Xanthine Oxidase (XO)
(Xanthine: oxygen oxidoreductase; EC 188.8.131.52)
Xanthine oxidase (XO) is a complex enzyme containing flavins,
molybdenum, iron and sulfide cofactors. The reaction catalyzed
by Xanthine oxidase is shown below:
Xanthine + O2 + H2O ------> Uric Acid + H2O2
Xanthine oxidase from cow milk has been extensively
studied. It has a molecular weight of 275,000. Milk or
buttermilk are used for preparation of commercial quantities
of the enzyme.
In healthy individuals, Xanthine oxidase is present in
appreciable amounts only in the liver and jejunum. However, in
various liver disorders the enzyme is released in the
circulation. Therefore, determination of serum Xanthine
oxidase level serves as a sensitive indicator of acute liver
damage such as jaundice (Giller, S., Sperling, O., Brosh, S.,
Urca, I. and DeVries, A., Clinica Chim. Acta, 63, 37, 1975). A
sensitive and rapid assay for human serum Xanthine oxidase,
which is applicable in the clinical diagnosis of liver
disorders has been described by McHale, et al (McHale, A.;
Grimes, H. and Coughlan, M.P., Int. J. Biochem., 10, 317,
1979). Xanthine oxidase is also used for determination of the
activity of Superoxide dismutase (Forman, H.J. and Fridovich,
I., J. Biol. Chem., 248, 2648, 1973).
||Ammonium Sulfate Suspension
||Soluble in distilled water or dilute buffer
||Stable when stored at 4° C. Traces of heavy metals adversely affect stability
||1.0-4.0 U/mg protein
||10 mg/ml (biuret)
That amount of
enzyme which catalyzes the oxidation of one micromole of Xanthine to
uric acid per minute at 37°C and pH 7.5.
The increase in absorbance at 290 nm, caused by the oxidation
of Xanthine to uric acid, is a measure of the catalytic
activity of Xanthine oxidase (Kalckar, H.M., J. Biol. Chem.,
167, 429, 1947).
- 0.05 M Sodium phosphate buffer, pH 7.5.
- 0.0001 M Xanthine (substrate). Dissolve 1.8 mg Xanthine, sodium
salt in 100 ml of 0.05 M sodium phosphate buffer. Adjust pH to 7.5 if
necessary. Aerate for 5 minutes before use.
- Xanthine oxidase (enzyme) solution. Using the buffer as a
solvent, prepare an enzyme solution containing 0.06 U/ml. Must be
prepared fresh prior to assay.
- Set the spectrophotometer (equipped with strip chart recorder and
temperature control) at 290 nm and 37°C.
- Into a quartz cuvette pipette 2.9 ml Xanthine (substrate)
solution. Incubate cuvette in spectrophotometer at 37°C for 5 minutes
to achieve temperature equilibration.
- Record blank rate at 290 nm, if any.
- Add 0.1 ml enzyme solution to the cuvette. Mix and record the
increase in absorbance at 290 nm for 5 minutes.
- Calculate µE290 nm/min
|Activity (U/mg) =
||(ΔE290nm/min)(Total Vol.)(Enz. Diln.)
|(12.3)(Enz. Vol.)(mg Enz./ml)