J. Biochem. 110, 450-456 (1991)
Allopurinol-Insensitive Oxygen Radical Formation by Milk Xanthine Oxidase Systems1 Masao Nakamura Biophysics, Research Institute of Applied Electricity, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060 Received for publication, May 9, 1991
Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5 -dimethyl-1-pyrroline-./Voxide. No xanthine-dependent oxygen radical formation was observed when allopurinoltreated xanthine oxidase was used. The significant superoxide generation in the NADHoxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the K^ and Vmax of xanthine oxidase were estimated to be respectively 51 /tM and 5.5 s"1 for menadione and 12 /tM and 0.4 s"1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH* radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH* radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.
Xanthine oxidase is a metalloflavoprotein that contains 2 atoms of molybdenum, 8 atoms of iron, and 2 mol of FAD per mol of enzyme. It is believed that the enzyme accepts electrons from xanthine through molybdenum and from NADH through FAD (1). When xanthine acts as an electron donor, reducing equivalents are finally transferred to oxygen via the flavin (2). The enzyme is found to generate both superoxide (product of one-electron reduction) and hydrogen peroxide (3-5). It has been proposed that xanthine oxidase plays an important role in tissue-damaging reactions such as those induced by ischemia or inflammation (6). In these reactions, trace metals such as iron or copper function as catalysts in Fenton-type Haber-Weiss reactions, producing OH' radicals (7). Convincing evidence exists that superoxide produced enzymatically by the enzyme can release iron from ferritin and that released iron can catalyze lipid peroxidation in vitro (8). It has been suggested that during ischemia or inflammation, xanthine oxidase might be formed from xanthine dehydrogenase (6) as xanthine oxidase exists as xanthine dehydrogenase in mammalian tissues (9). This proposal is based on the observation that allopurinol, a potent inhibitor of xanthine oxidase, Himinishes injury in some model systems (10, 11). It has been demonstrated that xanthine oxidase also catalyzes the redox-cycling of quinones and that quinones compete with oxygen for the active site of the enzyme (5, 12, 13). Nakamura and YamazaM (23) have reported that the xanthine- and NADH-p-benzoquinone reductase activ1 This research was supported in part by a Grant-in-Aid for Scientific Research from the Akiyama Foundation. Abbreviations: DMPO, 5,5'-dimethyl-l-pyrroline-iV-oxide; DMPOOOH, 2,2'-dimethyl-5-hydroperoxyl-l-pvrrolidinyloxyl; DMPO-OH, 2,2'-dimetyl-5-hydroxyl-l-pyrrolidinyloxyl; DTPA, diethylenetriamine penta-acetic acid.
450
ities of the enzyme are nearly the same at pH 7.4. The present study reports that the oxygen radical generation in NADH-oxygen, -menadione, and -adriamycin reductase reactions by milk xanthine oxidase is insensitive to allopurinol. The relation between the internal electron transport of xanthine oxidase and oxygen radical generation is described. MATERIALS AND METHODS Xanthine oxidase reversibly convertible to dehydrogenase type was prepared from fresh cow's milk by the method of Nakamura and Yamazaki (14). The purified enzyme was passed through a Sephadex G-25 column to remove thiol compounds before use. About 96% of the enzyme existed as oxidase. Xanthine oxidase activity was measured at 292 run and xanthine dehydrogenase at 340 nm in the presence of NAD+ under aerobic conditions. NADH-quinone reductase activity of the enzyme was observed in the decrease of absorbance at 340 nm. The concentration of the enzyme was calculated on the basis of a value of 37 for c mM at 450 nm(2). Allopurinol-treated xanthine oxidase (treated xanthine oxidase) was prepared by the addition of 100 /*M allopurinol to the solution containing xanthine oxidase. Aliquots were withdrawn within 5 min as treated xanthine oxidase. The percentage of one-electron flux in NADH-oxygen reductase activity of the enzyme was calculated as described previously (5). Horse heart cytochrome c, xanthine, NADH, NADPH, bovine erythrocyte superoxide dismutase (SOD), bovine liver catalase, diethylenetriamine penta-acetic acid (DTPA), and the spin-trapping agent, 5,5'-dimethyl-l-pyrroline-.ZV-oxide (DMPO) were purchased from Sigma (St. Louis); 2-methyl-l,4-naphthoquinone(menadione) from Nakarai (Kyoto); adriamycin was J. Biochem.
451
Oxygen Radical Formation by Milk Xanthine Oxidase Systems
obtained from Farmitalia Carlo Erba, Italy. Optical measurements for kinetic experiments were recorded on a Hitachi 200-10 spectrophotometer. ESR spectra were recorded using a Varian E-109B spectrometer. All reactions and measurements were carried out at 25'C in 0.1 M potassium phosphate buffer, pH 7.4. E
RESULTS It has been proven that milk xanthine oxidase accepts electrons from xanthine through molybdenum and donates them to oxygen and quinones through FAD. The active-site molybdenum is blocked by allopurinol, making the enzyme
1 min
1 min
B
SOD
1 mm
1mm
Fig. 1. Xanthine-oxygen (A) and NADH-menadlone (B) reductase activity of xanthine oxidase (a) and treated xanthine oxidase (b). Allopurinol-treated xanthine oxidase (treated xanthine oxidase) was prepared as described under "MATERIALS AND METHODS." A: Xanthine-oxygen reductase activity. The reactions were carried out at pH 7.4 in the presence of 100 ii M xanthine and 50 nM xanthine oxidase (a) and treated xanthine oxidase (b). B: NADH-menadione reductase activity. The concentrations were 120 //M NADH, 20//M menadione, and 120 nM xanthine oxidase (a) or treated xanthine oxidase (b). The enzyme (E) was added at arrows.
Fig. 2. Formation of superoxide anion in NADH-oxygen reductase reaction by xanthine oxidase. Superoxide generation was measured at 550 nm (a) in the presence of 35 pM cytochrome c. The concentrations were 120 ^M NADH and 120 nM xanthine oxidase in the presence (a) or absence (b) of 35 ^M cytochrome c. Superoxide dismutase, 0.1 fiM, was added at the time indicated. The percentage of one-electron flux was calculated from the ratio of cytochrome c reduction rate (a) to NADH oxidation (b).
z
z o
Fig. 3. NADH-menadione and -adriamycin reductase activity of xanthine oxidase. A: NADH-menadione (a) and -adriamycin (b) reductase reaction of xanthine oxidase. The concentrations were: 120 ^MNADH, 120 nM xanthine oxidase, and menadione or adriamycin. The concentrations of menadione and adriamycin are indicated on the figure. B: Lineweaver-Burk plots for NADHmenadione (a) and -adriamycin (b) reductase reaction of xanthine oxidase. The Ka value of the enzyme for NADH in the reaction of NADHmenadione and -adriamycin was estimated to be 17 //M. The same activity was observed when the enzyme was replaced by treated xanthine oxidase. Vol. 110, No. 3, 1991
12 ^ 1
1 _, 2 1 /[Menadionel.KT'M
05 1 /[Adriamycin],l
452
M. Nakamura A «Adnamycin
B Treated xanthine oxidase *Adnamycin
Fig. 4. E8R spectra ofDMPO-OOH and DMPO-OH produced by the xanthine oxidase systems in the absence (A) or presence (B) of menadione. The instrumental conditions were: magnetic field, 3.387 G; modulation amplitude, 1 G; receiver gain, 2x10*; time constant, 0.126 s; microwave frequency, 9.512 GHz; microwave power, 20 mW; and scan speed, 25 G/min. A: NADH-oxygen reductase reaction. The concentrations were 200 //M NADH, 0.35 //M xanthine oxidase, 0.5 mM DTPA, and 100 mM DMPO. The spectra were recorded at 2 (a), 4 (b), and 8 (c) min after the addition of the enzyme, d and e indicate the formation of DMPO-OOH and DMPOOH, respectively. B: NADH-menadione reductase reaction. The concentrations were 200//M NADH, 0.35//M xanthine oxidase, 20 fiM menadione, 0.5 mM DTPA, and 100 mM DMPO. The spectra were recorded at 2 (a), 4 (b), and 8 (c) min after the addition of the enzyme.
inactive (15). Figure 1A shows that the xanthine oxidase activity (urate formation) was completely inhibited by allopurinol. On the contrary, no loss of NADH-menadione reductase activity of the enzyme was found after incubation of the enzyme in the presence of allopurinol (Fig. IB). Much attention has been paid to the oxygen radical generation in the xanthine oxidase system, since the radical chain reactions involved in tissue injury may be initiated by oxygen radicals. The reduction mechanism of oxygen by xanthine oxidase has been documented in detail and the product ratio of superoxide to hydrogen peroxide depends upon the concentration of substrate, oxygen or xanthine, and pH (3, 5). Superoxide generation in the NADH-oxygen reductase reaction of xanthine oxidase was demonstrated by monitoring the reduction of cytochrome c, indicating the scavenging of superoxide by cytochrome c (Fig. 2). The percentage of one-electron flux was 37% and the turnover rate of O2 was estimated to be 0.09 s"1. Flavoproteins show variable superoxide generating activity (16). The NADH-oxidase activity of xanthine oxidase increased with increasing menadione and adriamycin concentrations. Kinetic con-
Fig. 5. ESR spectra of DMPO-OOH and DMPO-OH produced in NADH-adriamycin reductase reaction by xanthine oxidase (A) and treated xanthine oxidase (B). The conditions were the same as in Fig. 4. The concentrations were 200 //M NADH, 0.35 //M xanthine oxidase or treated xanthine oxidase, 80//M adriamycin, 0.5 mM DTPA, and 100 mM DMPO. The spectra were recorded at 2 (a), 4 (b), and 8 (c) min after the addition of the enzyme.
stants (Km and Vmax) for NADH-menadione and -adriamycin reductase activity of the enzyme were estimated from Lineweaver-Burk plots (Fig. 3). While the K^ values of the enzyme for menadione and adriamycin were 51 and 12 fiM, the value for NADH was not significantly different for the two activities. Since the hydroquinone and semiquinoid form of menadione readily reduce cytochrome c (17), superoxide generation was followed by the formation of the spin adduct DMPO-OOH (28, 29). A significant superoxide DMPO adduct (DMPO-OOH) was observed in NADH-oxygen reductase reaction of xanthine oxidase (Fig. 4Aa) and the spectrum of DMPO-OOH changed to a combination of one characteristic of DMPO-OOH and one of DMPO-OH (Fig. 4A, b and c). The same sequence of events and signal intensity were observed with allopurinolinactivated enzyme (data not shown). A marked signal increase of DMPO-OOH was obtained in NADH-menadione and -adriamycin reductase reactions of native and inactivated xanthine oxidase (Figs. 4B and 5). The degree of activation depended upon the concentration of quinones, being compatible with quinone-dependent NADH oxidation (Fig. 3). The results indicate that the quinone-mediated oxygen reduction proceeds mainly by way of one-electron transfer. The DMPO-OOH signal was replaced by that of DMPO-OH in the presence of superoxide dismutase (Fig. 6A) and no signal was seen in the presence of both superoxide dismutase and catalase (Fig. 6C). The presence of catalase had no effect on the formation of DMPO-OOH; J. Biochem.
Oxygen Radical Formation by Milk Xanthine Oxidase Systems
453
A*Adnamycin.SOD
Time (min)
B
Adriamycin. SOD-Catalase
Fig. 6. Effect of SOD and catalase upon the ESR spectra produced in NADH-adriamycin reductase reaction by xanthine oxidase. The conditions were the same as in Fig. 4. The concentrations were 200//M NADH, 0.35 ^M xanthine oxidase, 80 fiU adriamycin, 0.5 mM DTPA, and 100 mM DMPO. The reactions were carried out in the presence of 0.2 ^M SOD (A), 0.3 //M catalase (B), and both SOD and catalase (C). The spectra were recorded at 2 (a) and 8 (b) min after the addition of the enzyme.
however no DMPO-OH signal was observed (Fig. 6B). The time courses of individual signals are summarized in Figs. 7B and 8. The difference in signal height between native and inactive xanthine oxidase was within experimental error (Fig. 7B). However, no significant DMPOOOH formation was observed in the xanthine-oxygen reductase reaction using inactive xanthine oxidase (Fig. 7Ab). The results indicate that the pathway for reducing equivalents traveling from NADH to acceptors remained intact on treatment with allopurinol. The DMPO-OOH signal reached a peak at 100 s after the start of reaction in the NADH-menadione reductase reaction, then decreased with time (Fig. 7Be). From the menadione reductase activity of the enzyme (Table I), almost all NADH was estimated to be exhausted at 100 s. It has been shown that the DMPO-OOH signal decays with a half-life of 66 s and nearly 3% of DMPO-OOH decomposes to DMPO-OH (20, 21). However, no significant DMPO-OH signal remained in the presence of catalase during the NADH-menadione and -adriamycin reductase reactions (Fig. 8B, c and d). Formation of adriamycin radicals has been reported in the xanthine-adriamycin reductase reaction of the enzyme when the reaction is carried out under anaerobic conditions (22). Figure 9 also shows the formation of adriamycin radicals with time using the NADH as an electron donor. Although the menadione reductase activity of the enzyme was higher than the adriamycin reductase activity (Table I), no menadione radicals were observed. Under anaerobic Vol. 110, No. 3, 1991
Time (mm)
10
Fig. 7. Time courses of the ESR spectra of DMPO-OOH produced by the xanthine-oxygen reductase (A) and NADH-oxygen, -menadione, and -adriamycin reductase reaction (B) by xanthine oxidase and treated xanthine oxidase. A: Time courses of DMPO-OOH produced by xanthine oxidase (O) and treated xanthine oxidase (•). The signal height denotes a peak of the ESR spectrum (d in Fig. 4). The concentrations were 100 //M xanthine, 50 nM xanthine oxidase or treated xanthine oxidase, 0.5 mM DTPA, and 100 mM DMPO. B: Time courses of DMPO-OOH; NADH-oxygen reductase reaction by xanthine oxidase (o) and treated xanthine oxidase (•), NADH-adriamycin reductase reaction by xanthine oxidase (a) and treated xanthine oxidase (•), NADH-menadione reductase reaction by xanthine oxidase (A) and treated xanthine oxidase (A). The concentrations were the same as in Figs. 4 and 5.
TABLE I. Xanthine- and NADH-dependent activities of xanthine oxidase and treated xanthine oxidase. Electron donor Xanthine NADH
Acceptor
o, o, Menadione Adriamycin
Treated Native Activity (Jmol/FAD-s) 5.2"
Allopurinol-Insensitive Oxygen Radical Formation by Milk Xanthine Oxidase Systems1 Masao Nakamura Biophysics, Research Institute of Applied Electricity, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060 Received for publication, May 9, 1991
Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5 -dimethyl-1-pyrroline-./Voxide. No xanthine-dependent oxygen radical formation was observed when allopurinoltreated xanthine oxidase was used. The significant superoxide generation in the NADHoxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the K^ and Vmax of xanthine oxidase were estimated to be respectively 51 /tM and 5.5 s"1 for menadione and 12 /tM and 0.4 s"1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH* radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH* radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.
Xanthine oxidase is a metalloflavoprotein that contains 2 atoms of molybdenum, 8 atoms of iron, and 2 mol of FAD per mol of enzyme. It is believed that the enzyme accepts electrons from xanthine through molybdenum and from NADH through FAD (1). When xanthine acts as an electron donor, reducing equivalents are finally transferred to oxygen via the flavin (2). The enzyme is found to generate both superoxide (product of one-electron reduction) and hydrogen peroxide (3-5). It has been proposed that xanthine oxidase plays an important role in tissue-damaging reactions such as those induced by ischemia or inflammation (6). In these reactions, trace metals such as iron or copper function as catalysts in Fenton-type Haber-Weiss reactions, producing OH' radicals (7). Convincing evidence exists that superoxide produced enzymatically by the enzyme can release iron from ferritin and that released iron can catalyze lipid peroxidation in vitro (8). It has been suggested that during ischemia or inflammation, xanthine oxidase might be formed from xanthine dehydrogenase (6) as xanthine oxidase exists as xanthine dehydrogenase in mammalian tissues (9). This proposal is based on the observation that allopurinol, a potent inhibitor of xanthine oxidase, Himinishes injury in some model systems (10, 11). It has been demonstrated that xanthine oxidase also catalyzes the redox-cycling of quinones and that quinones compete with oxygen for the active site of the enzyme (5, 12, 13). Nakamura and YamazaM (23) have reported that the xanthine- and NADH-p-benzoquinone reductase activ1 This research was supported in part by a Grant-in-Aid for Scientific Research from the Akiyama Foundation. Abbreviations: DMPO, 5,5'-dimethyl-l-pyrroline-iV-oxide; DMPOOOH, 2,2'-dimethyl-5-hydroperoxyl-l-pvrrolidinyloxyl; DMPO-OH, 2,2'-dimetyl-5-hydroxyl-l-pyrrolidinyloxyl; DTPA, diethylenetriamine penta-acetic acid.
450
ities of the enzyme are nearly the same at pH 7.4. The present study reports that the oxygen radical generation in NADH-oxygen, -menadione, and -adriamycin reductase reactions by milk xanthine oxidase is insensitive to allopurinol. The relation between the internal electron transport of xanthine oxidase and oxygen radical generation is described. MATERIALS AND METHODS Xanthine oxidase reversibly convertible to dehydrogenase type was prepared from fresh cow's milk by the method of Nakamura and Yamazaki (14). The purified enzyme was passed through a Sephadex G-25 column to remove thiol compounds before use. About 96% of the enzyme existed as oxidase. Xanthine oxidase activity was measured at 292 run and xanthine dehydrogenase at 340 nm in the presence of NAD+ under aerobic conditions. NADH-quinone reductase activity of the enzyme was observed in the decrease of absorbance at 340 nm. The concentration of the enzyme was calculated on the basis of a value of 37 for c mM at 450 nm(2). Allopurinol-treated xanthine oxidase (treated xanthine oxidase) was prepared by the addition of 100 /*M allopurinol to the solution containing xanthine oxidase. Aliquots were withdrawn within 5 min as treated xanthine oxidase. The percentage of one-electron flux in NADH-oxygen reductase activity of the enzyme was calculated as described previously (5). Horse heart cytochrome c, xanthine, NADH, NADPH, bovine erythrocyte superoxide dismutase (SOD), bovine liver catalase, diethylenetriamine penta-acetic acid (DTPA), and the spin-trapping agent, 5,5'-dimethyl-l-pyrroline-.ZV-oxide (DMPO) were purchased from Sigma (St. Louis); 2-methyl-l,4-naphthoquinone(menadione) from Nakarai (Kyoto); adriamycin was J. Biochem.
451
Oxygen Radical Formation by Milk Xanthine Oxidase Systems
obtained from Farmitalia Carlo Erba, Italy. Optical measurements for kinetic experiments were recorded on a Hitachi 200-10 spectrophotometer. ESR spectra were recorded using a Varian E-109B spectrometer. All reactions and measurements were carried out at 25'C in 0.1 M potassium phosphate buffer, pH 7.4. E
RESULTS It has been proven that milk xanthine oxidase accepts electrons from xanthine through molybdenum and donates them to oxygen and quinones through FAD. The active-site molybdenum is blocked by allopurinol, making the enzyme
1 min
1 min
B
SOD
1 mm
1mm
Fig. 1. Xanthine-oxygen (A) and NADH-menadlone (B) reductase activity of xanthine oxidase (a) and treated xanthine oxidase (b). Allopurinol-treated xanthine oxidase (treated xanthine oxidase) was prepared as described under "MATERIALS AND METHODS." A: Xanthine-oxygen reductase activity. The reactions were carried out at pH 7.4 in the presence of 100 ii M xanthine and 50 nM xanthine oxidase (a) and treated xanthine oxidase (b). B: NADH-menadione reductase activity. The concentrations were 120 //M NADH, 20//M menadione, and 120 nM xanthine oxidase (a) or treated xanthine oxidase (b). The enzyme (E) was added at arrows.
Fig. 2. Formation of superoxide anion in NADH-oxygen reductase reaction by xanthine oxidase. Superoxide generation was measured at 550 nm (a) in the presence of 35 pM cytochrome c. The concentrations were 120 ^M NADH and 120 nM xanthine oxidase in the presence (a) or absence (b) of 35 ^M cytochrome c. Superoxide dismutase, 0.1 fiM, was added at the time indicated. The percentage of one-electron flux was calculated from the ratio of cytochrome c reduction rate (a) to NADH oxidation (b).
z
z o
Fig. 3. NADH-menadione and -adriamycin reductase activity of xanthine oxidase. A: NADH-menadione (a) and -adriamycin (b) reductase reaction of xanthine oxidase. The concentrations were: 120 ^MNADH, 120 nM xanthine oxidase, and menadione or adriamycin. The concentrations of menadione and adriamycin are indicated on the figure. B: Lineweaver-Burk plots for NADHmenadione (a) and -adriamycin (b) reductase reaction of xanthine oxidase. The Ka value of the enzyme for NADH in the reaction of NADHmenadione and -adriamycin was estimated to be 17 //M. The same activity was observed when the enzyme was replaced by treated xanthine oxidase. Vol. 110, No. 3, 1991
12 ^ 1
1 _, 2 1 /[Menadionel.KT'M
05 1 /[Adriamycin],l
452
M. Nakamura A «Adnamycin
B Treated xanthine oxidase *Adnamycin
Fig. 4. E8R spectra ofDMPO-OOH and DMPO-OH produced by the xanthine oxidase systems in the absence (A) or presence (B) of menadione. The instrumental conditions were: magnetic field, 3.387 G; modulation amplitude, 1 G; receiver gain, 2x10*; time constant, 0.126 s; microwave frequency, 9.512 GHz; microwave power, 20 mW; and scan speed, 25 G/min. A: NADH-oxygen reductase reaction. The concentrations were 200 //M NADH, 0.35 //M xanthine oxidase, 0.5 mM DTPA, and 100 mM DMPO. The spectra were recorded at 2 (a), 4 (b), and 8 (c) min after the addition of the enzyme, d and e indicate the formation of DMPO-OOH and DMPOOH, respectively. B: NADH-menadione reductase reaction. The concentrations were 200//M NADH, 0.35//M xanthine oxidase, 20 fiM menadione, 0.5 mM DTPA, and 100 mM DMPO. The spectra were recorded at 2 (a), 4 (b), and 8 (c) min after the addition of the enzyme.
inactive (15). Figure 1A shows that the xanthine oxidase activity (urate formation) was completely inhibited by allopurinol. On the contrary, no loss of NADH-menadione reductase activity of the enzyme was found after incubation of the enzyme in the presence of allopurinol (Fig. IB). Much attention has been paid to the oxygen radical generation in the xanthine oxidase system, since the radical chain reactions involved in tissue injury may be initiated by oxygen radicals. The reduction mechanism of oxygen by xanthine oxidase has been documented in detail and the product ratio of superoxide to hydrogen peroxide depends upon the concentration of substrate, oxygen or xanthine, and pH (3, 5). Superoxide generation in the NADH-oxygen reductase reaction of xanthine oxidase was demonstrated by monitoring the reduction of cytochrome c, indicating the scavenging of superoxide by cytochrome c (Fig. 2). The percentage of one-electron flux was 37% and the turnover rate of O2 was estimated to be 0.09 s"1. Flavoproteins show variable superoxide generating activity (16). The NADH-oxidase activity of xanthine oxidase increased with increasing menadione and adriamycin concentrations. Kinetic con-
Fig. 5. ESR spectra of DMPO-OOH and DMPO-OH produced in NADH-adriamycin reductase reaction by xanthine oxidase (A) and treated xanthine oxidase (B). The conditions were the same as in Fig. 4. The concentrations were 200 //M NADH, 0.35 //M xanthine oxidase or treated xanthine oxidase, 80//M adriamycin, 0.5 mM DTPA, and 100 mM DMPO. The spectra were recorded at 2 (a), 4 (b), and 8 (c) min after the addition of the enzyme.
stants (Km and Vmax) for NADH-menadione and -adriamycin reductase activity of the enzyme were estimated from Lineweaver-Burk plots (Fig. 3). While the K^ values of the enzyme for menadione and adriamycin were 51 and 12 fiM, the value for NADH was not significantly different for the two activities. Since the hydroquinone and semiquinoid form of menadione readily reduce cytochrome c (17), superoxide generation was followed by the formation of the spin adduct DMPO-OOH (28, 29). A significant superoxide DMPO adduct (DMPO-OOH) was observed in NADH-oxygen reductase reaction of xanthine oxidase (Fig. 4Aa) and the spectrum of DMPO-OOH changed to a combination of one characteristic of DMPO-OOH and one of DMPO-OH (Fig. 4A, b and c). The same sequence of events and signal intensity were observed with allopurinolinactivated enzyme (data not shown). A marked signal increase of DMPO-OOH was obtained in NADH-menadione and -adriamycin reductase reactions of native and inactivated xanthine oxidase (Figs. 4B and 5). The degree of activation depended upon the concentration of quinones, being compatible with quinone-dependent NADH oxidation (Fig. 3). The results indicate that the quinone-mediated oxygen reduction proceeds mainly by way of one-electron transfer. The DMPO-OOH signal was replaced by that of DMPO-OH in the presence of superoxide dismutase (Fig. 6A) and no signal was seen in the presence of both superoxide dismutase and catalase (Fig. 6C). The presence of catalase had no effect on the formation of DMPO-OOH; J. Biochem.
Oxygen Radical Formation by Milk Xanthine Oxidase Systems
453
A*Adnamycin.SOD
Time (min)
B
Adriamycin. SOD-Catalase
Fig. 6. Effect of SOD and catalase upon the ESR spectra produced in NADH-adriamycin reductase reaction by xanthine oxidase. The conditions were the same as in Fig. 4. The concentrations were 200//M NADH, 0.35 ^M xanthine oxidase, 80 fiU adriamycin, 0.5 mM DTPA, and 100 mM DMPO. The reactions were carried out in the presence of 0.2 ^M SOD (A), 0.3 //M catalase (B), and both SOD and catalase (C). The spectra were recorded at 2 (a) and 8 (b) min after the addition of the enzyme.
however no DMPO-OH signal was observed (Fig. 6B). The time courses of individual signals are summarized in Figs. 7B and 8. The difference in signal height between native and inactive xanthine oxidase was within experimental error (Fig. 7B). However, no significant DMPOOOH formation was observed in the xanthine-oxygen reductase reaction using inactive xanthine oxidase (Fig. 7Ab). The results indicate that the pathway for reducing equivalents traveling from NADH to acceptors remained intact on treatment with allopurinol. The DMPO-OOH signal reached a peak at 100 s after the start of reaction in the NADH-menadione reductase reaction, then decreased with time (Fig. 7Be). From the menadione reductase activity of the enzyme (Table I), almost all NADH was estimated to be exhausted at 100 s. It has been shown that the DMPO-OOH signal decays with a half-life of 66 s and nearly 3% of DMPO-OOH decomposes to DMPO-OH (20, 21). However, no significant DMPO-OH signal remained in the presence of catalase during the NADH-menadione and -adriamycin reductase reactions (Fig. 8B, c and d). Formation of adriamycin radicals has been reported in the xanthine-adriamycin reductase reaction of the enzyme when the reaction is carried out under anaerobic conditions (22). Figure 9 also shows the formation of adriamycin radicals with time using the NADH as an electron donor. Although the menadione reductase activity of the enzyme was higher than the adriamycin reductase activity (Table I), no menadione radicals were observed. Under anaerobic Vol. 110, No. 3, 1991
Time (mm)
10
Fig. 7. Time courses of the ESR spectra of DMPO-OOH produced by the xanthine-oxygen reductase (A) and NADH-oxygen, -menadione, and -adriamycin reductase reaction (B) by xanthine oxidase and treated xanthine oxidase. A: Time courses of DMPO-OOH produced by xanthine oxidase (O) and treated xanthine oxidase (•). The signal height denotes a peak of the ESR spectrum (d in Fig. 4). The concentrations were 100 //M xanthine, 50 nM xanthine oxidase or treated xanthine oxidase, 0.5 mM DTPA, and 100 mM DMPO. B: Time courses of DMPO-OOH; NADH-oxygen reductase reaction by xanthine oxidase (o) and treated xanthine oxidase (•), NADH-adriamycin reductase reaction by xanthine oxidase (a) and treated xanthine oxidase (•), NADH-menadione reductase reaction by xanthine oxidase (A) and treated xanthine oxidase (A). The concentrations were the same as in Figs. 4 and 5.
TABLE I. Xanthine- and NADH-dependent activities of xanthine oxidase and treated xanthine oxidase. Electron donor Xanthine NADH
Acceptor
o, o, Menadione Adriamycin
Treated Native Activity (Jmol/FAD-s) 5.2"
