ELECTROCHEMICAL ASSAYS: THE OXYGEN ELECTRODE

electrode-system to be constructed (4). Such adaptations may well be of considerable use in

continuous industrial processes.

7.2.1 Glucose oxidase assay

Glucose oxidase (EC 1.1.3.4) catalyses the oxidation of D-glucose to gluconolactone as follows:

glucose ⫹ O2 ⫹ H2O 씮 gluconolactone ⫹ H2O2

The oxygen utilized during the reaction may be used as a measure of the enzyme’s activity
or alternatively, in the presence of non-saturating glucose concentrations, as a measure of
glucose concentration (12). The assay is very simple and may be set up optimally in 50 mM Na
acetate buffer, pH 5.6, although other buffers and a wider range of pH may be used without
too much loss of activity (pH 3.5–9). Such a system was found to show a linear relationship
between either glucose concentration (70 ␮M–30 mM) or enzyme concentration (0.1–2 units)
(12).

7.2.2 Catalase assay

Catalase (EC 1.11.1.6) catalyses the breakdown of hydrogen peroxide as follows:

2H2O2 씮 2H2O ⫹ O2

It is a widely distributed enzyme across the animal and plant world and successful analysis of
its kinetics was difficult before the establishment of the oxygen electrode system. All assay
procedures involve the setting-up of an electrode chamber at constant temperature and with
constant stirring of the reaction medium, together with the ability to close-off the reaction
system to the atmosphere save for a small injection port for the addition of substrate or
enzyme. Such a system has been described (13), the essentials of which are outlined below:

Protocol 2
Assay of catalase
Equipment and reagents
● Oxygen electrode ● 50 mM Na2HPO4-KH2PO4, pH 7.0
● Recorder ● H2O2
● Nitrogen ● Catalase

1 Set up the electrode and add 1 ml of 50 mM Na2HPO4–KH2PO4, pH 7, buffer.

2 Allow to equilibrate to 25 ⬚C and establish recorder setting near 100%.
3 Bubble buffer system with N2 gas until zero setting is established and close-off reaction chamber
to atmosphere.
4 Add 10 ␮l H2O2 (33.5 mM final concentration) through injection port and allow to equilibrate.
5 Add 50 ␮l of suitably diluted catalase sample and the initial velocity of reaction may be estab-
lished from the kinetics of the O2 production rate. (Note that any non-enzymatic production of
oxygen should be deducted.)

It was found (13) that an activity range of 0.01–8.4 ␮mol O2/min could be measured and
further increases in sensitivity could be achieved by improving the electrode/recorder amplifi-
cation (down to 0.002 ␮mol O2/min). This permitted the measurement of catalase activities
over almost a 1000-fold concentration range and was 20 times more sensitive than other
assay systems.

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J. B. CLARK

7.2.3 Other systems

Considerable use has been made of the oxygen electrode to set up continuous assay pro-
cedures for metabolic intermediates along similar lines to those described for the glucose
electrode. These are particularly useful in clinical laboratories and include measurement of
free and esterified cholesterol by the use of impregnated cholesterol oxidase and hydrolase
(14), tyrosine (15), prostaglandin synthesis by the measurement of arachidonic acid (16), and
uric acid using urate oxidase and monitoring the oxygen consumption (17). These systems
are, however, outside the scope of this article and are only mentioned for completeness.

References
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medicine and technology CRC Press, Cleveland, USA.
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New York.
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438.
12. Hertz, R. and Barenholz, Y. (1973). Biochim. Biophys. Acta, 330, 1.
13. Del Rio, L. A., Gomez Ortega, M., Lopez, A. L., and Lopez Gorge, J. (1977). Anal. Biochem., 80, 409.
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