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Evidence that the peroxidase of the fatty acid cyclooxygenase acts via a fenton type of reaction
Prostaglandins Leukotrienes and Medicine 12: 73-76, 1983
EVIDENCE THAT THE PEROXIDASE OF THE FATTY ACID CYCLOOXYGENASE ACTS VIA A FENTON TYPE OF REACTION D. A. Peterson* and J. M. Gerrard** *Clinical Pharmacology, Veterans Administration Hospital, Minneapolis and **Manitoba Institute of Cell Biology, Winnipeg, Manitoba, R3E OV9. Canada. ABSTRACT Addition of ferrous sulfate to a solution containing peroxy-methyl arachidonate resulted in cleavage of the peroxy group on the methyl arachidonate as assessed by absorption at 232nm. The results suggest that ferrous iron can be involved in the reduction of fatty acid peroxides and supports the possibility that the peroxidase component of the fatty acid cyclooxygenase occurs via a Fenton type reaction. INTRODUCTION The fatty acid cyclooxygenase which converts polyunsaturated fatty acids to prostaglandin endoperoxides has two separate enzymatic functions. The first, a prostaglandin synthase converts arachidonic acid to prostaglandin G2. The second, a peroxidase reduces the peroxy moiety to a hydroxyl group. The second step has been extensively studied and some interesting observations have been made. The peroxidase reaction is heme dependent (1). Hydroxyl radical is a byproduct produced during the peroxide decomposition (2). Spectral data with lipoxygenase, an enzyme similar to the cycle-oxygenase and with a similar capacity to reduce peroxides, suggests that during the peroxidase reaction ferrous ion is converted to ferric ion (3-5). During the peroxide reduction, other compounds can be cooxygenated with atmospheric oxygen acting as the source of oxygen (6).
~e~~~~o~~~or;e~~~~~~o~o~e~~~~e~ ;;5q ferrpe~~n~r~~~~~n~~er .
this reaction has come to light. Cohen et al have shown that the _ Fenton reaction involves the consumption of molecular oxygen (7) and that this oxygen can appear in a cooxygenated product (8) similar to the known cooxygenation which occurs during the peroxidase reaction of the cycloxygenase enzyme. A Fenton type reaction involving cyclooxygenase would be asymmetric producing both RO' and 'OH as well as
73
RO- and OH-. The RO' would be expected to be the radical produced in a majority of reactions. In previous studies, we have used the interaction of ferrous iron and arachidonate to probe the mechanism of the cyclooxygenase reaction (g-11), believing this to be a simplified model system in which the interaction of arachidonate with the ferrous iron in the heme of the cyclooxygenase can be evaluated. Ne therefore have now evaluated the peroxidase of the cyclooxygenase enzyme using this system. Since arachidonate itself will be peroxidized by ferrous iron to produce arachidonic acid peroxides as evaluated by absorbance at 232 nm, peroxy arachidonate could not be used to evaluate the peroxidase reaction, because any peroxidase activity might be masked by the formation of new peroxides. However, methyl arachidonate, because it lacks the carboxy group will not form new peroxides in the presence of ferrous iron (9). Thus peroxy methyl arachidonate could be used to evaluate the peroxidase activity of ferrous iron without alternative peroxy reactions occurring. METHODS Methyl arachidonate was obtained from Nu Check Prep (Elysian, Minn.). Hydroperoxy methyl arachidonate was prepared by auto-oxidation of methyl arachidonate in methanol. To study the peroxidase activity of ferrous iron toward peroxidized arachidonate, the peroxy methyl arachidonate in a few microliters of methanol was diluted in 50% ethanol, and combined with ferrous sulfate originally prepared in water and then diluted in the same 50% ethanol solution. The presence of peroxidized methyl arachidonate was confirmed by assessing absorbance at 232 nm. Thirty seconds after combining the ferrous iron and the peroxy methyl arachidonate, peroxides were again measured on the spectraphotometer using absorbance at 232 nm. Background absorbance due to ferrous sulfate was subtracted. RESULTS When ferrous iron was added to the methyl arachidonate there was a reduction in the total amount of peroxides present which was proportional to the amount of ferrous iron added (Figure 1). The result confirms that ferrous iron can interact with peroxidized arachidonic acid derivatives to reduce the peroxide. DISCUSSION The above results showing that ferrous iron can reduce the peroxy group of methyl arachidonic acid show that ferrous iron can reduce ROOH as well as HOOH and in particular can reduce the peroxy group when R is methyl arachidonate. This finding suggests that a Fenton type reaction can be involved in the reduction of the peroxy groups of arachidonic acid and provides support for the concept that it is this type of reaction which is the mechanism of th_eperoxidaee astivlty of the cyclooxygenase enzyme (ROOH+RO' + OH and ROOH-tRC + OH'). The findings are consistent with other facets of the reaction which have been already established (i.e. the requirement for heme, and the conversion
74
Figure 1.
i+ Reduction of peroxymethylarachidonateby Fe . Reactants were combined as described in Methods. The amount of hydroperoxy group remaining at 30 set was evaluated by assessing the absor ante at 232 nm. Background absorbance due to FeB+ was subtracted. Means and standard errors are shown for each group. Samples are based on nine determinations for all but the highest concentration of Fe2'where six separate determinations onlv were performed (* = ~~0.05 ** = p
of Fe2+ to Fe3+; the production of 'OH and 'OR radicals, and the cooxygenation of aromatic compounds using molecular oxygen (l-6). Cyclooxygenase is known to be destroyed by HOOH and lipid peroxides. This step could occur through a site specific Fenton model (8,lZ). This is a Fenton type reaction generating highly reactive species such as RO' and '0 would be expected to react at the site of genesis. This could explain the finding that a radical produced during the cyclooxygenase reaction can inactivate the enzyme (13). It would also explain why GSH protects cyclooxygenase during heme induced destabilization. (14). ACKNOWLEDGEMENTS We gratefully acknowledge the support of grant MA-7396 from the Medical Research Council of Canada and USPHS grant AM12466 to Dr. S. Schwartz. Jonathan M. Gerrard is the recipient of a Canadian MRC scholarship.
76
REFERENCES 1. O'Brien PH, Rahimtula A. The possible involvement of a peroxidase in prostaglandin biosynthesis. Biochim Biophys Res Commun 70~832~838,
1976.
2. O'Brien PJ, Hawes FJ. Hydroxyl-radical formation during prostaglandin formation catalysed by prostaglandin cycooxygenase. Biochem Sot. Trans 6:1169-1171, 1978. 3. Egmond MR, Finazziagro A, Fasella PM, Veldirk GA, Vliegenthart JFG.
Changes in Flourescence and absorbance of lipoxygenase-1 induced by 13-LS-hydroperoxylinoleic acid and linoleic acid. Biochim Biophys Acta 397:43-49, 1976. 4. DeGroot JJMC, Veldink GA, Vliegenthart JFG, Boldingh J, Wever R, Van Gelder BF. Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase. Biochim Biophys Acta 377:71-79, 1971. 5. Axelrod B, Pistorius E, Palmer G. Temperature-dependent forms of lipoxygenase, Fed Proc 34:625, 1975. 6. Marnett LJ, Reed GA, Johnson JT.
Prostaglandin synthetase dependent benze(a)pyrene oxidation: products of the oxidation and inhibition of their formation by antioxidants. Biochim Biophys Res Commun. 79:569-576, 1977.
7. Cohen G, Lewis 0, Sinet PM. Oxygen consumption during the Fenton type reaction between hydrogen peroxide and ferrous chelate (Fe2+DTPA). J. Inoganic Biochem 15:143, 1981 8. Cohen G, Ofodile SE.
Activation of Molecular oxygen during the Fenton reaction: a study with 1802. Abstracts of the Third International Conference on Superoxide and Superoxide Dismutase p.22, 1982.
9. Peterson DA, Gerrard JM, Rao GHR, Krick TP, White JG. Ferrous iron mediated oxidation of arachidonic acid: studies employing nitroblue tetrazolium (NBT). Prostaglandins Med 1:304, 1978. 10. Peterson DA, Gerrard JM, Rao GHR, White JG, Salicylic acid inhibition of the irreversible effect of acetylsalicylic acid on prostaglandin synthetase may be due to competition for the enzyme cationic binding site. Prostaglandins Med 6:161, 1981. 12. Samuni A, Aronovitch J, Clevion M, Czapski G. Cytoxicity of vitamin C and metal irons: a "site-specific Fenton chemistrv" mechanism. Abstract of the Third International Conference on Superoxide and Superoxide Dismutase p. 25, 1982. 13.
Smith WL; Lands WEM. Oxygenation of polyunsaturated fatty acids during prostaglandin biosynthesis by sheep vesicular gland.
14.
Hemler ME, Lands WEM. Protection of cyclooxygenase activity during heme-induced destabilization. Arch Biochem Biophys 201:586-593, 1980. 76
EVIDENCE THAT THE PEROXIDASE OF THE FATTY ACID CYCLOOXYGENASE ACTS VIA A FENTON TYPE OF REACTION D. A. Peterson* and J. M. Gerrard** *Clinical Pharmacology, Veterans Administration Hospital, Minneapolis and **Manitoba Institute of Cell Biology, Winnipeg, Manitoba, R3E OV9. Canada. ABSTRACT Addition of ferrous sulfate to a solution containing peroxy-methyl arachidonate resulted in cleavage of the peroxy group on the methyl arachidonate as assessed by absorption at 232nm. The results suggest that ferrous iron can be involved in the reduction of fatty acid peroxides and supports the possibility that the peroxidase component of the fatty acid cyclooxygenase occurs via a Fenton type reaction. INTRODUCTION The fatty acid cyclooxygenase which converts polyunsaturated fatty acids to prostaglandin endoperoxides has two separate enzymatic functions. The first, a prostaglandin synthase converts arachidonic acid to prostaglandin G2. The second, a peroxidase reduces the peroxy moiety to a hydroxyl group. The second step has been extensively studied and some interesting observations have been made. The peroxidase reaction is heme dependent (1). Hydroxyl radical is a byproduct produced during the peroxide decomposition (2). Spectral data with lipoxygenase, an enzyme similar to the cycle-oxygenase and with a similar capacity to reduce peroxides, suggests that during the peroxidase reaction ferrous ion is converted to ferric ion (3-5). During the peroxide reduction, other compounds can be cooxygenated with atmospheric oxygen acting as the source of oxygen (6).
~e~~~~o~~~or;e~~~~~~o~o~e~~~~e~ ;;5q ferrpe~~n~r~~~~~n~~er .
this reaction has come to light. Cohen et al have shown that the _ Fenton reaction involves the consumption of molecular oxygen (7) and that this oxygen can appear in a cooxygenated product (8) similar to the known cooxygenation which occurs during the peroxidase reaction of the cycloxygenase enzyme. A Fenton type reaction involving cyclooxygenase would be asymmetric producing both RO' and 'OH as well as
73
RO- and OH-. The RO' would be expected to be the radical produced in a majority of reactions. In previous studies, we have used the interaction of ferrous iron and arachidonate to probe the mechanism of the cyclooxygenase reaction (g-11), believing this to be a simplified model system in which the interaction of arachidonate with the ferrous iron in the heme of the cyclooxygenase can be evaluated. Ne therefore have now evaluated the peroxidase of the cyclooxygenase enzyme using this system. Since arachidonate itself will be peroxidized by ferrous iron to produce arachidonic acid peroxides as evaluated by absorbance at 232 nm, peroxy arachidonate could not be used to evaluate the peroxidase reaction, because any peroxidase activity might be masked by the formation of new peroxides. However, methyl arachidonate, because it lacks the carboxy group will not form new peroxides in the presence of ferrous iron (9). Thus peroxy methyl arachidonate could be used to evaluate the peroxidase activity of ferrous iron without alternative peroxy reactions occurring. METHODS Methyl arachidonate was obtained from Nu Check Prep (Elysian, Minn.). Hydroperoxy methyl arachidonate was prepared by auto-oxidation of methyl arachidonate in methanol. To study the peroxidase activity of ferrous iron toward peroxidized arachidonate, the peroxy methyl arachidonate in a few microliters of methanol was diluted in 50% ethanol, and combined with ferrous sulfate originally prepared in water and then diluted in the same 50% ethanol solution. The presence of peroxidized methyl arachidonate was confirmed by assessing absorbance at 232 nm. Thirty seconds after combining the ferrous iron and the peroxy methyl arachidonate, peroxides were again measured on the spectraphotometer using absorbance at 232 nm. Background absorbance due to ferrous sulfate was subtracted. RESULTS When ferrous iron was added to the methyl arachidonate there was a reduction in the total amount of peroxides present which was proportional to the amount of ferrous iron added (Figure 1). The result confirms that ferrous iron can interact with peroxidized arachidonic acid derivatives to reduce the peroxide. DISCUSSION The above results showing that ferrous iron can reduce the peroxy group of methyl arachidonic acid show that ferrous iron can reduce ROOH as well as HOOH and in particular can reduce the peroxy group when R is methyl arachidonate. This finding suggests that a Fenton type reaction can be involved in the reduction of the peroxy groups of arachidonic acid and provides support for the concept that it is this type of reaction which is the mechanism of th_eperoxidaee astivlty of the cyclooxygenase enzyme (ROOH+RO' + OH and ROOH-tRC + OH'). The findings are consistent with other facets of the reaction which have been already established (i.e. the requirement for heme, and the conversion
74
Figure 1.
i+ Reduction of peroxymethylarachidonateby Fe . Reactants were combined as described in Methods. The amount of hydroperoxy group remaining at 30 set was evaluated by assessing the absor ante at 232 nm. Background absorbance due to FeB+ was subtracted. Means and standard errors are shown for each group. Samples are based on nine determinations for all but the highest concentration of Fe2'where six separate determinations onlv were performed (* = ~~0.05 ** = p
of Fe2+ to Fe3+; the production of 'OH and 'OR radicals, and the cooxygenation of aromatic compounds using molecular oxygen (l-6). Cyclooxygenase is known to be destroyed by HOOH and lipid peroxides. This step could occur through a site specific Fenton model (8,lZ). This is a Fenton type reaction generating highly reactive species such as RO' and '0 would be expected to react at the site of genesis. This could explain the finding that a radical produced during the cyclooxygenase reaction can inactivate the enzyme (13). It would also explain why GSH protects cyclooxygenase during heme induced destabilization. (14). ACKNOWLEDGEMENTS We gratefully acknowledge the support of grant MA-7396 from the Medical Research Council of Canada and USPHS grant AM12466 to Dr. S. Schwartz. Jonathan M. Gerrard is the recipient of a Canadian MRC scholarship.
76
REFERENCES 1. O'Brien PH, Rahimtula A. The possible involvement of a peroxidase in prostaglandin biosynthesis. Biochim Biophys Res Commun 70~832~838,
1976.
2. O'Brien PJ, Hawes FJ. Hydroxyl-radical formation during prostaglandin formation catalysed by prostaglandin cycooxygenase. Biochem Sot. Trans 6:1169-1171, 1978. 3. Egmond MR, Finazziagro A, Fasella PM, Veldirk GA, Vliegenthart JFG.
Changes in Flourescence and absorbance of lipoxygenase-1 induced by 13-LS-hydroperoxylinoleic acid and linoleic acid. Biochim Biophys Acta 397:43-49, 1976. 4. DeGroot JJMC, Veldink GA, Vliegenthart JFG, Boldingh J, Wever R, Van Gelder BF. Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase. Biochim Biophys Acta 377:71-79, 1971. 5. Axelrod B, Pistorius E, Palmer G. Temperature-dependent forms of lipoxygenase, Fed Proc 34:625, 1975. 6. Marnett LJ, Reed GA, Johnson JT.
Prostaglandin synthetase dependent benze(a)pyrene oxidation: products of the oxidation and inhibition of their formation by antioxidants. Biochim Biophys Res Commun. 79:569-576, 1977.
7. Cohen G, Lewis 0, Sinet PM. Oxygen consumption during the Fenton type reaction between hydrogen peroxide and ferrous chelate (Fe2+DTPA). J. Inoganic Biochem 15:143, 1981 8. Cohen G, Ofodile SE.
Activation of Molecular oxygen during the Fenton reaction: a study with 1802. Abstracts of the Third International Conference on Superoxide and Superoxide Dismutase p.22, 1982.
9. Peterson DA, Gerrard JM, Rao GHR, Krick TP, White JG. Ferrous iron mediated oxidation of arachidonic acid: studies employing nitroblue tetrazolium (NBT). Prostaglandins Med 1:304, 1978. 10. Peterson DA, Gerrard JM, Rao GHR, White JG, Salicylic acid inhibition of the irreversible effect of acetylsalicylic acid on prostaglandin synthetase may be due to competition for the enzyme cationic binding site. Prostaglandins Med 6:161, 1981. 12. Samuni A, Aronovitch J, Clevion M, Czapski G. Cytoxicity of vitamin C and metal irons: a "site-specific Fenton chemistrv" mechanism. Abstract of the Third International Conference on Superoxide and Superoxide Dismutase p. 25, 1982. 13.
Smith WL; Lands WEM. Oxygenation of polyunsaturated fatty acids during prostaglandin biosynthesis by sheep vesicular gland.
14.
Hemler ME, Lands WEM. Protection of cyclooxygenase activity during heme-induced destabilization. Arch Biochem Biophys 201:586-593, 1980. 76