/ . Biochem., 80, 397-399 (1976).
PRELIMINARY COMMUNICATION
Generation of the Superoxide Radical during Autoxidation of Oxymyoglobin Toshio GOTOH* and Keiji SHIKAMA** *Departraent of Biology, College of General Education, University of Tokushima, Tokushima, Tokushima 770, and **Biological Institute, Faculty of Science, Tohoku University, Sendai, Miyagi 980 Received for publication, March 19, 1976
Autoxidation of bovine oxymyoglobin to metmyoglobin induces co-oxidation of epinephrine to adrenochrome. This co-oxidation is markedly inhibited by superoxide dismutase [EC 1.15.1.1]. Electron transfer from oxymyoglobin to ferricytochrome c is partially inhibited by superoxide dismutase. These results indicate that autoxidation of oxymyoglobin results in generation of superoxide radicals. Autoxidation of oxymyoglobin is accelerated by superoxide dismutase and partially inhibited by catalase [EC 1.11.1.6].
Weiss first proposed that oxyhemoglobin could be described as a superoxo-ferriheme complex (Fes+Os~) formed by electron transfer from iron to molecular oxygen (7). Therefore, it has been suggested that a superoxide free radical anion, O£~ may be split off directly from the iron during the slow autoxidation of oxyhemoglobin to methemoglobin and in fact, there are recent reports of the generation of O2~ during autoxidation of shark hemoglobin (2), bovine hemoglobin (3), and isolated a-, and ^-chains of human hemoglobin (4). Previously, we reported that native oxymyoglobin (MbOi) is readily autoxidized to metmyoglobin (MbFe3+) with a half-life of less than 1.5 days at a physiological pH and temperature under air saturated conditions (5). However, there seems to be no proof that O r is liberated during autoxidation of
MbOj.
Abbreviations : MbO,, oxymyoglobin ; MbFeJ+, metmyoglobin. Vol. 80, No. 2, 1976
397
Therefore, in this work we examined whether O2" is actually produced during autoxidation of MbOi using superoxide dismutase [EC 1.15.1.1], which catalyzes the disproportionation of O r anions; 2 0 r + 2H + ->0,+Hi0i, and so inhibits reactions caused by O r (6). Native MbOj was isolated from bovine heart muscle (5, 7). Superoxide dismutase was purified from bovine red blood cells (6). Horse heart cytochrome c, type VI, was a product of Sigma Chemical Co. This protein was completely oxidized with KsFe(CN)j in distilled water and the products of the oxidizing agent were removed by passing the solution through Sephadex G-25 and a mixed bed ion-exchange resin (Bio-Rad AG 501-X8). The concentrations of the metal proteins were determined using the following extinction coefficients: 15.5 mM"1-cm"1 at 581 nm for MbOi, 0.3 mM-'-cm" 1 at 680 nm for superoxide dismutase, and 27.5 mM' 1 -cm' 1 at 550nm for
398
ferrocytochrome c. Bovine liver catalase [EC 1.11.1.6] was obtained from Sigma Chemical Co. Protein concentration was determined by the method of Lowry et al. (8). Generation of 0j~ during autoxidation of MbOj was examined in two different ways. One was to test the effect of superoxide dismutase on reduction of ferricytochrome c by MbO2 in 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and saturated with air at 25" by recording the spectra of myoglobin (25 fiM) and cytochrome c (25 fiM) in the incubation mixture from 500 to 600 nm at intervals of 40 min. The other way was to test the effect of superoxide dismutase on oxidation of epinephrine coupled with autoxidation of MbO2 by incubating MbOj (50 fiM) with epinephrine (0.5 mM) in 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and saturated with air at 37° and following the formation of adrenochrome by measuring the change in absorbancy at 525 nm, the isosbestic point of MbO2 and MbFe*+. Spectroscopic measurements were carried out with a Hitachi model 124 Spectrophotometer. MbO2 was oxidized very fast in the presence of ferricytochrome c with concomitant reduction of the latter, as reported previously (9, 10), and this could be clearly followed as a decrease in absorbancy at 581 nm (a-maximum of MbOj) with increase in absorbancy at 550 nm (a-maximum of ferrocytochrome c). The results in Fig. 1 show that superoxide dismutase (1 fiM) partially inhibited the coreduction of ferricytochrome c by MbO 2 . No further inhibition was observed in the presence of 10 ^M superoxide dismutase, and superoxide dismutase did not oxidize ferrocytochrome c in the absence of myoglobin. These results clearly indicate that O r is generated during the autoxidation of MbO,, and that there are two pathways for electron transfer from MbOj to ferricytochrome c: one that is sensitive to superoxide dismutase, and so mediated by O r generated during autoxidation of MbOi, and the other that is insensitive to superoxide dismutase, and that may proceed through the direct interaction of myoglobin with cytochrome c. It is interesting that the lower
PRELIMINARY COMMUNICATION
line in Fig. 1 began to curve 2 hr after the start of the reaction. This suggests that after 2 hr most of the cytochrome c reduction was mediated by Ot". Further studies are in progress on this phenomenon. The co-reduction of cytochrome c was not affected significantly by catalase (0.2 fiM) either in the presence or absence of superoxide dismutase. It is known that Oi~ initiates the oxidation of epinephrine to adrenochrome (6). Figure 2 shows that autoxidation of MbOj caused concomitant oxidation of epinephrine to adrenochrome, and that this co-oxidation was markedly inhibited by superoxide dismutase (1 f^M). Under the experimental conditions used, autoxidation of epinephrine was negligible. These results again indicate that O2~ is generated during the autoxidation of MbO 2 . The lag phase seen from the curve for cooxidation seems to be due mainly to complicated chain reactions involved in the oxidation of epinephrine to adrenochrome ( / / ) . The co-oxidation of epinephrine was inhibited by catalase (0.2 ftM) to almost the same extent as by superoxide dismutase (1 pM) and it was inhibited almost completely by a combination of these enzymes. The explanation of the effect of catalase on the co-oxidation of
Fig. 1. Effect of superoxide dismutase on reduction of cytochrome c during MbO2 autoxidation. Reaction mixtures consisted of 25 pM ferricytochrome c and 25 fiM MbO, in 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and saturated with air at 25°. The absorbancies at 550 nm and 581 nm were recorded at intervals of 40 min. O, without superoxide dismutase, • , with ouperoxide dismutase (1 I'M). /. Biochem.
GENERATION OF O r DURING AUTOXIDATION OF MbO,
40
Fig. 2. Effect of superoxide dismutase on oxidation of epinephrine during MbOj autoxidation. Reaction mixtures contained 0.5 mM epinephrine (Control), and where indicated 50 fiM MbO, without or with 1 fM superoxide dismutase (SOD) at 37°. Formation of adrenochrome was followed at 525 nm. Other experimental conditions were as for Fig. 1.
399
pH 7.0. It was also found that at pH 10.2 the initial rate of accumulation of adrenochrome during the autoxidation of epinephrine was not affected by catalase. The direct effects of superoxide dismutase and catalase on autoxidation of MbO2 are shown in Fig. 3. The autoxidation of MbOi was accelerated by superoxide dismutase and partially inhibited by catalase. Superoxide dismutase seems to act as a scavenger of O r , accelerating the following over-all reaction: MbO 2 ^MbFe 8 + + O r . On the other hand, the effect of catalase suggests that the conversion of MbO2 to MbFe3+ is partly due to HtOt. Recently, interest has increased in reactions resulting in formation of O2~ that may be toxic to living cells (12, 13). Since native MbOj is much more readily autoxidizable than hemoglobin, the cytotoxic effects of O2~ generated from MbO2 should be considered, especially in myoglobin-rich tissues such as cardiac muscle and tissues of aquatic mammals. REFERENCES
-03 •
T 9 Time(hr) Fig. 3. Effects of superoxide dismutase and catalase on MbO2 autoxidation. Reaction mixtures contained 50 ptM MbO, in 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and saturated with air at 37°. O, control; • , with superoxide dismutase (1 ftM); e , with catalase (0.2 //M).
epinephrine is unknown. The possibility that H2O2 as well as O2~ propagates the chain reactions in the oxidation of epinephrine is excluded by the fact that H2Oi did not stimulate the autoxidation of epinephrine at
Vol. 80, No. 2, 1976
1. Weiss, J J . (1964) Nature 202, 83-84 2. Misra, H.P. & Fridovich, I. (1972) /. Biol. Chetn. 247, 6960-6962 3. Wever, R., Oudega, B., & Van Gelder, B.F. (1973) Biochim. Biophys. Ada 302, 475-478 4. Brunori, M., Falcioni, G., Fioretti, E., Giardina, B., & Rotilio, G. (1975) Eur. J. Biochem. 53, 99-104 5. Gotoh, T. & Shikama, K. (1974) Arch. Biochem. Biophys. 163, 476-481 6. McCord, J.M. & Fridovich, I. (1969) /. Biol. Chem. 244, 6049-6055 7. Gotoh, T., Ochiai, T., & Shikama, K. (1971) / . Chromatogr. 60, 260-264 8. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 9. Yamazaki, I., Yokota, K., & Shikama, K. (1964) / . Bid. Chem. 239, 4151-4153 10. Wu, C.C., Duffy, P., & Brown, W.D. (1972) /. Biol. Chem. 247, 1899-1903 11. Misra, H.P. & Fridovich, I. (1972) /. Biol. Chem. 247, 3170-3175 12. McCord, J.M., Keele, B.B., Jr., & Fridovich, I. (1971) Proc. Nail. Acad. Sci. U.S. 68, 1024-1027 13. Fridovich, I. (1975) Ann. Rev. Biochem. 44, 147-159
PRELIMINARY COMMUNICATION
Generation of the Superoxide Radical during Autoxidation of Oxymyoglobin Toshio GOTOH* and Keiji SHIKAMA** *Departraent of Biology, College of General Education, University of Tokushima, Tokushima, Tokushima 770, and **Biological Institute, Faculty of Science, Tohoku University, Sendai, Miyagi 980 Received for publication, March 19, 1976
Autoxidation of bovine oxymyoglobin to metmyoglobin induces co-oxidation of epinephrine to adrenochrome. This co-oxidation is markedly inhibited by superoxide dismutase [EC 1.15.1.1]. Electron transfer from oxymyoglobin to ferricytochrome c is partially inhibited by superoxide dismutase. These results indicate that autoxidation of oxymyoglobin results in generation of superoxide radicals. Autoxidation of oxymyoglobin is accelerated by superoxide dismutase and partially inhibited by catalase [EC 1.11.1.6].
Weiss first proposed that oxyhemoglobin could be described as a superoxo-ferriheme complex (Fes+Os~) formed by electron transfer from iron to molecular oxygen (7). Therefore, it has been suggested that a superoxide free radical anion, O£~ may be split off directly from the iron during the slow autoxidation of oxyhemoglobin to methemoglobin and in fact, there are recent reports of the generation of O2~ during autoxidation of shark hemoglobin (2), bovine hemoglobin (3), and isolated a-, and ^-chains of human hemoglobin (4). Previously, we reported that native oxymyoglobin (MbOi) is readily autoxidized to metmyoglobin (MbFe3+) with a half-life of less than 1.5 days at a physiological pH and temperature under air saturated conditions (5). However, there seems to be no proof that O r is liberated during autoxidation of
MbOj.
Abbreviations : MbO,, oxymyoglobin ; MbFeJ+, metmyoglobin. Vol. 80, No. 2, 1976
397
Therefore, in this work we examined whether O2" is actually produced during autoxidation of MbOi using superoxide dismutase [EC 1.15.1.1], which catalyzes the disproportionation of O r anions; 2 0 r + 2H + ->0,+Hi0i, and so inhibits reactions caused by O r (6). Native MbOj was isolated from bovine heart muscle (5, 7). Superoxide dismutase was purified from bovine red blood cells (6). Horse heart cytochrome c, type VI, was a product of Sigma Chemical Co. This protein was completely oxidized with KsFe(CN)j in distilled water and the products of the oxidizing agent were removed by passing the solution through Sephadex G-25 and a mixed bed ion-exchange resin (Bio-Rad AG 501-X8). The concentrations of the metal proteins were determined using the following extinction coefficients: 15.5 mM"1-cm"1 at 581 nm for MbOi, 0.3 mM-'-cm" 1 at 680 nm for superoxide dismutase, and 27.5 mM' 1 -cm' 1 at 550nm for
398
ferrocytochrome c. Bovine liver catalase [EC 1.11.1.6] was obtained from Sigma Chemical Co. Protein concentration was determined by the method of Lowry et al. (8). Generation of 0j~ during autoxidation of MbOj was examined in two different ways. One was to test the effect of superoxide dismutase on reduction of ferricytochrome c by MbO2 in 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and saturated with air at 25" by recording the spectra of myoglobin (25 fiM) and cytochrome c (25 fiM) in the incubation mixture from 500 to 600 nm at intervals of 40 min. The other way was to test the effect of superoxide dismutase on oxidation of epinephrine coupled with autoxidation of MbO2 by incubating MbOj (50 fiM) with epinephrine (0.5 mM) in 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and saturated with air at 37° and following the formation of adrenochrome by measuring the change in absorbancy at 525 nm, the isosbestic point of MbO2 and MbFe*+. Spectroscopic measurements were carried out with a Hitachi model 124 Spectrophotometer. MbO2 was oxidized very fast in the presence of ferricytochrome c with concomitant reduction of the latter, as reported previously (9, 10), and this could be clearly followed as a decrease in absorbancy at 581 nm (a-maximum of MbOj) with increase in absorbancy at 550 nm (a-maximum of ferrocytochrome c). The results in Fig. 1 show that superoxide dismutase (1 fiM) partially inhibited the coreduction of ferricytochrome c by MbO 2 . No further inhibition was observed in the presence of 10 ^M superoxide dismutase, and superoxide dismutase did not oxidize ferrocytochrome c in the absence of myoglobin. These results clearly indicate that O r is generated during the autoxidation of MbO,, and that there are two pathways for electron transfer from MbOj to ferricytochrome c: one that is sensitive to superoxide dismutase, and so mediated by O r generated during autoxidation of MbOi, and the other that is insensitive to superoxide dismutase, and that may proceed through the direct interaction of myoglobin with cytochrome c. It is interesting that the lower
PRELIMINARY COMMUNICATION
line in Fig. 1 began to curve 2 hr after the start of the reaction. This suggests that after 2 hr most of the cytochrome c reduction was mediated by Ot". Further studies are in progress on this phenomenon. The co-reduction of cytochrome c was not affected significantly by catalase (0.2 fiM) either in the presence or absence of superoxide dismutase. It is known that Oi~ initiates the oxidation of epinephrine to adrenochrome (6). Figure 2 shows that autoxidation of MbOj caused concomitant oxidation of epinephrine to adrenochrome, and that this co-oxidation was markedly inhibited by superoxide dismutase (1 f^M). Under the experimental conditions used, autoxidation of epinephrine was negligible. These results again indicate that O2~ is generated during the autoxidation of MbO 2 . The lag phase seen from the curve for cooxidation seems to be due mainly to complicated chain reactions involved in the oxidation of epinephrine to adrenochrome ( / / ) . The co-oxidation of epinephrine was inhibited by catalase (0.2 ftM) to almost the same extent as by superoxide dismutase (1 pM) and it was inhibited almost completely by a combination of these enzymes. The explanation of the effect of catalase on the co-oxidation of
Fig. 1. Effect of superoxide dismutase on reduction of cytochrome c during MbO2 autoxidation. Reaction mixtures consisted of 25 pM ferricytochrome c and 25 fiM MbO, in 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and saturated with air at 25°. The absorbancies at 550 nm and 581 nm were recorded at intervals of 40 min. O, without superoxide dismutase, • , with ouperoxide dismutase (1 I'M). /. Biochem.
GENERATION OF O r DURING AUTOXIDATION OF MbO,
40
Fig. 2. Effect of superoxide dismutase on oxidation of epinephrine during MbOj autoxidation. Reaction mixtures contained 0.5 mM epinephrine (Control), and where indicated 50 fiM MbO, without or with 1 fM superoxide dismutase (SOD) at 37°. Formation of adrenochrome was followed at 525 nm. Other experimental conditions were as for Fig. 1.
399
pH 7.0. It was also found that at pH 10.2 the initial rate of accumulation of adrenochrome during the autoxidation of epinephrine was not affected by catalase. The direct effects of superoxide dismutase and catalase on autoxidation of MbO2 are shown in Fig. 3. The autoxidation of MbOi was accelerated by superoxide dismutase and partially inhibited by catalase. Superoxide dismutase seems to act as a scavenger of O r , accelerating the following over-all reaction: MbO 2 ^MbFe 8 + + O r . On the other hand, the effect of catalase suggests that the conversion of MbO2 to MbFe3+ is partly due to HtOt. Recently, interest has increased in reactions resulting in formation of O2~ that may be toxic to living cells (12, 13). Since native MbOj is much more readily autoxidizable than hemoglobin, the cytotoxic effects of O2~ generated from MbO2 should be considered, especially in myoglobin-rich tissues such as cardiac muscle and tissues of aquatic mammals. REFERENCES
-03 •
T 9 Time(hr) Fig. 3. Effects of superoxide dismutase and catalase on MbO2 autoxidation. Reaction mixtures contained 50 ptM MbO, in 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and saturated with air at 37°. O, control; • , with superoxide dismutase (1 ftM); e , with catalase (0.2 //M).
epinephrine is unknown. The possibility that H2O2 as well as O2~ propagates the chain reactions in the oxidation of epinephrine is excluded by the fact that H2Oi did not stimulate the autoxidation of epinephrine at
Vol. 80, No. 2, 1976
1. Weiss, J J . (1964) Nature 202, 83-84 2. Misra, H.P. & Fridovich, I. (1972) /. Biol. Chetn. 247, 6960-6962 3. Wever, R., Oudega, B., & Van Gelder, B.F. (1973) Biochim. Biophys. Ada 302, 475-478 4. Brunori, M., Falcioni, G., Fioretti, E., Giardina, B., & Rotilio, G. (1975) Eur. J. Biochem. 53, 99-104 5. Gotoh, T. & Shikama, K. (1974) Arch. Biochem. Biophys. 163, 476-481 6. McCord, J.M. & Fridovich, I. (1969) /. Biol. Chem. 244, 6049-6055 7. Gotoh, T., Ochiai, T., & Shikama, K. (1971) / . Chromatogr. 60, 260-264 8. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 9. Yamazaki, I., Yokota, K., & Shikama, K. (1964) / . Bid. Chem. 239, 4151-4153 10. Wu, C.C., Duffy, P., & Brown, W.D. (1972) /. Biol. Chem. 247, 1899-1903 11. Misra, H.P. & Fridovich, I. (1972) /. Biol. Chem. 247, 3170-3175 12. McCord, J.M., Keele, B.B., Jr., & Fridovich, I. (1971) Proc. Nail. Acad. Sci. U.S. 68, 1024-1027 13. Fridovich, I. (1975) Ann. Rev. Biochem. 44, 147-159
