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Direct spectrophotometric detection of ascorbate free radical formed by dopamine β-monooxygenase and by ascorbate oxidase
30
Biochimica et Biophysica Acta, 630 (1980) 30--35 © Elsevier/North-Holland Biomedical Press
BBA 29265
DIRECT SPECTROPHOTOMETRIC DETECTION OF ASCORBATE FREE RADICAL FORMED BY DOPAMINE fi-MONOOXYGENASE AND BY ASCORBATE OXIDASE
TORE SKOTLAND
and T O R B J ~ R N
LJONES
Department of Biochemistry, University of Bergen, ~rstadveien 19, N-5000 Bergen (Norway) (Received November 19th, 1979) Key words: Ascorbate free radical; Dopamine ~-monooxygenase ; Dopamine ~-hydrox ylase ; Ascorbate oxidase
Summary A direct spectrophotometric m e t h o d was used for detection o f the ascorbate free radical formed during enzyme catalysis with dopamine fi-monooxygenase and with ascorbate oxidase. The optical absorption spectra in the range of 330--390 nm for the free radical formed by either of these enzymes were quite similar to the previously reported spectrum from pulse radiolysis experiments. The second order rate constant for dismutation of the radical generated b y dopamine fi-monooxygenase at 23°C was estimated from the levels of radical in the steady state, and the values of 2 . 4 . 1 0 -6 M - l - s -1 at pH 7.0 and 9 . 7 . 10 -6 M -1" s -1 at pH 6.0 were in close agreement with reported values from experiments in which the radical had been generated with ascorbate oxidase or with pulse radiolysis. Moreover, the steady state radical levels at different levels of dopamine fi-monooxygenase or its substrate tyramine were also those predicted b y a mechanism of nonenzymic dismutation of the radical. We conclude, in agreement with our earlier report with the c y t o c h r o m e c scavenger m e t h o d , that the radical is n o t an enzyme-bound intermediate, but a p r o d u c t of dopamine ~-monooxygenase catalysis.
Introduction The biological importance of ascorbic acid is well known, b u t precise definition of its role in individual biochemical reactions is k n o w n only in a limited number of cases. One of these is the role of ascorbate as electron donor in the
Abbreviation: MES, 2-(N-morPholino)ethanesulfonic acid.
31 hydroxylation reaction catalyzed by dopamine ~-monooxygenase (3,4~lihydroxyphenylethylamine, ascorbate : oxygen oxidoreductase (~-hydroxylating), EC 1.14.17.1). Although this enzyme can also use other reductants in vitro, such a physiological role for ascorbate is supported by the high enzymic rates in the presence of this reductant and by the report on high levels of ascorbate within the catecholamine storage vesicles, where the enzyme reaction takes place. It is believed that the function of ascorbate is to reduce the enzyme-bound copper (see Ref. 1 for a review). Ascorbate may function in enzymic oxidations as either a one- or a two-elec tron donor. In the first case, each ascorbate ion transfers one electron to the enzyme, and the unstable free radical (semidehydroascorbate) is the product of enzyme catalysis; t w o radicals then dismutate rapidly to one ascorbate and one dehydroascorbate. In the second case, each ascorbate ion transfers two electrons to the enzyme, giving dehydroascorbate as the p r o d u c t of enzyme catalysis. The experimental m e t h o d s used so far to differentiate between these two mechanisms are EPR spectroscopy [2] and a scavenger m e t h o d [3], which measures the rapid reduction of c y t o c h r o m e c by ascorbate free radical. Yamazaki and coworkers [4] have shown with these t w o methods that ascorbate oxidation b y ascorbate oxidase and b y peroxidase are one-electron processes. We have recently used the c y t o c h r o m e c scavenger m e t h o d to show that the reduction of dopamine ~-monooxygenase b y ascorbate is a one-electron process [5]. The present report confirms this conclusion with results of direct spectrophotometric measurement of the ascorbate free radical formed during enzyme catalysis with both dopamine ~-monooxygenase and ascorbate oxidase (L-ascorbate : oxygen oxidoreductase, EC 1.10.3.3}. The m e t h o d is based on the observations of Bielski et al. [6], who found b y pulse radiolysis studies that the ascorbate radical has an absorption maximum at 360 nm. This simple spectrophotometric m e t h o d has not been used previously for detection of ascorbate free radical formed during enzyme catalysis. Materials and Methods The water-soluble form of dopamine ~-monooxygenase was purified from bovine adrenal medulla [7,8]. The apoenzyme of dopamine ~-monooxygenase was obtained b y dialysis against EDTA [9], and contained less than 0.04 copper atoms per enzyme tetramer of 290 000 daltons, as analyzed by the bathocuproine disulfonate m e t h o d [9]. The enzyme concentrations were estimated assuming an absorbance o f 1.24 for a solution of 1 mg/ml at 280 nm with a light path of 10 mm [10]. Ascorbate oxidase from squash was obtained from Boehringer Mannheim and catalase from bovine liver (C-100) was obtained from Sigma. Reaction mixtures with dopamine ~-monooxygenase contained, unless specified differently: 20 mM potassium phosphate, 1 mM tyramine (substrate to be hydroxylated), 20 mM fumarate (an activator), 2/~M CuSO4 (to give maximal stimulation of the enzyme [9]), 1 mM ascorbate and 3000 units of catalase; total vol. 1 ml; pH 7.0. Reaction mixtures with ascorbate oxidase contained: 20 mM potassium phosphate, 1 mM ascorbate, and 3000 units of catalase; total vol. 1 ml; pH 7.0. Ascorbate oxidation was followed at 300 nm in order to
32 keep the initial absorbance at an acceptable level. The rate of ascorbate oxidation was calculated by using e3oonm = 420 M -~ • cm -1 at pH 7.0. This coefficient was estimated from an absorption spectrum b y comparison with e26snm = 14 000 M -~ " cm -~ [11]. Ascorbate free radical concentrations were estimated using e36o,m = 5000 M -1 • cm -1 [6]. Absorbance measurements were carried o u t at 23°C with a Cary 219 recording spectrophotometer. The rate constant for the dismutation of the ascorbate free radical, k d i ~ n , w a s calculated from the following equation, assuming that nonenzymic dismutation is the only pathway for removal o f the radical Uascorbate oxidation
= i)radical dismutation
=
2
• kdisr a •
[ascorbate radical] 2.
Results Time courses for ascorbate oxidation and the ascorbate free radical level in the presence of dopamine /3-monooxygenase and ascorbate oxidase show (Fig. 1) that the level of ascorbate free radical falls to zero when the ascorbate oxidation ceases, that is, when all the oxygen has been used up. This happens after oxidation of a b o u t 0.3 mM ascorbate in the presence o f dopamine /3-monooxygenase (Fig. 1B), which uses one 02 molecule per ascorbate ion and, after oxidation of a b o u t 0.6 mM ascorbate in the presence of ascorbate oxidase (Fig. 1A), which uses one 02 molecule per t w o ascorbate ions. The steady-state level of the free radical could be maintained for a longer time by flushing the incubation mixture with 02 (data n o t shown). The different shapes of the curves in Fig. 1 show that the Michaelis constant for 02 is lower with dopamine 13-monooxygenase than with ascorbate oxidase, and the data in Fig. 1 indicate values of 7 and 70 pM, respectively. The value reported here for dopamine 13-monooxygenase is much lower than previously published values [12]. The rate constant for the dismutation of the ascorbate free radical was
03
0 006
A b
0.2
O.O04
< 0.t <3
0.002
< <~
Time (rain)
F i g . 1. S p e c t r o p h o t o m e t r i c m e a s u r e m e n t o f t h e levels o f a s c o r b a t e ( m e a s u r e d a t 3 0 0 n m , c u r v e s l a b e l l e d (a) a n d a s c o r b a t e f r e e r a d i c a l ( m e a s u r e d a t 3 6 0 n m , c u r v e s l a b e l l e d (b) d u r i n g e n z y m i c a s c o r b a t e o x i d a tion by ascorbate oxldase (A) or dopamine ~-monooxygc~e (B). T h e e x p e r i m e n t s w e r e c a r r i e d o u t a t 23°C and pH 7.0 with 1 unit (1/~g) per ml of ascorbate oxidue or 0.39/~M dopamine ~-monooxygenase ( t e t r a m e r ) ; t h e i n c u b a t i o n m i x t u r e s w e r e o t h e r w i s e as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e r e a c t i o n s were started by addition of ascorbate. The absorbances at 360 nm before addition of ascorbate correspond to zero on the figures.
33 TABLE I ESTIMATES OF THE SECOND-ORDER BATE FREE RADICAL Source of radical formation
RATE CONSTANT
Pulse r a d i o l y s i s
OF ASCOR-
kdism • 10-6 (M-I. s-l) pH 7.0
Dopamine ~-monooxygenase Ascorbate oxidase
FOR THE DISMUTATION
2.4 2.7 1.7 2.5
a c d e
pH 6.0 9.7 b 10.0 d 13.0 e
a M e a n o f six d e t e r m i n a t i o n s ( 1 . 7 , 2 . 1 , 2 . 4 , 2 . 6 , 2 . 6 a n d 2 . 7 ) c a r r i e d o u t w i t h 0 . 2 0 - - 0 . 4 5 f M e n z y m e tetramer i n 2 0 m M p o t a s s i u m p h o s p h a t e ; 2 3 ° C , b Mean of three determinations (9.3, 9.7 and 10.0) carried out with 0.45 fM enzyme tetramer in 20 mM M E S i n s t e a d of phosphate buffer; 2 3 ° C . c M e a n o f three determinations ( 2 . 2 , 2 . 5 a n d 3 . 3 ) c a r r i e d o u t w i t h 1 u n i t (1 fig) o f a s c o r b a t e o x i d a s e p e r ml 20 mM phosphate; 23°C. d Data from Yamazaki [3]. Experiments carried out in 8 mM phosphate at approx. 22°C. e D a t a o b t a i n e d b y interpolation o f t h e r e s u l t s r e p o r t e d b y Bielski et ai. [ 5 ] . E x p e r i m e n t s c a r r i e d o u t in 20 mM phosphate at 23.5°C.
calculated from experiments similar to those shown in Fig. 1. As shown in Table I, the rate constants at pH 6.0 or 7.0 are both similar to those reported for the radical formed by either ascorbate oxidase [3] or pulse radiolysis [6]. Reactivated apoenzyme of dopamine ~-monooxygenase produced ascorbate radical with the same apparent rate constant for dismutation as obtained with the native enzyme. The optical absorption spectrum of ascorbate free radical in the region 330-390 nm was obtained from experiments similar to those reported in Fig. 1. This spectrum, which is shown in Fig. 2, resembles rather closely the reported spectrum of ascorbate free radical produced b y pulse radiolysis [6]. The s t e a d y ~ t a t e level of ascorbate free radical formed b y dopamine ~-monooxygenase (Fig. 1B) is less than the active-site concentration in this experiment: 0.76 pM radical compared with 1.56/~M active sites, assuming one active site per enzyme subunit of 75 000 daltons [1]. The ratio of radical to active site concentrations would be expected to increase when the enzyme concentration is decreased, because of the second-order rate law for radical dismutation. We therefore performed experiments similar to those in Fig. 1B with only 0.11 pM enzyme tetramer and higher concentrations of tyramine and ascorbate (10 mM and 2 mM, respectively) and obtained a free radical level of 0.50/~M. Thus, there were 1.14 radicals per active site in the steady-state. Assays at lower tyramine concentrations gave lower rates of ascorbate oxidation and lower levels of ascorbate free radical. In an experiment similar to that in Fig. 1B the rate o f ascorbate oxidation with 0.5 mM tyramine was 28% of the rate with 10 mM tyramine, and the steady-state level of the radical decreased as predicted b y the dismutation mechanism, i.e. the calculated values for the dismutation rate constants were identical within experimental error. Dehydroascorbate reportedly shows an absorption maximum at 300 nm with an extinction coefficient of 720 M -1 • cm -1 [13], b u t we have found that this absorption must be due to a degradation p r o d u c t rather than dehydroascorbate
34
1.00
a c
0.75
2 P
~
©
0.50
<[
0.25
I
[ 340
I
I 360
Wavelength
[
] 380
[
(nm)
Fig. 2. O p t i c a l a b s o r p t i o n s p e c t r a o f a s c o r b a t e free r a d i c a l . (o): R e d r a w n f r o m the pulse radiolysis s t u d y b y Bielski et al. [ 5 ] ; I a r b i t r a r y u n i t c o r r e s p o n d s to e = 5 0 0 0 M - I • s - I , w h i c h is the e x t i n c t i o n coeffic i e n t at 3 6 0 n m . ~, The difference a b s o r p t i o n b e t w e e n the steady-state level a n d t h e final level o b t a i n e d w i t h a s e o r b a t e o x i d a s e f r o m t r a c e s s i m i l a r to t h a t o f Fig. 1 A , b . o, T h e d i f f e r e n c e a b s o r p t i o n b e t w e e n t h e steady-state level and the final level o b t a i n e d w i t h d o p a m i n e ~ - m o n o o x y g e n a s e f r o m t r a c e s similar t o that o f Fig. 1 B , b . The data for a s c o r b a t e o x i d a s e and d o p a m i n e ~ - m o n o o x y g e n a s e w e r e a d j u s t e d s u c h that the A A 3 6 0 n m o b t a i n e d w i t h these e n z y m e s c o r r e s p o n d s t o 1 a r b i t r a r y u n i t . T h e e x p e r i m e n t a l c o n d i t i o n s w e r e as for Fig. 1.
itself. A solution of 4 mM dehydroascorbate prepared by oxidation of 4 mM ascorbate (in 20 mM potassium phosphate, pH 7.0) with 02 catalyzed by ascorbate oxidase (the ascorbate concentration was measured at 265 nm until the oxidation was complete) showed very little absorbance in the range of 250--400 nm immediately after preparation. The absorbance increased with time, however, with absorption peaks at about 285 and 340 nm. Some values for the absorbance at 300 nm were 0.02 after 8 min, 0.04 after 30 min, 0.07 after 1 h, and 1.1 after 23 h at room temperature. Thus, the ea0Onm is less than 5 M -I • cm -I.
Discussion The results of direct spectrophotometric detection of ascorbate free radical agree with our previous conclusion that dopamine /3-monooxygenase releases ascorbate free radical as the product of ascorbate oxidation [5]. Because o f the slow turnover of dopamine ~-monooxygenase compared with ascorbate oxidase or peroxidase and the rapid dismutation of the radical, however, it is difficult to attain levels of the radical in excess of the concentration o f active sites. The ascorbate free radical t h a t is detected in the near ultraviolet range could then presumably be an enzyme-bound intermediate, rather than a product of enzyme catalysis t h a t dissociates from the enzyme followed by nonenzymic dismutation. Nevertheless, we feel t h a t the results from direct spectrophotometric measurement of ascorbate radical can also be interpreted as strong evidence for ascorbate radical being the product of dopamine ~-monooxygenase
35 catalysis rather than an enzyme-bound intermediate, for the following reasons: (a) A ratio of radical to active site concentration (assuming four active sites per tetramer) of 1.14 was observed. (b) The calculated value for the dismutation rate constant for the free radical formed by dopamine fi-monooxygenase is similar to values from experiments with ascorbate oxidase (which has rapid turnover and radical concentrations several times greater than the enzyme concentration) and to values from pulse radiolysis experiments (Table I). (c) The ratio between the steady-state levels of the radical at pH 7.0 and 6.0 is close to the ratio predicted from the known pH
Skotland, T. and Ljones, T. (1979) Inorg. Perspect. Biol. Med. 2 , 1 5 1 - - 1 8 0 Yamazaki, I. an d Piette, L.H. (1961) Bioehim. Biophys. Acta 50, 62--69 Yamazak i, I. (1962) J. Biol. Chem. 2 3 7 , 2 2 4 - - 2 2 9 Yamazak i, I. (1977) in Free Radicals in Biology (Pryor, W.A., ed.), Vol. 3, pp. 183--218, A c a d e m i c Press, New Yo rk Ljones, T. and Skotland0 T. (1979) FEBS Lett. 108, 25--27 Bielski, B . H J . , C o m s t o c k , D.A. and Bowen, R.A. (1971) J. Am. Chem. Soc. 93, 5 6 2 4 - - 5 6 2 9 Ljones, T., S k o t l a n d , T. and F l a t m a r k , T. (1976) EVEr.J. Biochem. 6 1 , 5 2 5 - - 5 3 3 Skotland, T. and Flatms.rk0 T. (1979) J. Neurochem. 32, 1 8 6 1 - - 1 8 6 3 Skotland, T. and L|ones, T. (1979) Euz. J. Bioehem. 9 4 , 1 4 5 - - 1 5 1 Sk otland, T. and Ljones, T. (1977) Int. J. Pept. Protein Res. 1 0 , 3 1 1 - - 3 1 4 Karayannis, M.I., Samios, D.N. and Gousetis, CJP. (1977) Anal. Chim. Acta 9 3 , 2 7 5 - - 2 7 9 Goldstein, M., J o h , T.H. and Garvey, T.Q. (1966) Biochemistry 7, 2 7 2 4 - - 2 7 3 0 M a t t o k , G.L. (1965) J. Chem. Soc. 4 7 2 8 - - 4 7 3 5
Biochimica et Biophysica Acta, 630 (1980) 30--35 © Elsevier/North-Holland Biomedical Press
BBA 29265
DIRECT SPECTROPHOTOMETRIC DETECTION OF ASCORBATE FREE RADICAL FORMED BY DOPAMINE fi-MONOOXYGENASE AND BY ASCORBATE OXIDASE
TORE SKOTLAND
and T O R B J ~ R N
LJONES
Department of Biochemistry, University of Bergen, ~rstadveien 19, N-5000 Bergen (Norway) (Received November 19th, 1979) Key words: Ascorbate free radical; Dopamine ~-monooxygenase ; Dopamine ~-hydrox ylase ; Ascorbate oxidase
Summary A direct spectrophotometric m e t h o d was used for detection o f the ascorbate free radical formed during enzyme catalysis with dopamine fi-monooxygenase and with ascorbate oxidase. The optical absorption spectra in the range of 330--390 nm for the free radical formed by either of these enzymes were quite similar to the previously reported spectrum from pulse radiolysis experiments. The second order rate constant for dismutation of the radical generated b y dopamine fi-monooxygenase at 23°C was estimated from the levels of radical in the steady state, and the values of 2 . 4 . 1 0 -6 M - l - s -1 at pH 7.0 and 9 . 7 . 10 -6 M -1" s -1 at pH 6.0 were in close agreement with reported values from experiments in which the radical had been generated with ascorbate oxidase or with pulse radiolysis. Moreover, the steady state radical levels at different levels of dopamine fi-monooxygenase or its substrate tyramine were also those predicted b y a mechanism of nonenzymic dismutation of the radical. We conclude, in agreement with our earlier report with the c y t o c h r o m e c scavenger m e t h o d , that the radical is n o t an enzyme-bound intermediate, but a p r o d u c t of dopamine ~-monooxygenase catalysis.
Introduction The biological importance of ascorbic acid is well known, b u t precise definition of its role in individual biochemical reactions is k n o w n only in a limited number of cases. One of these is the role of ascorbate as electron donor in the
Abbreviation: MES, 2-(N-morPholino)ethanesulfonic acid.
31 hydroxylation reaction catalyzed by dopamine ~-monooxygenase (3,4~lihydroxyphenylethylamine, ascorbate : oxygen oxidoreductase (~-hydroxylating), EC 1.14.17.1). Although this enzyme can also use other reductants in vitro, such a physiological role for ascorbate is supported by the high enzymic rates in the presence of this reductant and by the report on high levels of ascorbate within the catecholamine storage vesicles, where the enzyme reaction takes place. It is believed that the function of ascorbate is to reduce the enzyme-bound copper (see Ref. 1 for a review). Ascorbate may function in enzymic oxidations as either a one- or a two-elec tron donor. In the first case, each ascorbate ion transfers one electron to the enzyme, and the unstable free radical (semidehydroascorbate) is the product of enzyme catalysis; t w o radicals then dismutate rapidly to one ascorbate and one dehydroascorbate. In the second case, each ascorbate ion transfers two electrons to the enzyme, giving dehydroascorbate as the p r o d u c t of enzyme catalysis. The experimental m e t h o d s used so far to differentiate between these two mechanisms are EPR spectroscopy [2] and a scavenger m e t h o d [3], which measures the rapid reduction of c y t o c h r o m e c by ascorbate free radical. Yamazaki and coworkers [4] have shown with these t w o methods that ascorbate oxidation b y ascorbate oxidase and b y peroxidase are one-electron processes. We have recently used the c y t o c h r o m e c scavenger m e t h o d to show that the reduction of dopamine ~-monooxygenase b y ascorbate is a one-electron process [5]. The present report confirms this conclusion with results of direct spectrophotometric measurement of the ascorbate free radical formed during enzyme catalysis with both dopamine ~-monooxygenase and ascorbate oxidase (L-ascorbate : oxygen oxidoreductase, EC 1.10.3.3}. The m e t h o d is based on the observations of Bielski et al. [6], who found b y pulse radiolysis studies that the ascorbate radical has an absorption maximum at 360 nm. This simple spectrophotometric m e t h o d has not been used previously for detection of ascorbate free radical formed during enzyme catalysis. Materials and Methods The water-soluble form of dopamine ~-monooxygenase was purified from bovine adrenal medulla [7,8]. The apoenzyme of dopamine ~-monooxygenase was obtained b y dialysis against EDTA [9], and contained less than 0.04 copper atoms per enzyme tetramer of 290 000 daltons, as analyzed by the bathocuproine disulfonate m e t h o d [9]. The enzyme concentrations were estimated assuming an absorbance o f 1.24 for a solution of 1 mg/ml at 280 nm with a light path of 10 mm [10]. Ascorbate oxidase from squash was obtained from Boehringer Mannheim and catalase from bovine liver (C-100) was obtained from Sigma. Reaction mixtures with dopamine ~-monooxygenase contained, unless specified differently: 20 mM potassium phosphate, 1 mM tyramine (substrate to be hydroxylated), 20 mM fumarate (an activator), 2/~M CuSO4 (to give maximal stimulation of the enzyme [9]), 1 mM ascorbate and 3000 units of catalase; total vol. 1 ml; pH 7.0. Reaction mixtures with ascorbate oxidase contained: 20 mM potassium phosphate, 1 mM ascorbate, and 3000 units of catalase; total vol. 1 ml; pH 7.0. Ascorbate oxidation was followed at 300 nm in order to
32 keep the initial absorbance at an acceptable level. The rate of ascorbate oxidation was calculated by using e3oonm = 420 M -~ • cm -1 at pH 7.0. This coefficient was estimated from an absorption spectrum b y comparison with e26snm = 14 000 M -~ " cm -~ [11]. Ascorbate free radical concentrations were estimated using e36o,m = 5000 M -1 • cm -1 [6]. Absorbance measurements were carried o u t at 23°C with a Cary 219 recording spectrophotometer. The rate constant for the dismutation of the ascorbate free radical, k d i ~ n , w a s calculated from the following equation, assuming that nonenzymic dismutation is the only pathway for removal o f the radical Uascorbate oxidation
= i)radical dismutation
=
2
• kdisr a •
[ascorbate radical] 2.
Results Time courses for ascorbate oxidation and the ascorbate free radical level in the presence of dopamine /3-monooxygenase and ascorbate oxidase show (Fig. 1) that the level of ascorbate free radical falls to zero when the ascorbate oxidation ceases, that is, when all the oxygen has been used up. This happens after oxidation of a b o u t 0.3 mM ascorbate in the presence o f dopamine /3-monooxygenase (Fig. 1B), which uses one 02 molecule per ascorbate ion and, after oxidation of a b o u t 0.6 mM ascorbate in the presence of ascorbate oxidase (Fig. 1A), which uses one 02 molecule per t w o ascorbate ions. The steady-state level of the free radical could be maintained for a longer time by flushing the incubation mixture with 02 (data n o t shown). The different shapes of the curves in Fig. 1 show that the Michaelis constant for 02 is lower with dopamine 13-monooxygenase than with ascorbate oxidase, and the data in Fig. 1 indicate values of 7 and 70 pM, respectively. The value reported here for dopamine 13-monooxygenase is much lower than previously published values [12]. The rate constant for the dismutation of the ascorbate free radical was
03
0 006
A b
0.2
O.O04
< 0.t <3
0.002
< <~
Time (rain)
F i g . 1. S p e c t r o p h o t o m e t r i c m e a s u r e m e n t o f t h e levels o f a s c o r b a t e ( m e a s u r e d a t 3 0 0 n m , c u r v e s l a b e l l e d (a) a n d a s c o r b a t e f r e e r a d i c a l ( m e a s u r e d a t 3 6 0 n m , c u r v e s l a b e l l e d (b) d u r i n g e n z y m i c a s c o r b a t e o x i d a tion by ascorbate oxldase (A) or dopamine ~-monooxygc~e (B). T h e e x p e r i m e n t s w e r e c a r r i e d o u t a t 23°C and pH 7.0 with 1 unit (1/~g) per ml of ascorbate oxidue or 0.39/~M dopamine ~-monooxygenase ( t e t r a m e r ) ; t h e i n c u b a t i o n m i x t u r e s w e r e o t h e r w i s e as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e r e a c t i o n s were started by addition of ascorbate. The absorbances at 360 nm before addition of ascorbate correspond to zero on the figures.
33 TABLE I ESTIMATES OF THE SECOND-ORDER BATE FREE RADICAL Source of radical formation
RATE CONSTANT
Pulse r a d i o l y s i s
OF ASCOR-
kdism • 10-6 (M-I. s-l) pH 7.0
Dopamine ~-monooxygenase Ascorbate oxidase
FOR THE DISMUTATION
2.4 2.7 1.7 2.5
a c d e
pH 6.0 9.7 b 10.0 d 13.0 e
a M e a n o f six d e t e r m i n a t i o n s ( 1 . 7 , 2 . 1 , 2 . 4 , 2 . 6 , 2 . 6 a n d 2 . 7 ) c a r r i e d o u t w i t h 0 . 2 0 - - 0 . 4 5 f M e n z y m e tetramer i n 2 0 m M p o t a s s i u m p h o s p h a t e ; 2 3 ° C , b Mean of three determinations (9.3, 9.7 and 10.0) carried out with 0.45 fM enzyme tetramer in 20 mM M E S i n s t e a d of phosphate buffer; 2 3 ° C . c M e a n o f three determinations ( 2 . 2 , 2 . 5 a n d 3 . 3 ) c a r r i e d o u t w i t h 1 u n i t (1 fig) o f a s c o r b a t e o x i d a s e p e r ml 20 mM phosphate; 23°C. d Data from Yamazaki [3]. Experiments carried out in 8 mM phosphate at approx. 22°C. e D a t a o b t a i n e d b y interpolation o f t h e r e s u l t s r e p o r t e d b y Bielski et ai. [ 5 ] . E x p e r i m e n t s c a r r i e d o u t in 20 mM phosphate at 23.5°C.
calculated from experiments similar to those shown in Fig. 1. As shown in Table I, the rate constants at pH 6.0 or 7.0 are both similar to those reported for the radical formed by either ascorbate oxidase [3] or pulse radiolysis [6]. Reactivated apoenzyme of dopamine ~-monooxygenase produced ascorbate radical with the same apparent rate constant for dismutation as obtained with the native enzyme. The optical absorption spectrum of ascorbate free radical in the region 330-390 nm was obtained from experiments similar to those reported in Fig. 1. This spectrum, which is shown in Fig. 2, resembles rather closely the reported spectrum of ascorbate free radical produced b y pulse radiolysis [6]. The s t e a d y ~ t a t e level of ascorbate free radical formed b y dopamine ~-monooxygenase (Fig. 1B) is less than the active-site concentration in this experiment: 0.76 pM radical compared with 1.56/~M active sites, assuming one active site per enzyme subunit of 75 000 daltons [1]. The ratio of radical to active site concentrations would be expected to increase when the enzyme concentration is decreased, because of the second-order rate law for radical dismutation. We therefore performed experiments similar to those in Fig. 1B with only 0.11 pM enzyme tetramer and higher concentrations of tyramine and ascorbate (10 mM and 2 mM, respectively) and obtained a free radical level of 0.50/~M. Thus, there were 1.14 radicals per active site in the steady-state. Assays at lower tyramine concentrations gave lower rates of ascorbate oxidation and lower levels of ascorbate free radical. In an experiment similar to that in Fig. 1B the rate o f ascorbate oxidation with 0.5 mM tyramine was 28% of the rate with 10 mM tyramine, and the steady-state level of the radical decreased as predicted b y the dismutation mechanism, i.e. the calculated values for the dismutation rate constants were identical within experimental error. Dehydroascorbate reportedly shows an absorption maximum at 300 nm with an extinction coefficient of 720 M -1 • cm -1 [13], b u t we have found that this absorption must be due to a degradation p r o d u c t rather than dehydroascorbate
34
1.00
a c
0.75
2 P
~
©
0.50
<[
0.25
I
[ 340
I
I 360
Wavelength
[
] 380
[
(nm)
Fig. 2. O p t i c a l a b s o r p t i o n s p e c t r a o f a s c o r b a t e free r a d i c a l . (o): R e d r a w n f r o m the pulse radiolysis s t u d y b y Bielski et al. [ 5 ] ; I a r b i t r a r y u n i t c o r r e s p o n d s to e = 5 0 0 0 M - I • s - I , w h i c h is the e x t i n c t i o n coeffic i e n t at 3 6 0 n m . ~, The difference a b s o r p t i o n b e t w e e n the steady-state level a n d t h e final level o b t a i n e d w i t h a s e o r b a t e o x i d a s e f r o m t r a c e s s i m i l a r to t h a t o f Fig. 1 A , b . o, T h e d i f f e r e n c e a b s o r p t i o n b e t w e e n t h e steady-state level and the final level o b t a i n e d w i t h d o p a m i n e ~ - m o n o o x y g e n a s e f r o m t r a c e s similar t o that o f Fig. 1 B , b . The data for a s c o r b a t e o x i d a s e and d o p a m i n e ~ - m o n o o x y g e n a s e w e r e a d j u s t e d s u c h that the A A 3 6 0 n m o b t a i n e d w i t h these e n z y m e s c o r r e s p o n d s t o 1 a r b i t r a r y u n i t . T h e e x p e r i m e n t a l c o n d i t i o n s w e r e as for Fig. 1.
itself. A solution of 4 mM dehydroascorbate prepared by oxidation of 4 mM ascorbate (in 20 mM potassium phosphate, pH 7.0) with 02 catalyzed by ascorbate oxidase (the ascorbate concentration was measured at 265 nm until the oxidation was complete) showed very little absorbance in the range of 250--400 nm immediately after preparation. The absorbance increased with time, however, with absorption peaks at about 285 and 340 nm. Some values for the absorbance at 300 nm were 0.02 after 8 min, 0.04 after 30 min, 0.07 after 1 h, and 1.1 after 23 h at room temperature. Thus, the ea0Onm is less than 5 M -I • cm -I.
Discussion The results of direct spectrophotometric detection of ascorbate free radical agree with our previous conclusion that dopamine /3-monooxygenase releases ascorbate free radical as the product of ascorbate oxidation [5]. Because o f the slow turnover of dopamine ~-monooxygenase compared with ascorbate oxidase or peroxidase and the rapid dismutation of the radical, however, it is difficult to attain levels of the radical in excess of the concentration o f active sites. The ascorbate free radical t h a t is detected in the near ultraviolet range could then presumably be an enzyme-bound intermediate, rather than a product of enzyme catalysis t h a t dissociates from the enzyme followed by nonenzymic dismutation. Nevertheless, we feel t h a t the results from direct spectrophotometric measurement of ascorbate radical can also be interpreted as strong evidence for ascorbate radical being the product of dopamine ~-monooxygenase
35 catalysis rather than an enzyme-bound intermediate, for the following reasons: (a) A ratio of radical to active site concentration (assuming four active sites per tetramer) of 1.14 was observed. (b) The calculated value for the dismutation rate constant for the free radical formed by dopamine fi-monooxygenase is similar to values from experiments with ascorbate oxidase (which has rapid turnover and radical concentrations several times greater than the enzyme concentration) and to values from pulse radiolysis experiments (Table I). (c) The ratio between the steady-state levels of the radical at pH 7.0 and 6.0 is close to the ratio predicted from the known pH
Skotland, T. and Ljones, T. (1979) Inorg. Perspect. Biol. Med. 2 , 1 5 1 - - 1 8 0 Yamazaki, I. an d Piette, L.H. (1961) Bioehim. Biophys. Acta 50, 62--69 Yamazak i, I. (1962) J. Biol. Chem. 2 3 7 , 2 2 4 - - 2 2 9 Yamazak i, I. (1977) in Free Radicals in Biology (Pryor, W.A., ed.), Vol. 3, pp. 183--218, A c a d e m i c Press, New Yo rk Ljones, T. and Skotland0 T. (1979) FEBS Lett. 108, 25--27 Bielski, B . H J . , C o m s t o c k , D.A. and Bowen, R.A. (1971) J. Am. Chem. Soc. 93, 5 6 2 4 - - 5 6 2 9 Ljones, T., S k o t l a n d , T. and F l a t m a r k , T. (1976) EVEr.J. Biochem. 6 1 , 5 2 5 - - 5 3 3 Skotland, T. and Flatms.rk0 T. (1979) J. Neurochem. 32, 1 8 6 1 - - 1 8 6 3 Skotland, T. and L|ones, T. (1979) Euz. J. Bioehem. 9 4 , 1 4 5 - - 1 5 1 Sk otland, T. and Ljones, T. (1977) Int. J. Pept. Protein Res. 1 0 , 3 1 1 - - 3 1 4 Karayannis, M.I., Samios, D.N. and Gousetis, CJP. (1977) Anal. Chim. Acta 9 3 , 2 7 5 - - 2 7 9 Goldstein, M., J o h , T.H. and Garvey, T.Q. (1966) Biochemistry 7, 2 7 2 4 - - 2 7 3 0 M a t t o k , G.L. (1965) J. Chem. Soc. 4 7 2 8 - - 4 7 3 5