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Eur. J. Biochem.

47,469-474 (1974)

Involvement of the Superoxide Anion Radical in the Autoxidation of Pyrogallol and a Convenient Assay for Superoxide Dismutase
Stefan MARKLUND and Gudrun MARKLUND Department of Chemistry, Section of Physiological Chemistry, University of Umei (Received March 8 /June 8, 1974)

The autoxidation of pyrogallol was investigated in the presence ofEDTA in the pH range7.9- 10.6. The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99 by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide in anion radical, 02.-, the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O,.--independent mechanisms rapidly become dominant. Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H202is the stable product of oxygen and that H20, is not involved in the autoxidation mechanism. A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol. A plausible explanation is given for the non-competitive part of the inhibition of catechol 0-methyltransferase brought about by pyrogallol. Pyrogallol (1,2,3-benzenetriol) has long been known to autoxidize rapidly, especially in alkaline solution and the reaction has been employed for the removal of oxygen from gases. Molecular oxygen, carrying two unpaired electrons with parallel spins, has a preference for univalent reduction because spin restrictions arise when reduction with electron pairs is attempted [l]. The recently discovered enzyme superoxide dismutase [2] extremely rapidly dismutases univalently reduced oxygen 02.-, the superoxide anion radical (2 02.- 2 H + -+ 0, H,O,). The enzyme has proven to be a useful probe for studying the participation of the radical in reac- tions involving oxygen such as autoxidations. Thus 02.- been shown to be involved in the autoxidahas tion of e.g. sulphite [3], adrenalin [4] and 6-hydroxydopamine [ 5 ] . Bovine Cu-Zn superoxide dismutase (erythrocuprein) was the dismutase used in the present investigation. Its activity is essentially independent of pH in the range 5.5 to 9.5 [6,7] and it also has considerable activity at higher pH values. Another property of relevance in the present context is that it shows no saturation kinetics at the O,.- concentrations which have been feasible to produce [7] and hence induces a pseudo-first order dismutation of O,.-. The present paper describes studies of the autoxidation of pyrogallol under various conditions. The role of 02.- the reactions was investigated with the aid in of superoxide dismutase. The data obtained allotted suitable conditions for a convenient assay of superoxide dismutase.

MATERIALS AND METHODS All experiments were performed at 25 "C & 0.1. Pyrogallol (p.a. Merck, A.G.) was purified by sublimation. Stock solutions in 10 mM HCI are stable for weeks. Cacodylic acid, was obtained from British Drug Houses; Tris from Merck A.G. ; diethylenetriaminepentaacetic acid from Sigma Chemical Company; EDTA from Riedel-De HaZn A.-G.; NaCN (p.a.) from Merck, A.G. Water was double distilled in glass vessels and bovine Cu-Zn superoxide dismutase (erythrocuprein) was prepared from erythrocytes by the method of McCord and Fridovich [21 as modified by Weser et al. [8].

Enzymcv. Superoxide dismutase (EC 1.15.1.1); catalase (EC 1.11.1.6); catechol 0-methyltransferase (EC 2.1.1.6).

Eur. J. Bioc,hem.47 (1974)

470

Pyrogallol Autoxidation, Superoxide Dismutase

Crystalline bovine liver catalase was obtained from Boehringer Mannheim GmbH. The crystals were washed several times in distilled water before being dissolved. E~~~ was taken as 324mM-' cm-' [9]. 1 For spectrophotometry a Beckman Acta 1 1 was used. Oxygen consumption was determined polarographically with a Clark electrode using a Yellow Springs Instruments model 53 biological oxygen monitor. The different O2 saturations were obtained by mixing solutions carefully equilibrated with air, argon or pure oxygen. The latter two solutions were cautiously stratified under initially added air-equilibrated solution before mixing. The procedure was checked by the oxygen electrode and appeared to give the desired composition within 1 or 2 %.
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RESULTS AND DISCUSSION Pyrogallol autoxidizes rapidly in aqueous solution : the faster the higher the pH, and several intermediate products are apparently formed. Thus the solution first becomes yellow-brown with a spectrum showing a shoulder between 400 and 425 nm. After a number of minutes the colour begins to turn green and finally, after a few hours, a yellow colour appears. In the present investigation, the autoxidation was studied essentially during the first step(s) and the rate was taken from the linear increase in absorbance at 420 nm which is seen for a number of minutes after an induction period of some 10 s. The validity of this procedure is demonstrated in Fig. 1 by the constant relationship between increase in absorbance at 420 nm per min and oxygen consumption. However, this relationship was not tested at the higher pH values because of too slow a response of the oxygen electrode.

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Fig. 1. Effects o p H on the autoxidation o 0.2 mM pyrogallol f f in air-equilibrated buffer. Open symbols, 50 mM Tris-cacodylic acid buffer with 1 mM EDTA; filled symbols, 50 mM sodium carbonate buffer with 1 mM EDTA. (A) Rate of autoxidation as determined from the increase in &,. per , oxygen consumption, pM x min-' ; (0) maximal min; (0) inhibition of autoxidation by superoxide dismutase (> 40 pg x ml-I); (0) requirement of superoxide dismutase for 50% inhibition. (----) The part of the autoxidation which may be inhibited by superoxide dismutase

Effect of EDTA
In the absence of EDTA the autoxidation is faster than in its presence, and it tends to vary in rate and is less affected by superoxide dismutase. In the presence of EDTA the rate is independent of the concentration of the chelator (tested in the range 0.12 mM), which indicates that the effect of EDTA is only due to its binding of traces of metal ions. Most experiments in the present report were performed in the presence of 1 mM EDTA.

Effect of p H on the Autoxidation


Fig. 1 shows the effect of pH on a number of parameters of the autoxidation. The rate of autoxidation, taken as the initial rate of increase in absorbance at 420 nm, is increased by a factor of 2 per 0.3 pH

units around pH 8. When pH 9 is approached, the slope of the curve is decreased and a minimum is found around pH 9.5. At higher pH values, the slope again increases and finally corresponds to a doubling of the rate per 0.3 pH units. At pH 7.9 the autoxidation is inhibited to 99% by superoxide dismutase. The sensitivity to superoxide dismutase decreases when the pH is increased, but still amounts to 93% at pH 9. At higher pH values there is a strong decrease in the sensitivity to superoxide dismutase and at pH 10.6 the autoxidation is inhibited to only 15% by superoxide dismutase. The decrease in inhibition as the pH is raised can hardly be attributed to inactivation of the superoxide dismutase, as it was added in a large excess and addition of more superoxide dismutase had no further effect. The results indicate that there are at least two different mechanisms of autoxidation in the pH interval investigated. One mechanism, predominant at pH < 9.5 involves 02.-as a chain-propagating species. At higher pH values, another mechanism, not depending on 0 2 . -becomes predominant. ,
Eur. J. Biochem. 47 (1974)

S. Marklund and G. Marklund

47 1

The part of the autoxidation which may be inhibited by superoxide dismutase (dotted line in Fig. 1) reaches a plateau above pH 10 at a rate about twice the rate at pH 9. This fact and the decrease in slope of the curve describing the rate of autoxidation (Fig. 1) as pH 9 is approached, suggest that the first anion of pyrogallol (pK,, z 9, [lo]) has a central role in the 02.--dependent autoxidation of pyrogallol. The pH dependence of the involvement of 02.in the autoxidation of pyrogallol is opposite to that found for the autoxidation of adrenalin, where 02.-dependent paths increase in importance with increasing pH and predominate at pH > 9 [4].

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Effects oj'Pyrogallo1 and Oxygen Concentration on the Autoxidation


Fig. 2 shows that the rate of autoxidation increases linearly with pyrogallol concentration, but the straight line does not pass through the origin. The maximal inhibition of the autoxidation by superoxide dismutase remains unaltered at 97 % in the concentration interval. The effect of the other reactant, O,, is shown in Fig. 3. The rate increases with increasing 0, concentration, but not linearly. The maximal inhibition by superoxide dismutase increases from 97 % at 20 % O2 to 99% at 100% 0,.
[ Pyrogallol] (mM)

Fig. 2. Effects of pyrogallol concentration on the autoxidution. Air equilibrated 50 mM Tris-cacodylic acid buffer, pH 8.20 1 mM EDTA. (A) Rate of autoxidation as determined per from the increase in AdZ0 min; (0)requirement of superoxide dismutase for 50% inhibition of the autoxidation; (0) requirement of a different kind of superoxide dismutase with high molecular weight, probably containing Mn 1181, relative amount

Effects of Transition-Metal Ions Cuz' and Mn2+ do not significantly affect the autoxidation in the presence of 1 mM EDTA, until the chelating capacity is exceeded. Then both ions are powerful catalysts of the autoxidation and the reaction is not affected by superoxide dismutase. Fez', on the other hand, is active in micromolar concentrations in spite of the presence of EDTA as shown in Fig. 4. The reaction is apparently not , dependent on 0 2 . -as the increment in rate brought about by Fez+ is essentially identical in the presence and absence of superoxide dismutase. Superoxide dismutase is not inhibited by Fez+ [4]. The autoxidation of pyrogallol in the presence of EDTA may be initiated by, but is apparently not directly catalyzed by, traces of transition metal ions. Effect of Catalase
Catalase (0.2 pM) has no effect on the rate of autoxidation of pyrogallol (tested at pH 8.2 and 9.1) as determined spectrophotometrically, whereas the rate of oxygen consumption is halved by the enzyme. Thus, hydrogen peroxide is not involved in the autoxidation. A late product of pyrogallol autoxidation is purpurogallin [ l l ] which is formed from two pyrogallol
Eur. J. Biochem. 47 (1974)

02 saturation (%)

Fig. 3. Effects of oxygen saturation on autoxidation of pyrogallol. 0.2 mM pyrogallol in 50 mM Tris-cacodylicacid buffer pH 8.20 + 1 mM EDTA. (A) Rate of autoxidation as determined from the increase in A420 per min; (0)requirement of superoxide dismutase for 50 % inhibition of the autoxidation

molecules in which one carbon has been oxidized to CO, [12]. CO evolution has also been found during pyrogallol autoxidation [13]. However, the effect of catalase shows that hydrogen peroxide is the only significant product of oxygen during the initial part of the autoxidation. An old report of inhibition of the autoxidation by catalase [14] may be explained by the fact that

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Pyrogallol Autoxidation, Superoxide Dismutase

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Fig. 5. Inhibition 01'pyrogallol autoxidation by superoxide dismutase. 0.2 mM pyrogallol in air equilibrated 50 mM Tris-cacodylic acid buffer, pH 8.20 1 mM diethylenetriaminepentaacetic acid. The rate of autoxidation was taken from the increase in A,,, per min

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Fig. 4. Ejfects o j Felt on the uuto.uidation of py?yrogallol. 0.2 mM pyrogallol in air equilibrated 50 mM Tris-cacodylic acid buffer, pH 8.20 + 1 mM EDTA. (A) Rate of autoxidation in the absence of superoxide dismutase; (A) rate of autoxidation in the presence of 20 pg/ml superoxide dismutase

superoxide dismutase often contaminates catalase preparations [IS].

Determination o Superoxide-Dismutase Activity f by Means of Inhibition of Pyrogallol Autoxidation


The inhibition of pyrogallol autoxidation brought about by superoxide dismutase can be employed in a rapid and convenient method for the determination of the enzyme. After the above investigation we chose to use 0.2 mM pyrogallol in air-equilibrated 50 mM Tris-cacodylic acid buffer pH 8.20, containing 1 mM diethylenetriaminepentaacetic acid. Iron, even in trace amounts, accelerates pyrogallol autoxidation in spite of the presence of EDTA, Fig. 4. Diethylenetriaminepentaacetic acid was found to prevent interference from Fez+ (as well as from Cu2+ and M n Z +) and was therefore chosen as chelator in the assay medium. The rate of autoxidation is taken from the increase in absorbance at 420 nm, which is 0.020 min-' in the absence of superoxide dismutase. Fig. 5 shows the inhibition of autoxidation as a function of added superoxide dismutase. Superoxide dismutase may be added before or after the pyrogallol. The maximal inhibition which can be obtained is 97.5 %.

Most procedures for determining superoxide dismutase are based on the ability of the enzyme to inhibit O,.--dependent reactions. A unit of the enzyme is generally defined as the amount of the enzyme which inhibits the reaction by 50%. In the present method one unit corresponds to 100 ng bovine (CuZn) superoxide dismutase in a total volume of 1 ml; thus the method is about as sensitive as the method based on the reduction of cytochrome c by xanthine oxidase [2]. This sensitivity is for some purposes, e.g. monitoring column eluates, inconveniently high. Higher concentrations of pyrogallol decrease the sensitivity, cf Fig. 2. At 0.5 mM pyrogallol the sensitivity is decreased by a factor of 3.3. The pyrogallol used in the present experiments had been purified by sublimation. However, two reagent grade pyrogallol preparations (Merck AG ; British Drug Houses) were found to give the same result. HC1 can be used instead of cacodylic acid in the buffer but with some 30% loss of sensitivity. As can be deduced from Fig. 1 and 3, adequate pH control and air equilibration of the buffer are essential for high reproducibility. Cu-Zn-containing superoxide dismutases are efficien tly inhibited by cyanide [7] whereas those containing Mn [16] and Fe [17] are unaffected. Hence cyanide may be used as a means of differentiating between different types of superoxide dismutase. 1 mM cyanide decreases the rate of pyrogallol autoxidation by 25 % and the amount of superoxide dismutase (probably Mn containing [18]) required for 50% inhibition is decreased by 20 %. The increased sensitivity is probably due to a change in the characteristics of the autoxidation and not to an activation of the superoxide dismutase.
.Eur. J. Biochem. 47 (1974)

S. Marklund and G. Marklund

473

Reducing compounds interfere with pyrogallol autoxidation [19] by acting as scavengers. Thus 10 pM reduced glutathione and ascorbate inhibit the reaction by 20 /,. The method seems to be applicable for the assay of crude preparations ; bovine Cu-Zn superoxide dismutase exerted its full activity as internal standard in a bovine liver homogenate. The maximal inhibition that can be obtained with a liver homogenate is > 95 % and the presence of an amount of liver homogenate giving 80 % inhibition does not at all influence the maximal inhibition obtainable with pure superoxide dismutase. Peroxidases interfere by accelerating the autoxidation. Catalase in a 10 to 20-fold molar excess totally eliminates the interference by competing with peroxidase for H202.Thus, 0.2 pM catalase allows the assay of superoxide dismutase in horseradish homogenates.

Effects o Pyrogallol on Catechol 0-Methyltransferase f Pyrogallol is a mixed competitive and noncompetitive inhibitor of catechol 0-methyltransferase [27,28]. The noncompetitive inhibition increases with increasing purification of the enzyme and with increased preincubation with pyrogallol [27]. The assay of catechol 0-methyltransferase is performed at 37 C in a buffer of pH 7.9 for at least 10 min [27]. The pyrogallol will be rapidly autoxidized under these conditions. During autoxidation of pyrogallol 02.-, H 2 0 2 and possibly OH. are formed, the latter by the Haber-Weiss mechanism [29], 02.- H 2 0 2 O H . OH02. These reactive species may contribute significantly to the noncompetitive effects of pyrogallol on catechol 0-methyltransferase, which contains an essential sulfhydryl group [30]. It can also be questioned whether the competitive part of the inhibition is due to the pyrogallol or to its oxidation products.

--f

Possibility o Involvement o Singlet Oxygen f f in Pyrogallol Autoxidation

We wish to thank Professor K.-G. Paul for valuable discussions. The study was supported by Statens Medicinska A. Forskningsrcid, grant number B74-13X-4267-01

In addition to being a superoxide dismutase, erythrocuprein has .been stated to be an efficient quencher of singlet oxygen (dg) [20-221. Singlet oxygen has been suggested as forming during non[23] enzymatic dismutation of 02.- and the inhibitory effect of superoxide dismutase on pyrogallol autoxidation could consequently indicate a role of singlet oxygen in the reaction. However, there are several objections to this idea. The arguments for a role of erythrocuprein as a singlet oxygen quencher are questionable. In one paper, the quenching was studied using luminol chemiluminescence as a detector of singlet oxygen produced during perchromate decomposition [22]. However. luminol chemiluminescence may be brought about by a variety of reagents, and the mechanism(s) is composite [24,25]. In several cases erythrocuprein inhibits the luminescence and the inhibition could dismutation [25]. reasonably be ascribed to 02-The lifetime of singlet oxygen (dg) in aqueous solution has been given as 2 ps [26]. The quenching by erythrocuprein was stated to be observable already at 0.1 nM [22], requiring a rate constant for the quenching reaction of the order of 10l5 M- x sK1. This is physically impossible, and if erythrocuprein is a quencher, the lifetime of singlet oxygen must be at least lo5 times longer than reported [26]. Finally, if there is any significant reaction between the 0.2 mM pyrogallol and singlet oxygen possibly formed during autoxidation, an extraordinarily high rate constant is required, 2 lo9 M- x sK,
Eur. J. Biochem. 47 (1974)

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1. Taube, H. (1965) in Oxygen, Proc. Symp. Spons. New York Heurt Association, pp. 29- 52, Little Brown and Co. 2. Mc Cord, J. M. & Fridovich, I. (1969) J . Bid. Chem. 244,6049 - 6055. 3. Mc Cord, J. M. & Fridovich, I. (1969) J . Biol. Chem. 244,6056 - 6063. 4. Misra, H. P. & Fridovich, I. (1972) J . Bid. Chem. 247, 3170- 3175. 5. Heikkila, R. E. & Cohen, G. (1973) Science (Wash. D. C.) 181,456-457. 6. Klug, D., Rabani, J. & Fridovich, I. (1972) J . Biol. Chem. 247,4839 - 4842. 7. Rotilio, G., Bray, R. C. & Fielden, E. M. (1972) Biochim. Biophys. Acta, 268, 605 - 609. 8. Weser, U., Bunnenberg, E., Cammack, R., Djerassi, C., Flohe, L., Thomas, G. & Volter, W. (1971) Biochim. Biophys. Actu, 243, 203 - 213. 9. Samejima, T. & Tsi Yang, J. (1963) J . Biol. Chem. 238, 3256-3261. 10. Kortiim, G., Vogel, W. & Andrussow, K . (1961) Dissociution Constants o Organic Acids in Aqueous Solution, f p. 443, Butterworths, London. 11. Nierenstein, M. & Spiers, C. W. (1913) Ber. Dtsch. Chcm. Ges. 46, 3151 -3157. 12. Barltrop, J. A. & Nicholson, J. S. (1948) J . Chem. Soc. 116- 120. 13. Miyahara, S. & Takahashi, H. (1971)J . Biochem. (Tokyo) 69, 231 -233. 14. Siegel, S . M. & Siegel, B. Z. (1958) Nature (Lond.) 181, 1153- 1154. 15. Halliwell, B. (1973) Biochem. J . 135, 379-381. 16. Weisiger, R. A. & Fridovich, I. (1973) J. Biol. Chem. 248,3582- 3592.

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S. Marklund and G. Marklund : Pyrogallol Autoxidation, Superoxide Dismutase 24. Lee, K. D. & Seliger, H. H. (1972) Photochem. Photobiol. 15,227-237. 25. Hodgson, E. K. &Fridovich, I. (1973) Photochem. Photobiol. 18,451-455. 26. Merkel, P. B. & Kearns, D. R. (1972) J . Am. Chem. SOC. 94, 7244-7253. 27. Crout, J. R. (1961) Biochem. Pharmacol. 6,47-50. 28. Baldessarini, R. J. & Greiner, E. (1973) Biochem. Pharmacol. 22,247- 256. 29. Haber, F. & Weiss, J. (1934) Proc. SOC.Lond. A Math. Phys. Sci. 147,332-351. 30. Axelrod, J. & Tomchick, R. (1958) J. Biol. Chem. 233, 702 - 705.

17. Yost, F. J. & Fridovich, I. (1974) Arch. Biochem. Biophys. 161,395-401. 18. Marklund, S. (1973) Acta Chem. Scand. 27, 1458- 1460. 19. Ghosh, J. C. & Rakshit, P. C. (1937) Biochem. 2. 294, 330- 335. 20. Finazzi Agro, A,, Giovagnoli, C., De Sole, P., Calabrese, L., Rotilio, G. & Mondovi, B. (1972) FEBS Lett. 21, 183-185. 21. Weser, U. & Paschen, W. (1972) FEBS Lett. 27,248 - 250. 22. Paschen, W. & Weser, U. (1973) Biochim. Biophys. Acta, . . 327,217-222. 23. Khan, A. U. (1970) Science (Wash. D. C.) 168,476-477.

S. Marklund and G. Marklund, Kemiska Institutionen, Avdelningen for Medicinsk Kemi, Umeg Universitet, S-901 87 Ume%,Sweden

Eur. J. Biochem. 47 (1974)

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