ARCHIVES
OF BIOCHEMISTRY
Vol. 292, No. 2, February
AND
BIOPHYSICS
1, pp. 570-575,1992
Generation of Superoxide during the Enzymatic Action of Tyrosinase Satoshi Koga,*,t Minoru
Nakano,*Tl and Shozo Tero-Kubotaz
*Photon Medical Research Center, School of Medicine, Hamamatsu University, Hamamatsu, Handa-cho 3600, Shizuoka 431-31, Japan; TTaiho Pharmaceutical Co., Ltd., Kandanishiki-cho, Chiyoda-ku, Tokyo 101, Japan; and *Institute for Chemical Reaction Science, Tohoku University, Sendai 980, Japan
Received July 8, 1991, and in revised form October 4, 1991
Evidence for the generation of superoxide anion in an enzymatic action of tyrosinase is reported. In the dopatyrosinase reaction, 1 mol of Oz is required for the production of 2 mol of dopaquinone, 1 mol of dopachrome, and a mol of 0,. Superoxide dismutase and a-methyl6-phenyl-3,7-dihydroimidazo[l,2-alpyrazin-3-one (a chemiluminescence probe and Oz trap) do not inhibit the rate of dopachrome formation from dopa in the presence of tyrosinase, indicating that free 0, is not utilized for metabolizing dopa. ESR studies for the accumulation of semiquinone radicals generated from tyrosine and Nacetyltyrosine in the presence of tyrosinase imply that 0; is not generated by the semiquinone + O2 reaction. Since the addition of HzOz and dopa to tyrosinase promotes the release of 0, and formation of dopachrome, the Cu(II)O;Cu(I) complex could be formed as a intermediate (an active form of tyrosinase); [CU(II)]~ + H202 = CU(I)O~CU(II) f 2H’. 0 1992 Academic Press, Inc.
Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme widely distributed in nature and mainly involved in the biosynthesis of melanin and of other polyphenols (1). The copper-containing active site is binuclear in mushroom (2-4) and in human tyrosinase (5). Most of the enzyme is in the oxidized cupric form, which is activated by reduction by dihydric phenols (metabolites of monohydric phenols). The Cu(1) form then binds oxygen to form an oxytyrosinase (6). Three active oxygen-coordinated complexes have been proposed as active intermediates in the tyrosinase reaction (3). If active oxygen species (lo2 and 0;) are released from the active oxygen-coordinated complexes, they should be detectable by a specific and sensitive chemiluminescence method using a cypridina ’ To whom correspondence
should be addressed.
luciferin analog. The present work was undertaken to explore the generation of 0; in the tyrosinase reaction using 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2-u]pyrazin-3one (CLA)’ as a chemiluminescence probe and to confirm the generation of semiquinone radicals in these reactions, using a Zn2+-stabilizing method. MATERIALS
AND
METHODS
Chemicals. 3,4-Dihydroxy-L-phenylalanine (dopa), L-tyrosine (tyrosine), and N-acetyltyrosine were purchased from Sigma Chemical Co. 4-Hydroxyanisole and 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2alpyrazin-3-one (CLA) were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan) and Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), respectively. Partial purification of tymsinuse. A commercial mushroom tyrosinase (2200 units/mg, Sigma Chemicals) dissolved in 20 mM potassium phosphate buffer at pH 7.0 (250 ml containing 300 mg of protein) was applied to a column (2.5 X 30 cm) of DEAE-cellulose equilibrated with 20 mM potassium phosphate buffer at pH 7.0 (buffer A). The activity was eluted by application of a linear gradient of NaCl (mixing flask, 300 ml of buffer A; inlet flask, 300 ml of buffer A containing 0.5 M NaCl). Sevenmilliliter fractions were collected. The peak of tyrosinase activity (20 fractions) was pooled. The specific activity in the pooled solution was fourfold greater than that in the starting enzyme solution. Enzyme activity was measured in a system containing 1 mM dopa, 35 mM sodium phosphate buffer at pH 6.8, and enzyme, in a total volume of 3 ml at 35°C. One unit of enzyme activity was defined as that amount which produced 1 pmol of dopachrome/ml/min (7). Protein concentration was estimated by the method of Lowry et al. (8). Other enzymes. Catalase from bovine liver and superoxide dismutase from bovine erythrocytes were purchased from Sigma Chemicals. The former, dissolved in 50 mM potassium phosphate buffer at pH 7.0 (1 ml), was dialyzed against 3 liters of the same buffer overnight before use. The incubation systems and assays. The systems for investigating a relationship between CLA-dependent luminescence and cytochrome c reduction (0; generation) contained 43 PM hypoxanthine, 40 PM fer-
’ Abbreviations used: SOD, superoxide dismutase; CLA, 2-methyl-6phenyl-3,7-dihydroimidazo[l,2-a]pyrazin-3-one; dopa, 3,4-dihydroxy-Lphenylalanine.
570 All
0003.9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
GENER,$TION
OF SUPEROXIDE
DURING
THE
ricytochrome c (for the cytochrome c method) or 6 pM CLA (for the chemiluminescence method), a range of xanthine oxidase concentrations (162-486 units/ml), which were determined by the Roussos method (9), and 0.1 M potassium phosphate buffer at pH 6.5 in a total volume of 1 ml. The reaction was initiated by addition of the enzyme at 37°C. The initial rate (maximal rate) of cytochrome c reduction or the maximal luminescence intensity was determined by measuring the increase of absorption at 550 nm (t,,,. = 2.11 X lo4 Mm’ cm-‘) (10) or the maximal count/min in a luminescence re.ader (1 l), respectively. Calibration curves were then made by plotting maximal light intensity/min versus [enzyme] or maximal intensity/min versus initial rate of cytochrome c reduction (Fig. 1). All values were corrected for controls lacking xanthine oxidase. The standard reaction mixture contained 0.3 mM substrate (tyrosine, N-acetyltyrosine, dopa, 4-hydroxyanisole), 4 or 40 munits of tyrosinase/ ml, k6 FM CLA (for luminescence measurement), and 0.1 M potassium phosphate buffer at pH 6.5, in a total volume of 1 ml (for luminescence measurement), 0.6 ml (for o-dopaquinone measurement), 3.8 ml (for oxygen consumption), or 3.0 ml (for other assays). The reaction was initiated by the addition of tyrosinase and maintained at 37°C with a vigorous agitation. Production of o-dopaquinone was monitored in terms of ascorbate oxidation followed at 265 nm (t,., = 15.3 X lo3 Mm’ cm-‘) (12,13). Dopachrome formation was monitored by measuring A171m(~max = 3.7 X lo3 Mm’ cm-i) (14,15). Gxygen consumption was measured with a Clark-type electrode in an Instech oxygenometer (Model 102) assuming 217 nmol/ml for [O,] in the initial incubation mixture at 37°C. CLAdependent luminescence was measured in a luminescence reader (Aloka, BLR-102) as described previously (11). Integrated chemiluminescence intensity was obtained by tracing chemiluminescence intensity change (as a function of time) on homogeneous paper and weighing; it was expressed in terms of relative value. The HsOs concentration was calculated from the absorbance at 230 nm, assuming an extinction coefficient of 81 Mm’ cm-i (16). ESR spectra were observed with a Varian E-109 X-band spectrometer at 100 KHz field modulation, at 20°C. A magnetic field of 3360 G, a m:icrowave frequency of about 9.4 GHz, a microwave power of 5 mW, and a modulation width of 0.63 G were adopted. Semiquinone radicals were detected by the ESR-spin stabilization method (17).
RESULTS
(1) Generation of 0; in the Dopa-Tyrosinase
System
When dopa was incubated with tyrosinase in the presence of oxygen, O2 was generated and could be monitored
FIG. 1. Relationship between. CLA-dependent chemiluminescence and cytochrome c reduction (0; formation in hypoxanthine and xanthine oxidase). The reaction mixtures, incubation conditions, and assays were described under Materials and Methods. MAX.CL, maximal chemiluminescence; HX-XOD SYST, hypoxanthine-xanthine oxidase system.
ENZYMATIC
ACTION
INCUBATION
TIME,
571
OF TYROSINASE
min
TYROSINASE,
m unit/ml
FIG. 2. (A) O;-induced chemiluminescence in the dopa-tyrosinaseCLA system and the effect of possible inhibition on the chemiluminescence. The standard reaction mixture containing 0.3 mM dopa, 6 pM CLA, 4 munits of tyrosinase/ml, and 0.1 M potassium phosphate buffer at pH 6.5 was used. Additive were catalase (20 pg/ml), 20 mM histidine (HIS), 10 mM dimethylfuran (DMF), and 0.5 pM SOD. CL, chemiluminescence. (B) Double-reciprocal plot of the maximal light intensity against the concentration of dopa. The standard reaction mixtures containing 1, 2, and 6 munits of tyrosinase/ml, 6 pM CLA, and 0.1 M potassium phosphate buffer at pH 6.5 and various concentrations of dopa were used. (C) Relationship between 0, formation and tyrosinase activity. The standard reaction mixture was used, save that the enzyme concentration was varied from 0 to 10 munits/ml.
by CLA-dependent chemiluminescence. As shown in Fig. 2A, CLA-dependent luminescence appeared promptly just after the addition of enzyme, reached a maximum, and decreased exponentially thereafter. Since 0; reacts with CLA to emit light (18), CLA-dependent luminescence corresponds to the rate of 0; generation. Under the same experimental conditions, except for CLA, the rate of dopachrome formation reached a maximum within about 20 s and decreased to about 80% at 60 s, which is in good agreement with that for chemiluminescence intensities (data not shown). This luminescence was not influenced by histidine, dimethylfuran, or catalase, but was completely eliminated by a catalytic amount of SOD. The K,,, of tyrosinase for dopa was found to be 0.2 mM, as shown in Fig. 2B. Under the same experimental conditions, save that O2 consumption and dopachrome formation were used for the assays of the enzyme activities, K,,, values for the enzyme were found to be 0.5 mM with O2 consumption and 0.8 mM with dopachrome formation (data not shown). Korytowski et al. (17) have reported that the K,,, for dopa obtained with O2 consumption is 0.9 InM. With a fixed concentration of dopa, 0; generation was a linear function of enzyme concentration up to about 4 munits of the enzyme/ml and was calculated to be 0.55 nmol/min/ml (Fig. 2C). Cytochrome c reduction and chemiluminescence were linearly related, as shown in Fig. 1. The relationship between O2 consumption and product formation is shown in Table I. Under the standard reaction conditions, with 4 munits of tyrosinase/ml, 1 mol of Oz is required for the production of 2 mol of dopaquinone, 1 mol of dopachrome, and i mol of 0,. SOD at the
572
KOGA, TABLE
NAKANO,
AND
I
O2 Consumption and Metabolite Formation in the DopaTyrosinase System” Substance
[nmol/min/ml]b
0; Dopachrome Dopaquinone H,O, O2 consumption
0.55 2.12 4.00 Negligible 2.01
Note. Values were expressed as means of five experiments. ’ The standard reaction mixture with 4 m units of tyrosinase/ml 0.3 pmol of dopa/ml. * Initial rates.
catalytic amount had no effect on the dopachrome mation (data not shown).
and
for-
(2) Generation of 0; in the Tyrosine (or 4Hydroxyanisole)-Tyrosinase System To confirm the generation of 0, from tyrosinasemonohydric phenols, tyrosine and 4-hydroxyanisole, which possess a strong cytotoxicity on malignant melanoma (19), were used. Since CLA-dependent chemiluminescence was too weak to detect when 0.3 mM tyrosine was incubated with tyrosinase at an enzyme concentration of 4 munits/ml, a tyrosinase concentration of 40 munits/ ml was used in the tyrosine system. On the other hand, CLA-dependent chemiluminescence in the 4-hydroxyanisole system was detectable using tyrosinase at the lower concentration. As shown in Fig. 3A, the chemiluminescence from the tyrosine and 4-hydroxyanisole systems had lag periods. The lag was very much greater with tyrosine than with 4-hydroxyanisole. The CLA-dependent luminescence from both systems was completely quenched by a catalytic amount of SOD. Under the same experimental conditions, integrated chemiluminescence in the tyrosine system was not parallel to the dopachrome formation from tyrosine (Fig. 3B). This indicates that 0, generation is not directly related to the conventional enzyme activity. (3) ESR Spectrometry and O;-Induced Chemiluminescence It has been known that, in the tyrosinase-catalyzed reaction, tyrosine is oxidized to dopaquinone which rapidly cyclizes to generate dopa, while N-acetyltyrosine is oxidized by a noncyclizing pathway (14). Since both tyrosine and dopa are substrates for tyrosinase, tyrosine, when used as a substrate, would provide more electrons than would N-acetyltyrosine. Consequently 0; generation would be greater with tyrosine than with N-acetyltyrosine. To investigate this, two parameters such as semiquinone
TERO-KUBOTA
accumulation and quinone production were measured and compared with chemiluminescence. As shown in Fig. 4A, chemiluminescence with tyrosine and with N-acetyltyrosine had lag periods and similar kinetics, but was of significantly different intensity; i.e., the maximal light intensity in the tyrosine system was approximately 10 times that in the N-acetyltyrosine system. With the same systems, save that CLA was excluded, quinone accumulation detected in the presence of ascorbate in the tyrosine systems was approximately 1 that in the N-acetyltyrosine system (Fig. 4A, inset). Using higher enzyme concentrations at 2O”C, ESR signal heights (Fig. 4B) of Zn*+-stabilized radicals in N-acetyltyrosine and tyrosine systems were increased after lag periods, reached maximum at about 30 min, and then decreased, identical to the time course of chemiluminescence intensities in Fig. 4A. Such symmetric intensity curves for ESR study may indicate little or no production of their corresponding second radicals. Furthermore, the results showed that chemiluminescence and the semiquinone signal with tyrosine and N-acetyltyrosine were inversely related; i.e., high 0; generation correlated with low semiquinone accumulation. The ESR spectrum obtained during the incubation of tyrosine with tyrosinase in the presence of Zn2+ is shown in Fig. 5A. The hyperfine structure could be interpreted with four kinds of protons (a: = 4.0 G, a! = 0.63 G, a; = 2.25 and 4.25 G) as confirmed by computer simulation. These values are in good agreement with those of the Zn2+-stabilized dopaquinone anion radical, obtained by horseradish peroxidase-catalyzed dopa oxidation at pH 6.4 (20). Assignment of hyperfine splitting could reason-
A
z
-5% INCUBATION
TIME,min
4-HO-ANISOLE
TYR I
IO
I
20 30 INCUBATION TIME, mm
40
‘0 1
FIG. 3. (A) Time courses of CLA-dependent chemiluminescence in the tyrosine (or 4-hydroxyanisole)-tyrosinase system. The standard reaction mixture containing 0.3 mM tyrosine (or 4-hydroxyanisole), 6 pM CLA, 4 (. . . ) or 40 (-1 munits of tyrosinase/ml, and 0.1 M potassium phosphate buffer at pH 6.5 was used. (B) Relationship between relative integrated chemiluminescence (REL. ICLI) and dopachrome formation. The standard reaction mixtures containing 0.3 mM tyrosine, 40 munits of tyrosinase/ml, 6 pM CLA (for luminescence measurement) or none, and 0.1 M potassium phosphate buffer at pH 6.5 were used. The values at 40 min were taken as unity. TYR, tyrosine; 4-HO-ANISOLE, 4-hydroxyanisole; CL, chemiluminescence; muE, munits of enzyme.
GENERATION
OF SUPEROXIDE
DURING
ably be given by spin density calculation for the 4-alkylo-quinone anion radical (21). The hyperfine splitting due to the proton at the 3-plosition of the quinonoid ring is unresolved because of the smallness of its value (
OF BIOCHEMISTRY
Vol. 292, No. 2, February
AND
BIOPHYSICS
1, pp. 570-575,1992
Generation of Superoxide during the Enzymatic Action of Tyrosinase Satoshi Koga,*,t Minoru
Nakano,*Tl and Shozo Tero-Kubotaz
*Photon Medical Research Center, School of Medicine, Hamamatsu University, Hamamatsu, Handa-cho 3600, Shizuoka 431-31, Japan; TTaiho Pharmaceutical Co., Ltd., Kandanishiki-cho, Chiyoda-ku, Tokyo 101, Japan; and *Institute for Chemical Reaction Science, Tohoku University, Sendai 980, Japan
Received July 8, 1991, and in revised form October 4, 1991
Evidence for the generation of superoxide anion in an enzymatic action of tyrosinase is reported. In the dopatyrosinase reaction, 1 mol of Oz is required for the production of 2 mol of dopaquinone, 1 mol of dopachrome, and a mol of 0,. Superoxide dismutase and a-methyl6-phenyl-3,7-dihydroimidazo[l,2-alpyrazin-3-one (a chemiluminescence probe and Oz trap) do not inhibit the rate of dopachrome formation from dopa in the presence of tyrosinase, indicating that free 0, is not utilized for metabolizing dopa. ESR studies for the accumulation of semiquinone radicals generated from tyrosine and Nacetyltyrosine in the presence of tyrosinase imply that 0; is not generated by the semiquinone + O2 reaction. Since the addition of HzOz and dopa to tyrosinase promotes the release of 0, and formation of dopachrome, the Cu(II)O;Cu(I) complex could be formed as a intermediate (an active form of tyrosinase); [CU(II)]~ + H202 = CU(I)O~CU(II) f 2H’. 0 1992 Academic Press, Inc.
Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme widely distributed in nature and mainly involved in the biosynthesis of melanin and of other polyphenols (1). The copper-containing active site is binuclear in mushroom (2-4) and in human tyrosinase (5). Most of the enzyme is in the oxidized cupric form, which is activated by reduction by dihydric phenols (metabolites of monohydric phenols). The Cu(1) form then binds oxygen to form an oxytyrosinase (6). Three active oxygen-coordinated complexes have been proposed as active intermediates in the tyrosinase reaction (3). If active oxygen species (lo2 and 0;) are released from the active oxygen-coordinated complexes, they should be detectable by a specific and sensitive chemiluminescence method using a cypridina ’ To whom correspondence
should be addressed.
luciferin analog. The present work was undertaken to explore the generation of 0; in the tyrosinase reaction using 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2-u]pyrazin-3one (CLA)’ as a chemiluminescence probe and to confirm the generation of semiquinone radicals in these reactions, using a Zn2+-stabilizing method. MATERIALS
AND
METHODS
Chemicals. 3,4-Dihydroxy-L-phenylalanine (dopa), L-tyrosine (tyrosine), and N-acetyltyrosine were purchased from Sigma Chemical Co. 4-Hydroxyanisole and 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2alpyrazin-3-one (CLA) were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan) and Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), respectively. Partial purification of tymsinuse. A commercial mushroom tyrosinase (2200 units/mg, Sigma Chemicals) dissolved in 20 mM potassium phosphate buffer at pH 7.0 (250 ml containing 300 mg of protein) was applied to a column (2.5 X 30 cm) of DEAE-cellulose equilibrated with 20 mM potassium phosphate buffer at pH 7.0 (buffer A). The activity was eluted by application of a linear gradient of NaCl (mixing flask, 300 ml of buffer A; inlet flask, 300 ml of buffer A containing 0.5 M NaCl). Sevenmilliliter fractions were collected. The peak of tyrosinase activity (20 fractions) was pooled. The specific activity in the pooled solution was fourfold greater than that in the starting enzyme solution. Enzyme activity was measured in a system containing 1 mM dopa, 35 mM sodium phosphate buffer at pH 6.8, and enzyme, in a total volume of 3 ml at 35°C. One unit of enzyme activity was defined as that amount which produced 1 pmol of dopachrome/ml/min (7). Protein concentration was estimated by the method of Lowry et al. (8). Other enzymes. Catalase from bovine liver and superoxide dismutase from bovine erythrocytes were purchased from Sigma Chemicals. The former, dissolved in 50 mM potassium phosphate buffer at pH 7.0 (1 ml), was dialyzed against 3 liters of the same buffer overnight before use. The incubation systems and assays. The systems for investigating a relationship between CLA-dependent luminescence and cytochrome c reduction (0; generation) contained 43 PM hypoxanthine, 40 PM fer-
’ Abbreviations used: SOD, superoxide dismutase; CLA, 2-methyl-6phenyl-3,7-dihydroimidazo[l,2-a]pyrazin-3-one; dopa, 3,4-dihydroxy-Lphenylalanine.
570 All
0003.9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
GENER,$TION
OF SUPEROXIDE
DURING
THE
ricytochrome c (for the cytochrome c method) or 6 pM CLA (for the chemiluminescence method), a range of xanthine oxidase concentrations (162-486 units/ml), which were determined by the Roussos method (9), and 0.1 M potassium phosphate buffer at pH 6.5 in a total volume of 1 ml. The reaction was initiated by addition of the enzyme at 37°C. The initial rate (maximal rate) of cytochrome c reduction or the maximal luminescence intensity was determined by measuring the increase of absorption at 550 nm (t,,,. = 2.11 X lo4 Mm’ cm-‘) (10) or the maximal count/min in a luminescence re.ader (1 l), respectively. Calibration curves were then made by plotting maximal light intensity/min versus [enzyme] or maximal intensity/min versus initial rate of cytochrome c reduction (Fig. 1). All values were corrected for controls lacking xanthine oxidase. The standard reaction mixture contained 0.3 mM substrate (tyrosine, N-acetyltyrosine, dopa, 4-hydroxyanisole), 4 or 40 munits of tyrosinase/ ml, k6 FM CLA (for luminescence measurement), and 0.1 M potassium phosphate buffer at pH 6.5, in a total volume of 1 ml (for luminescence measurement), 0.6 ml (for o-dopaquinone measurement), 3.8 ml (for oxygen consumption), or 3.0 ml (for other assays). The reaction was initiated by the addition of tyrosinase and maintained at 37°C with a vigorous agitation. Production of o-dopaquinone was monitored in terms of ascorbate oxidation followed at 265 nm (t,., = 15.3 X lo3 Mm’ cm-‘) (12,13). Dopachrome formation was monitored by measuring A171m(~max = 3.7 X lo3 Mm’ cm-i) (14,15). Gxygen consumption was measured with a Clark-type electrode in an Instech oxygenometer (Model 102) assuming 217 nmol/ml for [O,] in the initial incubation mixture at 37°C. CLAdependent luminescence was measured in a luminescence reader (Aloka, BLR-102) as described previously (11). Integrated chemiluminescence intensity was obtained by tracing chemiluminescence intensity change (as a function of time) on homogeneous paper and weighing; it was expressed in terms of relative value. The HsOs concentration was calculated from the absorbance at 230 nm, assuming an extinction coefficient of 81 Mm’ cm-i (16). ESR spectra were observed with a Varian E-109 X-band spectrometer at 100 KHz field modulation, at 20°C. A magnetic field of 3360 G, a m:icrowave frequency of about 9.4 GHz, a microwave power of 5 mW, and a modulation width of 0.63 G were adopted. Semiquinone radicals were detected by the ESR-spin stabilization method (17).
RESULTS
(1) Generation of 0; in the Dopa-Tyrosinase
System
When dopa was incubated with tyrosinase in the presence of oxygen, O2 was generated and could be monitored
FIG. 1. Relationship between. CLA-dependent chemiluminescence and cytochrome c reduction (0; formation in hypoxanthine and xanthine oxidase). The reaction mixtures, incubation conditions, and assays were described under Materials and Methods. MAX.CL, maximal chemiluminescence; HX-XOD SYST, hypoxanthine-xanthine oxidase system.
ENZYMATIC
ACTION
INCUBATION
TIME,
571
OF TYROSINASE
min
TYROSINASE,
m unit/ml
FIG. 2. (A) O;-induced chemiluminescence in the dopa-tyrosinaseCLA system and the effect of possible inhibition on the chemiluminescence. The standard reaction mixture containing 0.3 mM dopa, 6 pM CLA, 4 munits of tyrosinase/ml, and 0.1 M potassium phosphate buffer at pH 6.5 was used. Additive were catalase (20 pg/ml), 20 mM histidine (HIS), 10 mM dimethylfuran (DMF), and 0.5 pM SOD. CL, chemiluminescence. (B) Double-reciprocal plot of the maximal light intensity against the concentration of dopa. The standard reaction mixtures containing 1, 2, and 6 munits of tyrosinase/ml, 6 pM CLA, and 0.1 M potassium phosphate buffer at pH 6.5 and various concentrations of dopa were used. (C) Relationship between 0, formation and tyrosinase activity. The standard reaction mixture was used, save that the enzyme concentration was varied from 0 to 10 munits/ml.
by CLA-dependent chemiluminescence. As shown in Fig. 2A, CLA-dependent luminescence appeared promptly just after the addition of enzyme, reached a maximum, and decreased exponentially thereafter. Since 0; reacts with CLA to emit light (18), CLA-dependent luminescence corresponds to the rate of 0; generation. Under the same experimental conditions, except for CLA, the rate of dopachrome formation reached a maximum within about 20 s and decreased to about 80% at 60 s, which is in good agreement with that for chemiluminescence intensities (data not shown). This luminescence was not influenced by histidine, dimethylfuran, or catalase, but was completely eliminated by a catalytic amount of SOD. The K,,, of tyrosinase for dopa was found to be 0.2 mM, as shown in Fig. 2B. Under the same experimental conditions, save that O2 consumption and dopachrome formation were used for the assays of the enzyme activities, K,,, values for the enzyme were found to be 0.5 mM with O2 consumption and 0.8 mM with dopachrome formation (data not shown). Korytowski et al. (17) have reported that the K,,, for dopa obtained with O2 consumption is 0.9 InM. With a fixed concentration of dopa, 0; generation was a linear function of enzyme concentration up to about 4 munits of the enzyme/ml and was calculated to be 0.55 nmol/min/ml (Fig. 2C). Cytochrome c reduction and chemiluminescence were linearly related, as shown in Fig. 1. The relationship between O2 consumption and product formation is shown in Table I. Under the standard reaction conditions, with 4 munits of tyrosinase/ml, 1 mol of Oz is required for the production of 2 mol of dopaquinone, 1 mol of dopachrome, and i mol of 0,. SOD at the
572
KOGA, TABLE
NAKANO,
AND
I
O2 Consumption and Metabolite Formation in the DopaTyrosinase System” Substance
[nmol/min/ml]b
0; Dopachrome Dopaquinone H,O, O2 consumption
0.55 2.12 4.00 Negligible 2.01
Note. Values were expressed as means of five experiments. ’ The standard reaction mixture with 4 m units of tyrosinase/ml 0.3 pmol of dopa/ml. * Initial rates.
catalytic amount had no effect on the dopachrome mation (data not shown).
and
for-
(2) Generation of 0; in the Tyrosine (or 4Hydroxyanisole)-Tyrosinase System To confirm the generation of 0, from tyrosinasemonohydric phenols, tyrosine and 4-hydroxyanisole, which possess a strong cytotoxicity on malignant melanoma (19), were used. Since CLA-dependent chemiluminescence was too weak to detect when 0.3 mM tyrosine was incubated with tyrosinase at an enzyme concentration of 4 munits/ml, a tyrosinase concentration of 40 munits/ ml was used in the tyrosine system. On the other hand, CLA-dependent chemiluminescence in the 4-hydroxyanisole system was detectable using tyrosinase at the lower concentration. As shown in Fig. 3A, the chemiluminescence from the tyrosine and 4-hydroxyanisole systems had lag periods. The lag was very much greater with tyrosine than with 4-hydroxyanisole. The CLA-dependent luminescence from both systems was completely quenched by a catalytic amount of SOD. Under the same experimental conditions, integrated chemiluminescence in the tyrosine system was not parallel to the dopachrome formation from tyrosine (Fig. 3B). This indicates that 0, generation is not directly related to the conventional enzyme activity. (3) ESR Spectrometry and O;-Induced Chemiluminescence It has been known that, in the tyrosinase-catalyzed reaction, tyrosine is oxidized to dopaquinone which rapidly cyclizes to generate dopa, while N-acetyltyrosine is oxidized by a noncyclizing pathway (14). Since both tyrosine and dopa are substrates for tyrosinase, tyrosine, when used as a substrate, would provide more electrons than would N-acetyltyrosine. Consequently 0; generation would be greater with tyrosine than with N-acetyltyrosine. To investigate this, two parameters such as semiquinone
TERO-KUBOTA
accumulation and quinone production were measured and compared with chemiluminescence. As shown in Fig. 4A, chemiluminescence with tyrosine and with N-acetyltyrosine had lag periods and similar kinetics, but was of significantly different intensity; i.e., the maximal light intensity in the tyrosine system was approximately 10 times that in the N-acetyltyrosine system. With the same systems, save that CLA was excluded, quinone accumulation detected in the presence of ascorbate in the tyrosine systems was approximately 1 that in the N-acetyltyrosine system (Fig. 4A, inset). Using higher enzyme concentrations at 2O”C, ESR signal heights (Fig. 4B) of Zn*+-stabilized radicals in N-acetyltyrosine and tyrosine systems were increased after lag periods, reached maximum at about 30 min, and then decreased, identical to the time course of chemiluminescence intensities in Fig. 4A. Such symmetric intensity curves for ESR study may indicate little or no production of their corresponding second radicals. Furthermore, the results showed that chemiluminescence and the semiquinone signal with tyrosine and N-acetyltyrosine were inversely related; i.e., high 0; generation correlated with low semiquinone accumulation. The ESR spectrum obtained during the incubation of tyrosine with tyrosinase in the presence of Zn2+ is shown in Fig. 5A. The hyperfine structure could be interpreted with four kinds of protons (a: = 4.0 G, a! = 0.63 G, a; = 2.25 and 4.25 G) as confirmed by computer simulation. These values are in good agreement with those of the Zn2+-stabilized dopaquinone anion radical, obtained by horseradish peroxidase-catalyzed dopa oxidation at pH 6.4 (20). Assignment of hyperfine splitting could reason-
A
z
-5% INCUBATION
TIME,min
4-HO-ANISOLE
TYR I
IO
I
20 30 INCUBATION TIME, mm
40
‘0 1
FIG. 3. (A) Time courses of CLA-dependent chemiluminescence in the tyrosine (or 4-hydroxyanisole)-tyrosinase system. The standard reaction mixture containing 0.3 mM tyrosine (or 4-hydroxyanisole), 6 pM CLA, 4 (. . . ) or 40 (-1 munits of tyrosinase/ml, and 0.1 M potassium phosphate buffer at pH 6.5 was used. (B) Relationship between relative integrated chemiluminescence (REL. ICLI) and dopachrome formation. The standard reaction mixtures containing 0.3 mM tyrosine, 40 munits of tyrosinase/ml, 6 pM CLA (for luminescence measurement) or none, and 0.1 M potassium phosphate buffer at pH 6.5 were used. The values at 40 min were taken as unity. TYR, tyrosine; 4-HO-ANISOLE, 4-hydroxyanisole; CL, chemiluminescence; muE, munits of enzyme.
GENERATION
OF SUPEROXIDE
DURING
ably be given by spin density calculation for the 4-alkylo-quinone anion radical (21). The hyperfine splitting due to the proton at the 3-plosition of the quinonoid ring is unresolved because of the smallness of its value (
