX
½5
2A þ 2O2 ! 2Aþ þ 2O 2
½6
where POX is a plant peroxidase, AH is a monoamine, A is a monoamine-derived free radical species and Aþ is the two-electron oxidized intermediate product of monoamines. During the peroxidase-catalysed reaction, monoamines and H2 O2 may act as the donor and acceptor of electron exchange, respectively, finally yielding monoamine-derived free radical species. Compounds I and II are considered to possess haems at FeIV states with and without additional porphyrin radicals, respectively (32), thus the compounds I and II are analogous to ferryl Hb with and without additional globin radicals, respectively. We hypothesize that O 2 Is possibly generated in Hb solution by the mechanism coupled to the oxidation of aromatic monoamines as follows: Hb–FeIV ðþglobin radicalsÞ þ AH ! Hb–FeIV þ A
½7
Hb–FeIV þ AH ! Hb–FeIII þ A
½8
A þ O2 ! Aþ þ O 2
½9
ª 2002 Elsevier Science Ltd. All rights reserved.
Oxidative burst by acellular haemoglobin and neurotransmitters
13
There H2 O2 , the product of concomitant O 2 dismutation, may reproduce the globin radical-associated Hb– FeIV from Hb–FeII and Hb–FeIII by two-electron and one-electron oxidation, respectively. Thus O 2 can be produced continuously until the substrate is run out or the reaction is terminated by addition of scavengers of reactive oxygen species such as thiols. Asc AS AN INDICATOR FOR MONOAMINEDERIVED FREE RADICAL SPECIES Here we propose the possible experimental procedure for examination of our hypotheses that Hb catalyses the oxidation of aromatic amines, by using electron spin resonance (ESR) spectroscopic analysis. We have previously observed the aromatic monoamine-dependent production of Asc in a model system for the haemoprotein-catalysed reactions, employing tobacco peroxidase and HRP (27,33). Haemoproteins like plant peroxidases are considered to catalyse the generation of organic free radicals derived from aromatic monoamines, although only indirect evidence has been obtained (29–31). The lifetime of such monoamine-derived free radicals may be too short to detect their signals by conventional ESR measurements. Since such radical species are highly reactive against ascorbate, ascorbatemediated ESR method may be applicable to detect the monoamine-derived free radicals. Formation of such radicals may be detectable in the reaction mixture containing Hb and aromatic monoamines, by measuring the ESR signals reflecting the production of Asc , according to the following reaction: Ascorbate þ A ! Asc þ AH
½10
where A and AH are aromatic monoamine-derived free radical species and regenerated aromatic monoamines, respectively. Here we speculate that Asc formation is observable as a consequence of Hb–FeIV -catalysed oxidation of aromatic monoamines consequently releasing the monoamine-derived radicals. In the near future, this experiment should be carried out to examine our hypothesis. MODEL MECHANISM Lastly, we illustrate the pathways forming metHb and possible reactions to be catalysed by metHb (Fig. 1). DeoxyHb reversibly binds O2 and forms oxyHb, and O2 is released at the site of low pH and high CO2 concentrations (Fig. 1A). By auto-oxidation, oxyHb is converted to metHb and bound O2 is monovalently reduced to O 2 (Fig. 1B). This process is similar to the mechanism for release of O from plant peroxidase Compound III 2 formed in the presence of plant hormone, indole-3-aceª 2002 Elsevier Science Ltd. All rights reserved.
Fig. 1 Models for the mechanism of Hb-catalysed oxidation of aromatic monoamines, and generation of superoxide anion. A, oxygen affinity greatly affected by CO2 concentration; B, generation of superoxide (O 2 ) coupled to auto-oxidation of oxyHb to met Hb; C, pseudoperoxidase cycle involved in oxidation of monoamines; D, the O 2 generating reaction involving O2 and aromatic monoaminederived free radicals. Numbers indicate the formal oxidation state of Hb. Disproportionation of O 2 to H2 O2 shown in (B) may be catalysed by haem iron of Hb and also by SOD in vivo.
tic acid (33). DeoxyHb is also oxidized and yield metHb. Then resultant metHb can enter the pseudoperoxidase cycle in the presence of trace of H2 O2 produced via autooxidation of oxyHb (Fig. 1C). Then ferryl Hb associated with globin radicals (FeIV : O [globin]þ ) equivalent to compound I of peroxidase is formed. Thus, without addition of H2 O2 to the system, a certain amount of metHb may be converted to the FeIV : O [globin]þ form of Hb in the presence of trace H2 O2 derived from auto-oxidation. Addition of H2 O2 to the system may enhance the conversion of metHb to the FeIV : O½globinþ form of Hb, and thus potentiates the pseudoperoxidative substrate oxidation. This form of Hb (FeIV : O [globin]þ ) may monovalentiy oxidize monoamines (AH) to monoamine radicals (A ) which may further react with O2 to yield O 2 (Fig. 1D). In the above reaction, Ferry Hb without globin radicals, equivalent to peroxidase compound II may be formed. This form of Hb (FeIV : O) also catalyses the monovalent oxidation of aromatic monoamines, again Medical Hypotheses (2002) 59(1), 11–15
14 Kawano and Hosoya
yielding O 2 (Fig. 1D). Consequently metHb is yielded again and it may re-enter the cycle in the presence of H2 O2 derived from O 2 . CONCLUSION Based on our previous works on plant peroxidase-catalysed oxidation of aromatic monoamines and production of O 2 and ascorbate free radicals (8,9), we propose that the production of O 2 and ascorbate free radicals are possibly catalysed by the pseudoperoxidase activity of Hb in the presence of aromatic monoamines and related substances, when acellular Hb is artificially introduced to the vascular system and conveyed to the monoaminerich neural tissues. As a consequence, formation of aromatic monoamine-derived free radicals may be formed and react with molecular oxygen (thus reactive oxygen species are formed) or ascorbate (thus ascorbate free radicals are formed). The former may potentially cause damage to the tissues rich in aromatic monoamines, if the Hb-based blood substitutes were circulated without addition of free radical scavengers. REFERENCES 1. Alayash A. J. Hemoglobin-based blood substitutes: oxygen carriers, pressor agents, or oxidants. Nat Biotechnol 1999; 17: 545–549. 2. Darley-Usmar V. W., Radomski R. Free radicals in the vasculature – the good, the bad, and the ugly. Biochemist (Bull Biochem Soc) 1994: 13–17. 3. Motterlini R., Foresti R., Vandegriff K. D. et al. Oxidative stress response in vascular endothelial cells exposed to acellular hemoglobin solution. Am J Physiol 1995; 269: H648–H655. 4. Bunn H. F., Forget B. G. Hemoglobin: Molecular Genetic and Clinical Aspects. Philadelphia, PA: W.B. Saunders, 1986. 5. King N. K., Winfield M. E. The mechanism of metmyogloblin oxidation. J Biol Chem 1963; 238: 1520– 1528. 6. Patel R. P., Svistunenko D. A., Darley-Usmar V. M. et al. Redox cycling of human methaemoglobin by H2 O2 yields persistent ferryl iron and protein based radicals. Free Radic Res 1996; 25: 117–123. 7. Yamada T., Volkmer C., Grisham M. B. The effects of sulfasalazine metabolites on hemoglobin-catalyzed lipid peroxidation. Free Radic Biol Med 1991; 10: 41–49. 8. Miller Y. I., Altamentova S. M., Shaklai N. Oxidation of lowdensity lipoprotein by hemoglobin stems from a hemeinitiated globin radical: antioxidant role of heptoglobin. Biochemistry 1997; 36: 12189–12198. 9. Dunford H. B. One-electron oxidations by peroxidases. Xenobiotica 1995; 25: 725–733. 10. Matsuura T., Miyai K., Trakulnaleamsai S. et al. Evolutionary molecular engineering by random elongation mutagenesis. Nat Biotechnol 1999; 17: 58–61. 11. Renganathan V., Gold M. H. Spectoral characterization of the oxidised status of lignin peroxidase, an extracellular heme enzyme from the white rot basidiomycete
Medical Hypotheses (2002) 59(1), 11–15
Phanerochaete chrysosporium. Biochemistry 1986; 25: 1626–1631. 12. Schulz C. E., Rutter R., Sage J. T. et al. Mossbauer and electron paramagnetic resonance studies of horseradish peroxidase and its catalytic intermediates. Biochemistry 1984; 23: 4743–4754. 13. Rutter R., Hager L. P. The detection of two electron paramagnetic resonance radical signals associated with chloroperoxidase compound I. J Biol Chem 1982; 257: 7958–7961. 14. Marquez L. A., Quitoriano M., Zilinskas B. A. et al. Kinetic and spectral properties of pea cytosolic ascorbate peroxidase. FEBS Lett 1996; 389: 153–156. 15. Hsuanyu Y. C., Dunford H. B. Reduction of prostaglandin H synthase compound II by phenol and hydroquinone, and the effect of indomethacin. Arch Biochem Biophys 1992; 292: 213–220. 16. Lang G., Spartalian K., Yonetani T. Mossbauer spectroscopic study of compound ES of cytochrome c peroxidase. Biochim Biophys Acta 1976; 451: 250–258. 17. Ronnberg M., Lambeir A. M., Ellfolk N. et al. A rapid-scan spectrometric and stopped-flow study of compound I and compound II of Pseudomonas cytochrome c peroxidase. Arch Biochem Biophys 1985; 236: 714–719. 18. Davies M. J. Identification of a globin free radical in equine myoglobin treated with peroxides. Biochim Biophys Acta 1991; 1077: 86–90. 19. Davies M. J., Puppo A. Identification of the site of the globin-derived radical in leghaemoglobins. Biochim Biophys Acta 1993; 1202: 182–188. 20. Svistunenko D. A., Patel R. P., Voloshenko S. V. et al. The globin-based radical of ferryl hemoglobin is detected in normal human blood. J Biol Chem 1997; 272: 7114–7121. 21. Ivancich A., Jouve H. M., Sartor B. et al. EPR investigation of compound I in Proteus mirabilis and bovine liver catalases: formation of porphyrin and tyrosyl radical intermediates. Biochemistry 1997; 36: 9356–9364. 22. Moore K. P., Holt S. G., Patel R. P. et al. A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure. J Biol Chem 1998; 273: 31731–31737. 23. Goldman D. W., Breyer R. J., Yeh D. et al. Acellular hemoglobin-mediated oxidative stress toward endothelium: a role for ferryl iron. Am J Physiol 1998; 44: H1046–1053. 24. Golly I., Hlavica P. The role of hemoglobin in the Noxidation of 4-chloroaniline. Biochim Biophys Acta 1983; 760: 69–76. 25. Kuo C. F., Fridovich I. Stimulation of the activity of horseradish peroxidase by nitrogenous compounds. J Biol Chem 1988; 263: 3811–3817. 26. Kawano T., Sahashi N., Takahashi K. et al. Salicylic acid induces extracellular superoxide generation followed by an increase in cytosolic calcium ion in tobacco suspension culture: The earliest events in salicylic acid signal transduction. Plant Cell Physiol 1998; 39: 721–730. 27. Kawano T., Muto S. Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture. J Exp Bot 2000; 51: 685–693. 28. Mori I. C., Pinontoan R., Kawano T. et al. Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba. Plant Cell Physiol 2001; 42: 1383–1388.
ª 2002 Elsevier Science Ltd. All rights reserved.
Oxidative burst by acellular haemoglobin and neurotransmitters
29. Kawano T., Pinontoan R., Uozumi N. et al. Aromatic monoamine-induced immediate oxidative burst leading to an increase in cytosolic Ca2þ concentration in tobacco suspension culture. Plant Cell Physiol 2000; 41: 1251– 1258. 30. Kawano T., Pinontoan R., Uozumi N. et al. Phenylethylamine-induced generation of reactive oxygen species and ascorbate free radicals in tobacco suspension culture: mechanism for oxidative burst mediating Ca2þ movements. Plant Cell Physiol 2000; 41: 1259–1266. 31. Kawano T., Sahashi N., Uozumi N. et al. Involvement of apoplastic peroxidase in the chitosaccharide-induced
ª 2002 Elsevier Science Ltd. All rights reserved.
15
immediate oxidative burst and a cytosolic Ca2þ increase in tobacco suspension culture. Plant Peroxidase Newslett 2000; 14: 117–124. 32. Rutter R., Valentine M., Hendrich M. P. et al. Chemical nature of the porphyrin pi cation radical in horseradish peroxidase compound I. Biochemistry 2000; 22: 4769–4774 (1983). 33. Kawano T., Kawano N., Hosoya H. et al. A fungal auxin antagonist, hypaphorine competitively inhibits indole-3acetic acid-dependent superoxide generation by horseradish peroxidase. Biochem Biophys Res Commun 2001; 288: 546–551.
Medical Hypotheses (2002) 59(1), 11–15
Oxidative burst by acellular haemoglobin and neurotransmitters T. Kawano,1 H. Hosoya2 1
Hiroshima University, Higashi-Hiroshima, Japan; 2PRESTO, Japan Science and Technology Corporation (JST), Japan
Summary Acellular haemoglobin (Hb) has intrinsic toxicity to the tissues since harmful reactive oxygen species are readily produced during auto-oxidation of Hb. On the other hand, Hb is known to have peroxidase-like activity monovalently oxidizing various peroxidase substrates. Thus, monovalently oxidized organic free radical species may be produced. This may relay the radical reactions leading to the production of reactive oxygen species such as superoxide. Such substrates possibly generating superoxide, include aromatic monoamines such as neurotransmitters and their precursors rich in neural a tissues. Based on our knowledge on the reactivity of haemoproteins against phenolics and aromatic monoamines, we proposed a hindered danger in use of Hb as a reperfusion agent. Clinical use of recently developing Hb-based blood substitutes must be reconsidered. ª 2002 Elsevier Science Ltd. All rights reserved. INTRODUCTION
Hb–FeII þ O2 $ Hb–FeIII O2
½1
Human haemoglobin (Hb) consists of a- and b-polypeptide chains, and a non-protein component within each polypeptide chain containing a prosthetic haem group that reversibly binds one oxygen molecule. Recently Hb solution-based blood substitutes are developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock (1). However, cell-free Hb has intrinsic toxicity to tissues since it causes distortion in the vascular oxidant/antioxidant balance (1,2), and it readily produces the reactive oxygen species during its auto-oxidation from ferrous state to the ferric state (3). There are some reports concerning the generation of reactive oxygen species due to the auto-oxidation of Hb (1). It is well known that superoxide anion (O 2 ) is produced by spontaneous oxidation of the ferrous haem iron of oxyHb (FeII –O2 ) as the following:
Hb–FeII O2 ! Hb–FeII þ O 2
½2
Received 22 October 2001 Accepted 20 December 2001 Correspondence to: Dr. Tomonori Kawano, Department of Biological Science, Graduate School of Science, Hiroshima University, HigashiHiroshima 739-8526, Japan. Phone: +81 824 24 7443; Fax: +81 824 24 0734; E-mail: [email protected]
Basic problems are associated with the use of Hb outside the erythrocytes. Erythrocytes provide abundant antioxidant enzymes such as catalase and superoxide dismutase (SOD), that catalyse the breakdown of H2 O2 and O 2 , respectively, and the reductase systems that catalyse the reduction of ferric iron (FeIII ) back to the ferrous state, the only form that reversibly binds O2 (4). In addition to auto-oxidation of Hb, there may be another mechanism that produces reactive oxygen species. When Hb is circulated in the vascular system as a blood substitute, H2 O2 naturally supplied by platelets, neutrophils and macrophages may react with the deoxy or ferric forms of Hb and consequently the highly reactive ferryl Hb intermediates are readily formed (5). Formation of ferryl Hb is initially accompanied with globincentred free radicals (6). Formation of the ferryl Hb is problematic since its pseudoperoxidase activity results in peroxidation of lipids, degradation of carbohydrates, and modification of proteins despite its short half-life (7). In this paper, we propound a hindered danger of Hb’s pseudoperoxidase activity in clinical use of Hb as reperfusion agent. Based on our knowledge of the chemistry
11
12 Kawano and Hosoya
of haemoproteins, we assume that the Hb’s peroxidatic reactivity against neurotransmitters and their precursors such as b-phenylethylamine (PEA) may lead to burst of O 2 and/or monovalent oxidation of ascorbate to ascorbate free radicals (Asc ). PSEUDOPEROXIDASE CYCLE Hb has been known to possess a weak peroxidase activity since it degrades H2 O2 in peroxidase-like manner (8), but it has no catalase-like activity (7). Other haemoproteins possess the properties similar to Hb, and thus studies on various haemoproteins such as myoglobin (Mb) and horseradhish peroxidase (HRP) provide us with the general knowledge about the redox chemistry of haems. When reacted with H2 O2 , both peroxidase and catalase primarily form the oxidized intermediate Compound I (9,10). This intermediate is formed by accepting both oxidizing equivalents of H2 O2 , thus compound I contains two additional oxidizing equivalents over the native ferric form of the enzyme (9,11). The heam iron of Compound I is known to be at ferryl (FeIV ¼ O) state (12), but the absorption maxima of Compounds I of various proteins display varied features. This has been attributed to the localization site of the second oxidizing equivalent (11). For HRP (12), chloroperoxidase (13), plant ascorbate perodixdase (14), prostaglandin H synthase (15) and lignin peroxidase (11), the second oxidizing equivalent is stored as a porphyrin p-cation radical. However, for compound ESs of yeast and Pseudomonas cytochrome c peroxidase (16,17), horse myoglobin (18), leghaemoglobin (Lb) (19) and human Hb (20), the second oxidizing equivalent is on an amino acid side chain of the protein. Recently it has been shown that catalase compound I has a porphyrin p-cation radical and it is likely being converted to tyrosyl radical on protein depending on pH and temperature (21). The FeIV haemoproteins produced by reaction of metHb with H2 O2 , has highly reactive nature and may cause oxidative damages to vascular systems (1). Although there is no in vivo evidence in support of the cytotoxicity of cell-free Hb directly associated with the ferryl iron, a causative role for the redox cycling of Mb in rhabdomyolysis-induced renal failure has recently been reported (22). In human Hb (4,22) and sperm whale Mb (1), ferryl intermediates are formed in a similar manner. The oxidative power of the ferryl haemoproteins lies within its high redox potential (E0 ¼ þ1:4 V for FeIV compared with +1.5 V and +0.04 V for FeII –O2 and FeIII , respectively) (7). Thus, the transition of Hb to a redoxactive compound sets Hb aside from other known bloodderived biologics and may also explain some of the unique toxicological effects associated with its use in clinical medicine. This can be best illustrated by a recent
Medical Hypotheses (2002) 59(1), 11–15
study by Goldman and Breyer (23), in which a modified Hb prepared from cross-linking of a-chains by bis(3,5dibromosalicyl)fumarate was shown to produce both necrotic and apoptotic cell deaths in the presence of trace H2 O2 in bovine endothelial cell culture. A persistent ferryl form of this cross-linked Hb was detected in an endothelial cell model of ischemia/reperfusion without the addition of H2 O2 to the system. The long-lived ferryl intermediate of the cross-linked Hb was correlated with the increases in release of lactate dehydrogenase and in the extent of DNA fragmentation in these cells. There has been an implication that the ferryl state of Hb is involved in oxidation of aromatic amines in erythrocytes (24), supporting our hypothesis that Hb can oxidize PEA and related amines. However, the mechanism and involvement of the Hb-catalysed amine oxidation in reactive oxygen species production have not been reported. It has been intensively studied how the ferryl state of HRP (Compounds I and II) oxidizes the anilino substrates (25). We have been studying the biological significance of plant peroxidases in production of reactive oxygen species coupled to oxidation of salicylates (26– 28) or various amines including PEA (29–31), and proposed a mechanism of monoamine-dependent production of O by plant peroxidases such as HRP and 2 guaiacol-utilizing peroxidase from tobacco cell culture as follows: POX þ H2 O2 ! Compound I þ H2 O ½3 Compound I þ AH ! A þ Compound II
½4
Compound II þ AH ! A þ POX
½5
2A þ 2O2 ! 2Aþ þ 2O 2
½6
where POX is a plant peroxidase, AH is a monoamine, A is a monoamine-derived free radical species and Aþ is the two-electron oxidized intermediate product of monoamines. During the peroxidase-catalysed reaction, monoamines and H2 O2 may act as the donor and acceptor of electron exchange, respectively, finally yielding monoamine-derived free radical species. Compounds I and II are considered to possess haems at FeIV states with and without additional porphyrin radicals, respectively (32), thus the compounds I and II are analogous to ferryl Hb with and without additional globin radicals, respectively. We hypothesize that O 2 Is possibly generated in Hb solution by the mechanism coupled to the oxidation of aromatic monoamines as follows: Hb–FeIV ðþglobin radicalsÞ þ AH ! Hb–FeIV þ A
½7
Hb–FeIV þ AH ! Hb–FeIII þ A
½8
A þ O2 ! Aþ þ O 2
½9
ª 2002 Elsevier Science Ltd. All rights reserved.
Oxidative burst by acellular haemoglobin and neurotransmitters
13
There H2 O2 , the product of concomitant O 2 dismutation, may reproduce the globin radical-associated Hb– FeIV from Hb–FeII and Hb–FeIII by two-electron and one-electron oxidation, respectively. Thus O 2 can be produced continuously until the substrate is run out or the reaction is terminated by addition of scavengers of reactive oxygen species such as thiols. Asc AS AN INDICATOR FOR MONOAMINEDERIVED FREE RADICAL SPECIES Here we propose the possible experimental procedure for examination of our hypotheses that Hb catalyses the oxidation of aromatic amines, by using electron spin resonance (ESR) spectroscopic analysis. We have previously observed the aromatic monoamine-dependent production of Asc in a model system for the haemoprotein-catalysed reactions, employing tobacco peroxidase and HRP (27,33). Haemoproteins like plant peroxidases are considered to catalyse the generation of organic free radicals derived from aromatic monoamines, although only indirect evidence has been obtained (29–31). The lifetime of such monoamine-derived free radicals may be too short to detect their signals by conventional ESR measurements. Since such radical species are highly reactive against ascorbate, ascorbatemediated ESR method may be applicable to detect the monoamine-derived free radicals. Formation of such radicals may be detectable in the reaction mixture containing Hb and aromatic monoamines, by measuring the ESR signals reflecting the production of Asc , according to the following reaction: Ascorbate þ A ! Asc þ AH
½10
where A and AH are aromatic monoamine-derived free radical species and regenerated aromatic monoamines, respectively. Here we speculate that Asc formation is observable as a consequence of Hb–FeIV -catalysed oxidation of aromatic monoamines consequently releasing the monoamine-derived radicals. In the near future, this experiment should be carried out to examine our hypothesis. MODEL MECHANISM Lastly, we illustrate the pathways forming metHb and possible reactions to be catalysed by metHb (Fig. 1). DeoxyHb reversibly binds O2 and forms oxyHb, and O2 is released at the site of low pH and high CO2 concentrations (Fig. 1A). By auto-oxidation, oxyHb is converted to metHb and bound O2 is monovalently reduced to O 2 (Fig. 1B). This process is similar to the mechanism for release of O from plant peroxidase Compound III 2 formed in the presence of plant hormone, indole-3-aceª 2002 Elsevier Science Ltd. All rights reserved.
Fig. 1 Models for the mechanism of Hb-catalysed oxidation of aromatic monoamines, and generation of superoxide anion. A, oxygen affinity greatly affected by CO2 concentration; B, generation of superoxide (O 2 ) coupled to auto-oxidation of oxyHb to met Hb; C, pseudoperoxidase cycle involved in oxidation of monoamines; D, the O 2 generating reaction involving O2 and aromatic monoaminederived free radicals. Numbers indicate the formal oxidation state of Hb. Disproportionation of O 2 to H2 O2 shown in (B) may be catalysed by haem iron of Hb and also by SOD in vivo.
tic acid (33). DeoxyHb is also oxidized and yield metHb. Then resultant metHb can enter the pseudoperoxidase cycle in the presence of trace of H2 O2 produced via autooxidation of oxyHb (Fig. 1C). Then ferryl Hb associated with globin radicals (FeIV : O [globin]þ ) equivalent to compound I of peroxidase is formed. Thus, without addition of H2 O2 to the system, a certain amount of metHb may be converted to the FeIV : O [globin]þ form of Hb in the presence of trace H2 O2 derived from auto-oxidation. Addition of H2 O2 to the system may enhance the conversion of metHb to the FeIV : O½globinþ form of Hb, and thus potentiates the pseudoperoxidative substrate oxidation. This form of Hb (FeIV : O [globin]þ ) may monovalentiy oxidize monoamines (AH) to monoamine radicals (A ) which may further react with O2 to yield O 2 (Fig. 1D). In the above reaction, Ferry Hb without globin radicals, equivalent to peroxidase compound II may be formed. This form of Hb (FeIV : O) also catalyses the monovalent oxidation of aromatic monoamines, again Medical Hypotheses (2002) 59(1), 11–15
14 Kawano and Hosoya
yielding O 2 (Fig. 1D). Consequently metHb is yielded again and it may re-enter the cycle in the presence of H2 O2 derived from O 2 . CONCLUSION Based on our previous works on plant peroxidase-catalysed oxidation of aromatic monoamines and production of O 2 and ascorbate free radicals (8,9), we propose that the production of O 2 and ascorbate free radicals are possibly catalysed by the pseudoperoxidase activity of Hb in the presence of aromatic monoamines and related substances, when acellular Hb is artificially introduced to the vascular system and conveyed to the monoaminerich neural tissues. As a consequence, formation of aromatic monoamine-derived free radicals may be formed and react with molecular oxygen (thus reactive oxygen species are formed) or ascorbate (thus ascorbate free radicals are formed). The former may potentially cause damage to the tissues rich in aromatic monoamines, if the Hb-based blood substitutes were circulated without addition of free radical scavengers. REFERENCES 1. Alayash A. J. Hemoglobin-based blood substitutes: oxygen carriers, pressor agents, or oxidants. Nat Biotechnol 1999; 17: 545–549. 2. Darley-Usmar V. W., Radomski R. Free radicals in the vasculature – the good, the bad, and the ugly. Biochemist (Bull Biochem Soc) 1994: 13–17. 3. Motterlini R., Foresti R., Vandegriff K. D. et al. Oxidative stress response in vascular endothelial cells exposed to acellular hemoglobin solution. Am J Physiol 1995; 269: H648–H655. 4. Bunn H. F., Forget B. G. Hemoglobin: Molecular Genetic and Clinical Aspects. Philadelphia, PA: W.B. Saunders, 1986. 5. King N. K., Winfield M. E. The mechanism of metmyogloblin oxidation. J Biol Chem 1963; 238: 1520– 1528. 6. Patel R. P., Svistunenko D. A., Darley-Usmar V. M. et al. Redox cycling of human methaemoglobin by H2 O2 yields persistent ferryl iron and protein based radicals. Free Radic Res 1996; 25: 117–123. 7. Yamada T., Volkmer C., Grisham M. B. The effects of sulfasalazine metabolites on hemoglobin-catalyzed lipid peroxidation. Free Radic Biol Med 1991; 10: 41–49. 8. Miller Y. I., Altamentova S. M., Shaklai N. Oxidation of lowdensity lipoprotein by hemoglobin stems from a hemeinitiated globin radical: antioxidant role of heptoglobin. Biochemistry 1997; 36: 12189–12198. 9. Dunford H. B. One-electron oxidations by peroxidases. Xenobiotica 1995; 25: 725–733. 10. Matsuura T., Miyai K., Trakulnaleamsai S. et al. Evolutionary molecular engineering by random elongation mutagenesis. Nat Biotechnol 1999; 17: 58–61. 11. Renganathan V., Gold M. H. Spectoral characterization of the oxidised status of lignin peroxidase, an extracellular heme enzyme from the white rot basidiomycete
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