Bioelectrochemistry and Bioenergetics 45 1998.

4145

Mechanism of pyrogallol autoxidation and determination of superoxide

dismutase enzyme activity
Ruomei Gao a , Zhuobin Yuan
a

a,)

, Zhiqiang Zhao b, Xiurui Gao

Department of Chemistry, Graduate School, Uniersity of Science and Technology of China, Academia Sinica, Beijing 100039, China
b
Department of Chemistry, Hebei Normal Uniersity, Shijiazhuang 050016, China
Received 14 October 1997; revised 15 December 1997; accepted 9 January 1998

Abstract
The autoxidation of pyrogallol was investigated in the presence of EDTA or DETAPAC diethylenetriaminepentaacetic acid. in the pH
range 7.87 to 9.10. Pyrogallol reacts with dioxygen in weakly alkaline solutions to form several intermediate products which are
electroactive substance and can be detected by electroanalytical methods. The focus here was putted on the effect of pH on the
autoxidation rate of some intermediate products produced in pyrogallol autoxidation, which gives sensitive second-order derivative
cathodic waves at y0.20, y0.96 and y1.45 V vs. SCE, respectively. Reaction mechanism was discussed. The paper also presented a
convenient electroanalytical assay for superoxide dismutase enzyme activity. q 1998 Elsevier Science S.A.
Keywords: Pyrogallol autoxidation; SOD activity; Single-sweep oscillopolarography

1. Introduction

following equation w3x:

Oxygen is an essential element for aerobes as it is the

terminal acceptor of the electrons during respiration, which
is the main source of energy in these organisms. However,
oxygen is toxic when supplied at concentrations greater
than those in air. In 1954, Gershman et al. w1x proposed
that known harmful effect of oxygen were due to the
formation of free radicals derived from it. This idea was
not completely accepted until the superoxide dismutase
SOD., an enzyme that catalyzes the superoxide radical
dismutation to hydrogen peroxide, was discovered in 1968
w2x. All the analytical methods for the determination of
SOD are based on this ability to accelerate the dismutation
of O 2 P and require a source of superoxide ion and a system
for detecting it. Methods currently used for assaying SOD
are time consuming and labor intensive. A rapid, simple
enzymatic assay for SOD could be useful clinically and
experimentally.
Superoxide dismutase play a central role in modulating
O 2 P concentrations by catalyzing the reaction shown in the

O 2y q O 2y q 2Hq H 2 O 2 q O 2

Corresponding author.

0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved.

PII S 0 3 0 2 - 4 5 9 8 9 8 . 0 0 0 7 2 - 5

SOD

1.

Therefore, the enzyme has proven to be a useful probe for

studying the participation of the radical in reactions involving oxygen such as autoxidations. O 2 P has been shown to
be involved in the autoxidation of, e.g., sulphite w4x,
adrenalin w5x, pyrogallol w6,7x and 6-hydroxy-dopamine w8x.
Pyrogallol indirect spectrophotometric assay has been developed for measuring SOD w6,7x. The main advantage of
the pyrogallol method is that the autoxidizing substance,
pyrogallol, serves both as the source of O 2 P and as the
indicating scavenger for O 2 P. Although pyrogallol methods
were widely used for measuring SOD activity, the kinetics
of intermediate products is yet unknown.
In previous studies w911x, we investigated the mechanism and the kinetics of the autoxidation of pyrogallol by
means of single-sweep oscillopolarography. The electrochemical system of pyrogallol can also be employed to
study scavenging effect on O 2 P and enzymatic determination. The present paper describes studies the autoxidation
of pyrogallol under various conditions. The role of O 2 P in
the reactions was investigated with the aid of superoxide
dismutase. Pyrogallol reacts with dioxygen in weakly alka-

42

R. Gao et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 4145

line solutions to form several intermediate products which

are electroactive substance and can be detected by electroanalytical methods. The focus here was putted on the
effect of pH on the rate of autoxidation of pyrogallol
measured by single-sweep oscillopolarography at y0.20,
y0.96 and y1.45 V vs. SCE, respectively. Reaction
mechanism was discussed. The precision of the enzymatic
assay was estimated using bovine blood copperzinc containing superoxide dismutase. The data obtained allotted
suitable conditions for a convenient assay of superoxide
dismutase.

2. Experimental
All experiments were performed at 13 1. Bovine CuZn
SOD specific activity of 13000 unitsrmg protein. was
dissolved in 10 mM sodium phosphate buffer pH 7.4. and
the solution was maintained in an ice bath. Pyrogallol
Beijing Chemical Reagent. was purified by sublimation.
All other solutions were prepared using double-distilled
water and analytical grade reagents.
An polarographic unit from The Seventh Telecommunication Equipment Plant of ShanDong, Model JP3-1 was
employed for the polarographic measurements. Measurements were made using single-sweep oscillopolarography
in a three-electrode polarographic cell fitted with a dropping mercury electrode, a platinum counter electrode and a
saturated calomel electrode SCE. as reference.
Electrochemical system of pyrogallol autoxidation was
employed to study reaction kinetics, scavenging effect on
O 2 P and enzymatic determination. Reaction mixtures contained, in a final volume of 10.00 ml, the following
reagents at the final concentrations stated: EDTA or DETAPAC diethylenetriaminepentaacetic acid. 1.00 mM.
and air equilibrated TrisHCl 45 mM. buffer, pH 8.14.
SOD was added at appropriate concentrations. The mixed
solution was stirred and then pyrogallol 0.20 mM or 0.04
mM. was added to reaction mixtures. The wave heights
corresponding to the intermediate products in pyrogallol
autoxidation were recorded following addition of pyrogallol start reagent to reaction mixtures. The electrosignals
were measured by single-sweep oscillopolarography at
y0.20, y0.96 and y1.45 V vs. SCE, respectively.

3. Results and discussion

3.1. Effect of pH on autoxidation
Pyrogallol 1. reacts with dioxygen in alkaline solutions
to form purpurogallin 2., which then forms a blue transient species, thought to be the dianion of purpurogalloquinone 3. and then, via peroxide oxidation, species 4.
w12,13x. We have used single-sweep oscillopolarography to

study behaviours of primary peaks in polarogram w9,10x.

Our results show that most intermediate and final products
of reactions are electroactive substance and can be measured by electroanalytical methods. Fig. 1 shows the second-order derivative cathodic waves of primary intermediate products in pyrogallol autoxidation.
Reaction course scheme is expressed in Fig. 2.
The autoxidation of pyrogallol was investigated in the
presence of EDTA or DETAPAC in the pH range 7.87 to
9.10. The rate of autoxidation D hrmin. was taken as the
initial rate of increase in peak current height at y0.20,
y0.96 and y1.45 V corresponding to the semiquinone of
pyrogallol, 3. and 2., respectively see above reaction
course scheme.. The semiquinone of pyrogallol gave second-order derivative cathodic wave at y0.20 V which
confirmed the one-electron redox potential of pyrogallol
reported in Refs. w14,15x. y1.45 and y0.96 V correspond
to the intermediate products of 2. and 3., respectively. A
number of kinetic systems and their rate constants have
been tested in Ref. w13x. Our results show that SOD is an
effective inhibitor of intermediate oxidation at y0.20,
y0.96 and y1.45 V, which confirmed an involvement of
the superoxide radical in the autoxidation of pyrogallol
w13x.
For y0.20 V, the slope of curve was maximal and
constant when pH was varied from 8.00 to 8.30; for y0.96
V and y1.45 V, from 8.10 to 8.40 see Fig. 3.. At higher
pH values there is a decrease in the rate of autoxidation as
the pH is raised which can be attributed to the increase in
the rate of decay of corresponding intermediate products.
In addition to these spontaneous decays, 3. reacts with
pyrogallol w13x. Therefore, when 3. forms during the
autoxidation, it may be consumed partly by a reaction with
pyrogallol. A more rapid rate decrease at y0.20 or y0.96
V may result from this extra consumption. Same reason
can account for the difference between y0.96 and y1.45
V in Figs. 46 .
Pyrogallol autoxidation can be inhibited by SOD. The
percent inhibition I%. depends on both the quantity of
SOD and the pH of the system. I% can be calculated
according to the following equation:

I% s

h 0 h1
h0

100%

where h1 represents the average peak height in the presence of various concentration of SOD, and h 0 average
peak height in the absence of SOD.
Fig. 4 shows effects of pH on maximal percent inhibition of pyrogallol autoxidation by SOD. At pH 7.87, the
reaction is inhibited to over 99% by SOD, indicating an
almost totally participating O 2 P, the intermediate product of
autoxidation, in the reaction. SOD concentrations at maximal percent inhibition increase with the pH increasing. The

R. Gao et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 4145

43

Fig. 1. Second-order derivative cathodic waves of pyrogallol. pH 8.14 TrisHCl buffer solution; pH 8.14 TrisHCl buffer solutionq 0.04 mM pyrogallol.
Recording timermin: 1, 1; 2, 3; 3, 5; 4, 10.

sensitivity to SOD decreases when the pH is increased, but

still amounts to 93% at pH 9.10.
3.2. Effects of pyrogallol on the autoxidation
Fig. 5 shows that the rate of autoxidation increases
linearly with pyrogallol concentration, but the straight lines
do not pass through the origin. The second-order derivative
cathodic wave at y0.20 V is so sensitive that the electrosignals go beyond detectable scope when pyrogallol
concentrations are up to 0.10 mM.

3.3. Effects of some metal ions

The rate of pyrogallol autoxidation is catalyzed obviously by some metal ions, such as, Fe 2q, Cu2q, Mn2q,
Co 2q, Zn2q, etc. After adding EDTA this influence can be
eliminated, but the interference of Fe 2q even in micromolar. still exists w5,6x. In the presence of EDTA, the rate is
independent of the concentration of the chelator and metal
ions do not significantly affect the autoxidation until the
chelating capacity is exceeded. Most of experiments in the
present report were performed in the presence of 1.00 mM

Fig. 2. Reaction course scheme.

44

R. Gao et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 4145

Fig. 3. Effect of pH on autoxidation rate of pyrogallol. 0.20 mM

pyrogallol for y1.45 and y0.96 V, and 0.04 mM for 0.20 V, respectively, in air-equilibrated 45 mM TrisHCl bufferq1.00 mM EDTA.
Scanning rate: 300 mVrs, hsquare. s 50 i p m A..

EDTA. DETAPAC was found to prevent interference from

Fe 2q as well as from other metal ions, such as Cu2q,
Zn2q, etc.. and was therefore chosen as chelator in the
enzymatic assay medium w6x.
3.4. Determination of SOD actiity by means of inhibition
of pyrogallol autoxidation
Since SOD reduces the O 2 P concentration by catalyzing
the reaction in Eq. 1., there is measurable inhibition in
initial reaction rates with the detector molecule w4x. The
percent inhibition of initial reaction rates depends on the
pH and the quantity of SOD present in the reaction mixtures see Fig. 6.. The quantity of enzyme inhibiting the
reaction by 50% IC 50 . is defined as one unit of SOD w5x.
This values can be calculated from Fig. 6.
In the present method, one unit corresponds to 0.17
m grml SOD in pH 7.87 TrisHCl buffer and 0.88 m grml
in pH 9.10 TrisHCl buffer at y1.45 V; 0.17 m grml in
pH 7.87 and 0.80 m grml in pH 9.10 at y0.96 V; and 0.02
m grml in pH 7.87 and 0.13 m grml in pH 9.10 at y0.20
V.
Table 1 shows results for 11 same samples of CuZn
SOD assayed on the same day using this method. In Table
1, one unit corresponds to 0.35 m g and 0.33 m g SOD in

Fig. 4. Effects of pH on maximal percent inhibition by SOD. Up two

lines: maximal percent inhibition by SOD %.; down two lines: SOD
concentrations at maximal inhibition. y0.96 V; y1.45 V. 0.20 mM
pyrogallol in air-equilibrated 45 mM TrisHCl bufferq1.00 mM DETAPAC. Scanning rate: 300 mVrs, hsquare. s 50 i p m A..

Fig. 5. Effects of pyrogallol concentration on the autoxidation. Pyrogallol

at appropriate concentrations in air-equilibrated 45 mM TrisHCl buffer
q1.00 mM EDTA. Scanning rate: 300 mVrs, hsquare. s 50 i p m A..

total volume of 1.00 ml at y1.45 and y0.96 V, respectively. The method gives similar sensitivity at both electric
potentials. However, y0.20 V is the most sensitive electric potential for SOD assay. Determination results in 0.05
m grunit at y0.20 V. The CV in Table 1 indicates reasonable precision for this assay.
The rate of pyrogallol autoxidation strongly depends on
pH as well as pyrogallol concentration. Therefore, the key
to obtain satisfactory results lies in precise control of both
values. The pyrogallol used in the present experiments had
been purified by sublimation. Another complicating factor
may be low molecular weight redox compounds that react
directly with O 2 P by acting as scavengers. A 0.09 mM
ascorbic acid inhibits the reaction by 50%. Cell extracts
are routinely dialyzed to remove redox compounds. Bovine
blood CuZn SOD is often used as the standard in enzyme
determination since it is readily available w16,17x, but it
may not react identically to SOD from other sources.
These factors are all possible sources of error in the
determination of SOD in biological media.
The decrease of SOD activity which resulted from the
alkali denaturation of protein had already been investigated
by other methods w18,19x. The reversible decrease of SOD
activity below pH 12.5 w18x and pH 11.3 w19x was observed, while above pH 12.5 there was an irreversible loss
of the activity due to the alkali denaturation of the protein.

Fig. 6. Inhibition of pyrogallol autoxidation by SOD. 0.20 mM pyrogallol

in air equilibrated 45 mM TrisHCl buffer pH 8.14.q1.00 mM DETAPAC. Scanning rate: 300 mVrs, hsquare. s 50 i p m A..

R. Gao et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 4145

Table 1
Bovine CuZn SOD determination on the same sample assayed repetitively on the same day ns11.
Sample

m grunit
CuZn SOD

Standard
deviation

CVr%

ErV

Bovine CuZn SOD

0.35a
0.33 a
0.05 b

0.02
0.02
0.003

5.7
6.1
6.0

y1.45
y0.96
y0.20

0.20 mM pyrogallol in air equilibrated 45 mM TrisHCl buffer, pH

8.14.
b
0.04 mM pyrogallol in air equilibrated 45 mM TrisHCl buffer, pH
8.14.

In the work presented here, no dependence of SOD activity

on this factor was observed in the range of pH 7.87 to
9.10.
We investigated the suitability of this method in the
presence of some potential denaturants such as urea, guanidine and sodium dodecyl sulphate SDS., which had been
used in the epinephrine oxidation assay to test the stability
of bovine SOD w20x. Guanidinium chloride and urea do not
modify the polarographic waves in the potential region
determined. The activity of SOD is lowered by about 30
and 40% in 3.0 and 9.5 M urea solutions, respectively,
which was similar to the data reported by Forman and
Fridovich w20x. The decrease of SOD activity observed in
the presence of guanidinium chloride and SDS can be
attributed to the effect of Cly ionic strength and the
inhibition of SDS on the mean limiting current. The electrosignal was lowered by about 50% in 0.1 M SDS solution in absence of SOD.
Acknowledgements
This investigation was supported by a grant from the
National Natural Science Foundation of China and Electroanalytical Chemistry Opening Laboratory Foundation of
ChangChun Applied Chemistry Institute, Academia Sinica.
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