Reduction
of ferrylmyoglobin
LYNNE Institute
EDDY,
LYNNE,
in rat diaphragm
EDDY, ARDUINO ARDUINI, AND PAUL HOCHSTEIN for Toxicology, University of Southern California, Los Angeles, California
ARDUINO
ARDUINI,
AND
PAUL
HOCHSTEIN.
Reduction of ferrylmyoglobin in rat diaphragm. Am. J. Physiol. 259 (Cell Physiol. 28): C995-C997, 1990.-The oxidation of myoglobin was monitored by transmission spectroscopy in isolated, superfused preparations of rat diaphragms. In its deoxygenated form, during anoxia, myoglobin was oxidized by adding hydrogen peroxide (1.0 mM) to its ferry1 form (Fe’“). On the other hand, peroxide-induced formation of ferrylmyoglobin was not observed when the perfusate contained oxygen. Ferrylmyoglobin was visualized after its derivatization with NazS to form sulfmyoglobin. Depending on the time of addition, ascorbate (4.0 mM) or ergothioneine (2.0 mM) either prevented the formation of or dissipated ferrylmyoglobin. These agents are known to be reductants of this hypervalent form of myoglobin. In addition to providing the first demonstration of ferrylmyoglobin in skeletal muscle, these observations are consistent with the concept that the oxidation of myoglobin to hypervalent states might be an important event in the initiation of muscle damage associated with anoxia and reoxygenation. The rapid reduction of myoglobin would prevent peroxidatic alterations of essential cellular constituents by ferrylmyoglobin. myoglobin;
ascorbate; ergothioneine
HAVE PREVIOUSLY SUGGESTED that the oxidation of myoglobin to its ferry1 state may be a critical event in cardiac muscle damage associated with episodes of ischemia and reperfusion (8). In part, the basis for this suggestion is the association of hydrogen peroxide formation, as well as other oxidants, with such episodes (13), the special sensitivity of deoxymyoglobin (Fe”) to oxidation by hydrogen peroxide to form ferrylmyoglobin (FeIV) (8, 17), and the capacity of reductants of ferrylmyoglobin to protect the isolated heart against reperfusion injury (8). As a result of these observations on the facile redox cycling of myoglobin, we have called attention to the potential therapeutic value of one-electron agents such as ascorbate and ergothioneine in preventing damage associated with the formation of hydrogen peroxide and the accumulation of ferrylmyoglobin during postischemic oxygenation of muscle (8). The redox chemistry and functions of myoglobin have been investigated for many years (5, 16). However, the detection of ferrylmyoglobin in situ is often complicated by the presence of cytochromes that absorb light at interfering wavelengths. A potentially useful solution to this problem has been to derivatize ferrylmyoglobin with WE
0363-6143/90 $1.50 Copyright
0
90033
Na2S (2). This agent forms a spectrally distinct species, sulfmyoglobin, with an absorption maximum at 618 nm (2). We have utilized this method to detect the formation of ferrylmyoglobin in postischemic cardiac muscle by reflectance spectroscopy (7). The aim of the experiments described in this paper was to determine whether a similar approach could be utilized along with transmission rather than reflectance spectroscopy to identify peroxide-dependent formation of ferrylmyoglobin in the isolated rat diaphragm. The hemoglobin-free, superfused diaphragm was used because of its high myoglobin-to-cytochrome c ratio (6) and because this tissue is sufficiently thin to permit transmission spectroscopy. The formation of ferrylmyoglobin has not been demonstrated previously in skeletal muscle. METHODS
After anesthesia with pentobarbital sodium (30 mg/kg intraperitoneally) and heparinization (500 U intravenously), rats were perfused with buffer through the aorta by a pump to remove blood from the tissues. KrebsHenseleit buffer of the following composition was used throughout the experiments (final concentration in mM): 118 NaCl, 4.7 KCl, 2.5 CaCIZ, 1.2 MgS04, 1.2 KH2P04, 0.5 CaEDTA, 25 NaHCOs, and 10 glucose, pH 7.4. The diaphragm was removed and bisected. The isolated hemidiaphragm was sutured to a small plastic frame and placed within a covered 3-ml cuvette. The cuvette was superfused from an enclosed reservoir with buffer using a peristaltic pump at a flow rate of 2.5 ml/min. Oxygenation was maintained by bubbling the reservoir with 95% 02-5% C02. Anoxia was initiated by changing the gas to 95% Nz-5% CO,. Twenty-five minutes of anoxia were used to convert oxymyoglobin to deoxymyoglobin. Hydrogen peroxide, 1.0 mM, was added to the reservoir and circulation was continued for 20 min. In some experiments, catalase (400 U/ml) was then added to reduce any hydrogen peroxide still present. However, identical results were obtained when catalase was not present. Ferrylmyoglobin was indirectly determined as the ferrosulfmyoglobin derivative after addition of 1.0 mM NazS (2). Ascorbic acid, 4.0 mM, and ergothioneine, 2.0 mM, when used, were added to the recirculating buffer either before hydrogen peroxide or 20 min after hydrogen peroxide addition. Spectra were recorded from 500-650 nm, using 540 nm as the reference wavelength (Shimadzu UV
1990 the American
Physiological
Society
c995
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 4, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
C996
FERRYLMYOGLOBIN
3000 dual wavelength-double beam spectrophotometer). Difference spectra were constructed using the SpectroCalc computer package (Galactic Industries, Salem, NH) and an IBM-PC computer. RESULTS
Although not illustrated, the difference spectra obtained between the oxygenated and the deoxygenated diaphragms were dominated by myoglobin and were similar to those observed by Tamura et al. (14) for purified myoglobin and in the isolated rat heart. In addition, the inclusion of hydrogen peroxide in the perfusion medium caused no alterations in the spectra of oxygenated diaphragms, either in the absence of Na2S or after its inclusion in the perfusate. On the other hand, there was a striking increase in absorption at 617 nm when Na2S was added to the perfusate buffer after treatment of deoxygenated diaphragms with hydrogen peroxide. This effect is illustrated in curue B of Fig. 1. The appearance of this new peak in the difference spectrum between peroxidetreated and untreated diaphragms strongly implicates the formation of ferrylmyoglobin from deoxymyoglobin and its derivatization by Na2S to sulfmyoglobin. Using the wavelength pair 550-620 nm (EmM = 11) for deoxymyoglobin, derived from Ref. 1 and the difference spectra (617-600 nm) after the addition of Na2S (EmM = 10.5) (l5), we calculated that -35% of the deoxymyoglobin was converted to ferrylmyoglobin in these experiments. Curve A of Fig. I also shows that, when either ascorbate (4.0 mM) or ergothioneine (2.0 mM) was present in the perfusate, the difference spectra between deoxygenated and oxygenated diaphragms were unaltered despite the presence of hydrogen peroxide and NaS. Identical results were obtained when these reducing agents were added before the peroxide or 20 min after the peroxide but 10 min before NaS. It should be mentioned that the shoulder in the difference spectra below 600 nm is unexplained.
600
650 Nanometers
FIG. 1. Curve B: difference spectrum (600-650 nm) obtained after addition of Na2S (1.0 mM) to deoxygenated diaphragm exposed to hydrogen peroxide (1.0 mM). Na2S was added 20 min after peroxide, and spectrum was recorded within 5 min. Curve A: difference spectrum obtained when ascorbate (4.0 mM) was included in perfusion medium before addition of peroxide. Identical spectra were observed when ascorbate was added after addition of peroxide or when ergothioneine (2.0 mM) was substituted for ascorbate. Spectral determinations were carried out on at least triplicate diaphragm preparations with nearly identical results.
REDUCTION
DISCUSSION
The experiments described above illustrate the u.sefulness of the rodent diaphragm in studies of the redox state of myoglobi n. The thin .ness of the diap h.ragm and the high ratio of myoglobin to cytochromes (6) permit such studies by transmission spectroscopy rather than by more sophisticated reflectance techniques. We previously have utilized the latter method to demonstrate the accumulation of ferrylmyoglobin from deoxymyoglobin in the perfused heart after brief (30 min) periods of ischemia (7). The detection of ferrylmyoglobin in situ in the diaphragm only after anoxia affirms the previously reported in vitro sensitivity of deoxymyoglobin, as compared with oxymyoglobin, to oxidation by peroxide (8, 17). As was the case in the heart, reducing agents that rapidly convert ferrylmyoglobin to metmyoglobin and ferrousmyoglobin either prevent the formation of or apparently dissipate ferrylmyoglobin in the diaphragm, depending on the time of their addition in relation to peroxide. It should be noted that the amounts of hydrogen peroxide utilized in these experiments are probably severa1 orders of magnitude higher than those that might be generated endogenously as a response to reoxygenation of occluded or hypoxic muscle. For this reason, these experiments are not evidence for a pathophysiological role for hydrogen peroxide in postanoxic injury. The high concentrations of peroxide utilized are apparently necessary because of endogenous catalase activity. It is reasonable and more likely that endogenously generated peroxide, in more intimate association with myoglobin, might be more effective in the oxidation of myoglobin than that added in a perfusing buffer. It is of special interest that reducing agents have been used as cardioprotective agents in various animal models of ischemia-reperfusion. Mitsos et al. have shown with N-2-mercaptopropionyl glycine (MPG) that not only does this agent salvage potentially necrotic cardiac tissue in a canine model of ischemia-reperfusion (11) but that it can inhibit myoglobin-hydrogen peroxide-induced peroxidation of uric acid and arachidonic acid (12). Bolli et al. have demonstrated improved cardiac function after ischemia-reperfusion in dogs treated with dimethylthiourea (4) and MPG (3). However, they credited the protective response to hydroxyl radical scavenging. We suggest that the favorable responses to reducing agents may be related to reduction of oxidized myoglobin. Such a concept is based on the well-known peroxidatic activity of ferrylmyoglobin toward a variety of essential cellular constituents (9, 10). A. Arduini is a Visiting Scientist from the University of Chieti and was supported, in part, by Sigma Tau, SPA, Pomezia, Italy. This work was supported also by grants from the Ciba Geigy Corporation and the Los Angeles Affiliate of the American Heart Association. Received
20 February
1990; accepted
in final
form
3 August
1990.
REFERENCES 1. ANTONINI, E., AND M. BRUNORI. Hemoglobin and Myoglobin in Their Reactions with Ligands. Amsterdam: North Holland, 1971, p. 17. 2. BERZOFSKY, J. A.,J. PEISACH,AND W. E. BLUMBERG. Sulfheme
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 4, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
FERRYLMYOGLOBIN
3.
4.
5. 6.
7.
8.
9.
10.
proteins. 1. Optical and magnetic properties of sulfmyoglobin and its derivatives. J. Biol. Chem. 246: 3367-3377, 1971. BOLLI, R., M. 0. JEROUDI, B. S. PATEL, 0. I. ARUOMA, B. HALLIWELL, E. K. LAI, AND P. B. MCCAY. Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion. Circ. Res. 65: 607-622, 1989. BOLLI, R., W.-X. ZHU, C. J. HARTLEY, L. H. MICHAEL, J. E. REPINE, M. L. HESS, R. C. KUKREJA, AND R. ROBERTS. Attenuation of dysfunction in the postischemic “stunned” myocardium by dimethylthiourea. Circulation 76: 458-468, 1987. COLE, R. P. Myoglobin function in exercising skeletal muscle. Science Wash. DC 216: 523-525, 1982. DRABKIN, D. L. The distribution of the chromoproteins, hemoglobin, myoglobin, and cytochrome c in the tissues of different species, and the relationship of the total content of each chromoprotein to body mass. J. Biol. Chem. 182: 317-333, 1950. EDDY, L., A. ARDUINI, F. OKONSKI, AND P. HOCHSTEIN. The detection of hypervalent states of myoglobin by reflectance spectrophotometry in cardiac ischemic/reperfusion injury (Abstract). FASEB J. 4: A1219,1990. GALARIS, D., L. EDDY, A. ARDUINI, E. CADENAS, AND P. HOCHSTEIN. Mechanisms of reoxygenation injury in myocardial infarction: implications of a myoglobin redox cycle. Biochem. Biophys. Res. Commun. 160: 1162-1168,1989. GRISHAM, M. B. Myoglobin-catalyzed hydrogen peroxide dependent arachidonic acid peroxidation. J. Free Radicals Biol. Med. 1: 227-232, 1985. KANNER. J.. AND S. HAREL. Initiation of membranal linid peroxi-
REDUCTION
11.
12 ’ 13 .
14.
15*
16.
17.
c997
dation by activated metmyoglobin and methemoglobin. Arch. Biochem. Biophys. 237: 314-321, 1985. MITSOS, S. E., J. C. FANTONE, K. P. GALLAGHER, K. M. WALDEN, P. J. SIMPSON, G. D. ABRAMS, M. A. SCHORK, AND B. R. LucCHESI. Canine myocardial reperfusion injury: protection by a free radical scavenger, N2-mercaptopropionyl glycine. J. Cardiovasc. Pharmacol. 8: 978-988, 1986. MITSOS, S. E., D. KIM, B. R. LUCCHESI, AND J. C. FANTONE. Modulation of myoglobin-HzOz-mediated peroxidation reactions by sulfhydryl compounds. Lab. Invest. 59: 824-830, 1988. SHLAFER, M., K. BROSAMER, J. R. FORDER, R. H. SIMON, P. A. WARD, AND C. M. GRUM. Cerium chloride as a histochemical marker of hydrogen peroxide in reperfused ischemic hearts. J. Mol. Cell. Cardiol. 22: 83-97, 1990. TAMURA, M., N. OSHINO, B. CHANCE, AND I. A. SILVER. Optical measurements of intracellular oxygen concentration of rat heart in vitro. Arch. Biochem. Biophys. 191: 8-22, 1978. WALTERS, F. P., F. G. KENNEDY, AND D. P. JONES. Oxidation of myoglobin in isolated adult rat cardiac myocytes by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. FEBS Lett. 163: 292-296, 1983. WITTENBERG, B. A., J. B. WITTENBERG, AND P. R. B. CALDWELL. Role of myoglobin in the oxygen supply to red skeletal muscle. J. Biol. Chem. 250: 9038-9043, 1975. YUSA, K., AND K. SHIKAMA. Oxidation of oxymyoglobin to metmyoglobin with hydrogen peroxide: involvement of the ferry1 intermediate. Biochemistry 26: 6684-6688. 1987.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 4, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
of ferrylmyoglobin
LYNNE Institute
EDDY,
LYNNE,
in rat diaphragm
EDDY, ARDUINO ARDUINI, AND PAUL HOCHSTEIN for Toxicology, University of Southern California, Los Angeles, California
ARDUINO
ARDUINI,
AND
PAUL
HOCHSTEIN.
Reduction of ferrylmyoglobin in rat diaphragm. Am. J. Physiol. 259 (Cell Physiol. 28): C995-C997, 1990.-The oxidation of myoglobin was monitored by transmission spectroscopy in isolated, superfused preparations of rat diaphragms. In its deoxygenated form, during anoxia, myoglobin was oxidized by adding hydrogen peroxide (1.0 mM) to its ferry1 form (Fe’“). On the other hand, peroxide-induced formation of ferrylmyoglobin was not observed when the perfusate contained oxygen. Ferrylmyoglobin was visualized after its derivatization with NazS to form sulfmyoglobin. Depending on the time of addition, ascorbate (4.0 mM) or ergothioneine (2.0 mM) either prevented the formation of or dissipated ferrylmyoglobin. These agents are known to be reductants of this hypervalent form of myoglobin. In addition to providing the first demonstration of ferrylmyoglobin in skeletal muscle, these observations are consistent with the concept that the oxidation of myoglobin to hypervalent states might be an important event in the initiation of muscle damage associated with anoxia and reoxygenation. The rapid reduction of myoglobin would prevent peroxidatic alterations of essential cellular constituents by ferrylmyoglobin. myoglobin;
ascorbate; ergothioneine
HAVE PREVIOUSLY SUGGESTED that the oxidation of myoglobin to its ferry1 state may be a critical event in cardiac muscle damage associated with episodes of ischemia and reperfusion (8). In part, the basis for this suggestion is the association of hydrogen peroxide formation, as well as other oxidants, with such episodes (13), the special sensitivity of deoxymyoglobin (Fe”) to oxidation by hydrogen peroxide to form ferrylmyoglobin (FeIV) (8, 17), and the capacity of reductants of ferrylmyoglobin to protect the isolated heart against reperfusion injury (8). As a result of these observations on the facile redox cycling of myoglobin, we have called attention to the potential therapeutic value of one-electron agents such as ascorbate and ergothioneine in preventing damage associated with the formation of hydrogen peroxide and the accumulation of ferrylmyoglobin during postischemic oxygenation of muscle (8). The redox chemistry and functions of myoglobin have been investigated for many years (5, 16). However, the detection of ferrylmyoglobin in situ is often complicated by the presence of cytochromes that absorb light at interfering wavelengths. A potentially useful solution to this problem has been to derivatize ferrylmyoglobin with WE
0363-6143/90 $1.50 Copyright
0
90033
Na2S (2). This agent forms a spectrally distinct species, sulfmyoglobin, with an absorption maximum at 618 nm (2). We have utilized this method to detect the formation of ferrylmyoglobin in postischemic cardiac muscle by reflectance spectroscopy (7). The aim of the experiments described in this paper was to determine whether a similar approach could be utilized along with transmission rather than reflectance spectroscopy to identify peroxide-dependent formation of ferrylmyoglobin in the isolated rat diaphragm. The hemoglobin-free, superfused diaphragm was used because of its high myoglobin-to-cytochrome c ratio (6) and because this tissue is sufficiently thin to permit transmission spectroscopy. The formation of ferrylmyoglobin has not been demonstrated previously in skeletal muscle. METHODS
After anesthesia with pentobarbital sodium (30 mg/kg intraperitoneally) and heparinization (500 U intravenously), rats were perfused with buffer through the aorta by a pump to remove blood from the tissues. KrebsHenseleit buffer of the following composition was used throughout the experiments (final concentration in mM): 118 NaCl, 4.7 KCl, 2.5 CaCIZ, 1.2 MgS04, 1.2 KH2P04, 0.5 CaEDTA, 25 NaHCOs, and 10 glucose, pH 7.4. The diaphragm was removed and bisected. The isolated hemidiaphragm was sutured to a small plastic frame and placed within a covered 3-ml cuvette. The cuvette was superfused from an enclosed reservoir with buffer using a peristaltic pump at a flow rate of 2.5 ml/min. Oxygenation was maintained by bubbling the reservoir with 95% 02-5% C02. Anoxia was initiated by changing the gas to 95% Nz-5% CO,. Twenty-five minutes of anoxia were used to convert oxymyoglobin to deoxymyoglobin. Hydrogen peroxide, 1.0 mM, was added to the reservoir and circulation was continued for 20 min. In some experiments, catalase (400 U/ml) was then added to reduce any hydrogen peroxide still present. However, identical results were obtained when catalase was not present. Ferrylmyoglobin was indirectly determined as the ferrosulfmyoglobin derivative after addition of 1.0 mM NazS (2). Ascorbic acid, 4.0 mM, and ergothioneine, 2.0 mM, when used, were added to the recirculating buffer either before hydrogen peroxide or 20 min after hydrogen peroxide addition. Spectra were recorded from 500-650 nm, using 540 nm as the reference wavelength (Shimadzu UV
1990 the American
Physiological
Society
c995
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 4, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
C996
FERRYLMYOGLOBIN
3000 dual wavelength-double beam spectrophotometer). Difference spectra were constructed using the SpectroCalc computer package (Galactic Industries, Salem, NH) and an IBM-PC computer. RESULTS
Although not illustrated, the difference spectra obtained between the oxygenated and the deoxygenated diaphragms were dominated by myoglobin and were similar to those observed by Tamura et al. (14) for purified myoglobin and in the isolated rat heart. In addition, the inclusion of hydrogen peroxide in the perfusion medium caused no alterations in the spectra of oxygenated diaphragms, either in the absence of Na2S or after its inclusion in the perfusate. On the other hand, there was a striking increase in absorption at 617 nm when Na2S was added to the perfusate buffer after treatment of deoxygenated diaphragms with hydrogen peroxide. This effect is illustrated in curue B of Fig. 1. The appearance of this new peak in the difference spectrum between peroxidetreated and untreated diaphragms strongly implicates the formation of ferrylmyoglobin from deoxymyoglobin and its derivatization by Na2S to sulfmyoglobin. Using the wavelength pair 550-620 nm (EmM = 11) for deoxymyoglobin, derived from Ref. 1 and the difference spectra (617-600 nm) after the addition of Na2S (EmM = 10.5) (l5), we calculated that -35% of the deoxymyoglobin was converted to ferrylmyoglobin in these experiments. Curve A of Fig. I also shows that, when either ascorbate (4.0 mM) or ergothioneine (2.0 mM) was present in the perfusate, the difference spectra between deoxygenated and oxygenated diaphragms were unaltered despite the presence of hydrogen peroxide and NaS. Identical results were obtained when these reducing agents were added before the peroxide or 20 min after the peroxide but 10 min before NaS. It should be mentioned that the shoulder in the difference spectra below 600 nm is unexplained.
600
650 Nanometers
FIG. 1. Curve B: difference spectrum (600-650 nm) obtained after addition of Na2S (1.0 mM) to deoxygenated diaphragm exposed to hydrogen peroxide (1.0 mM). Na2S was added 20 min after peroxide, and spectrum was recorded within 5 min. Curve A: difference spectrum obtained when ascorbate (4.0 mM) was included in perfusion medium before addition of peroxide. Identical spectra were observed when ascorbate was added after addition of peroxide or when ergothioneine (2.0 mM) was substituted for ascorbate. Spectral determinations were carried out on at least triplicate diaphragm preparations with nearly identical results.
REDUCTION
DISCUSSION
The experiments described above illustrate the u.sefulness of the rodent diaphragm in studies of the redox state of myoglobi n. The thin .ness of the diap h.ragm and the high ratio of myoglobin to cytochromes (6) permit such studies by transmission spectroscopy rather than by more sophisticated reflectance techniques. We previously have utilized the latter method to demonstrate the accumulation of ferrylmyoglobin from deoxymyoglobin in the perfused heart after brief (30 min) periods of ischemia (7). The detection of ferrylmyoglobin in situ in the diaphragm only after anoxia affirms the previously reported in vitro sensitivity of deoxymyoglobin, as compared with oxymyoglobin, to oxidation by peroxide (8, 17). As was the case in the heart, reducing agents that rapidly convert ferrylmyoglobin to metmyoglobin and ferrousmyoglobin either prevent the formation of or apparently dissipate ferrylmyoglobin in the diaphragm, depending on the time of their addition in relation to peroxide. It should be noted that the amounts of hydrogen peroxide utilized in these experiments are probably severa1 orders of magnitude higher than those that might be generated endogenously as a response to reoxygenation of occluded or hypoxic muscle. For this reason, these experiments are not evidence for a pathophysiological role for hydrogen peroxide in postanoxic injury. The high concentrations of peroxide utilized are apparently necessary because of endogenous catalase activity. It is reasonable and more likely that endogenously generated peroxide, in more intimate association with myoglobin, might be more effective in the oxidation of myoglobin than that added in a perfusing buffer. It is of special interest that reducing agents have been used as cardioprotective agents in various animal models of ischemia-reperfusion. Mitsos et al. have shown with N-2-mercaptopropionyl glycine (MPG) that not only does this agent salvage potentially necrotic cardiac tissue in a canine model of ischemia-reperfusion (11) but that it can inhibit myoglobin-hydrogen peroxide-induced peroxidation of uric acid and arachidonic acid (12). Bolli et al. have demonstrated improved cardiac function after ischemia-reperfusion in dogs treated with dimethylthiourea (4) and MPG (3). However, they credited the protective response to hydroxyl radical scavenging. We suggest that the favorable responses to reducing agents may be related to reduction of oxidized myoglobin. Such a concept is based on the well-known peroxidatic activity of ferrylmyoglobin toward a variety of essential cellular constituents (9, 10). A. Arduini is a Visiting Scientist from the University of Chieti and was supported, in part, by Sigma Tau, SPA, Pomezia, Italy. This work was supported also by grants from the Ciba Geigy Corporation and the Los Angeles Affiliate of the American Heart Association. Received
20 February
1990; accepted
in final
form
3 August
1990.
REFERENCES 1. ANTONINI, E., AND M. BRUNORI. Hemoglobin and Myoglobin in Their Reactions with Ligands. Amsterdam: North Holland, 1971, p. 17. 2. BERZOFSKY, J. A.,J. PEISACH,AND W. E. BLUMBERG. Sulfheme
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 4, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
FERRYLMYOGLOBIN
3.
4.
5. 6.
7.
8.
9.
10.
proteins. 1. Optical and magnetic properties of sulfmyoglobin and its derivatives. J. Biol. Chem. 246: 3367-3377, 1971. BOLLI, R., M. 0. JEROUDI, B. S. PATEL, 0. I. ARUOMA, B. HALLIWELL, E. K. LAI, AND P. B. MCCAY. Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion. Circ. Res. 65: 607-622, 1989. BOLLI, R., W.-X. ZHU, C. J. HARTLEY, L. H. MICHAEL, J. E. REPINE, M. L. HESS, R. C. KUKREJA, AND R. ROBERTS. Attenuation of dysfunction in the postischemic “stunned” myocardium by dimethylthiourea. Circulation 76: 458-468, 1987. COLE, R. P. Myoglobin function in exercising skeletal muscle. Science Wash. DC 216: 523-525, 1982. DRABKIN, D. L. The distribution of the chromoproteins, hemoglobin, myoglobin, and cytochrome c in the tissues of different species, and the relationship of the total content of each chromoprotein to body mass. J. Biol. Chem. 182: 317-333, 1950. EDDY, L., A. ARDUINI, F. OKONSKI, AND P. HOCHSTEIN. The detection of hypervalent states of myoglobin by reflectance spectrophotometry in cardiac ischemic/reperfusion injury (Abstract). FASEB J. 4: A1219,1990. GALARIS, D., L. EDDY, A. ARDUINI, E. CADENAS, AND P. HOCHSTEIN. Mechanisms of reoxygenation injury in myocardial infarction: implications of a myoglobin redox cycle. Biochem. Biophys. Res. Commun. 160: 1162-1168,1989. GRISHAM, M. B. Myoglobin-catalyzed hydrogen peroxide dependent arachidonic acid peroxidation. J. Free Radicals Biol. Med. 1: 227-232, 1985. KANNER. J.. AND S. HAREL. Initiation of membranal linid peroxi-
REDUCTION
11.
12 ’ 13 .
14.
15*
16.
17.
c997
dation by activated metmyoglobin and methemoglobin. Arch. Biochem. Biophys. 237: 314-321, 1985. MITSOS, S. E., J. C. FANTONE, K. P. GALLAGHER, K. M. WALDEN, P. J. SIMPSON, G. D. ABRAMS, M. A. SCHORK, AND B. R. LucCHESI. Canine myocardial reperfusion injury: protection by a free radical scavenger, N2-mercaptopropionyl glycine. J. Cardiovasc. Pharmacol. 8: 978-988, 1986. MITSOS, S. E., D. KIM, B. R. LUCCHESI, AND J. C. FANTONE. Modulation of myoglobin-HzOz-mediated peroxidation reactions by sulfhydryl compounds. Lab. Invest. 59: 824-830, 1988. SHLAFER, M., K. BROSAMER, J. R. FORDER, R. H. SIMON, P. A. WARD, AND C. M. GRUM. Cerium chloride as a histochemical marker of hydrogen peroxide in reperfused ischemic hearts. J. Mol. Cell. Cardiol. 22: 83-97, 1990. TAMURA, M., N. OSHINO, B. CHANCE, AND I. A. SILVER. Optical measurements of intracellular oxygen concentration of rat heart in vitro. Arch. Biochem. Biophys. 191: 8-22, 1978. WALTERS, F. P., F. G. KENNEDY, AND D. P. JONES. Oxidation of myoglobin in isolated adult rat cardiac myocytes by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. FEBS Lett. 163: 292-296, 1983. WITTENBERG, B. A., J. B. WITTENBERG, AND P. R. B. CALDWELL. Role of myoglobin in the oxygen supply to red skeletal muscle. J. Biol. Chem. 250: 9038-9043, 1975. YUSA, K., AND K. SHIKAMA. Oxidation of oxymyoglobin to metmyoglobin with hydrogen peroxide: involvement of the ferry1 intermediate. Biochemistry 26: 6684-6688. 1987.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 4, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
