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Mechanism of Pyrogallol Autoxidation and Determination of Superoxide
Mechanism of Pyrogallol Autoxidation and Determination of Superoxide
4145
a,)
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
O 2y q O 2y q 2Hq H 2 O 2 q O 2
Corresponding author.
SOD
1.
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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.
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
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.
44
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.
m grunit
CuZn SOD
Standard
deviation
CVr%
ErV
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
45