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Xanthine Oxidase (XO)

(Xanthine: oxygen oxidoreductase; EC

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).

1.Source: Buttermilk
   Form: Ammonium Sulfate Suspension 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: Stable when stored at 4 C. Traces of heavy metals adversely affect stability 
   Activity (approx.): 1.0-4.0 U/mg protein 
   Protein (approx.): 10 mg/ml (biuret) 
   Catalog No.: 076B0001 

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).

  1. 0.05 M Sodium phosphate buffer, pH 7.5.
  2. 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.
  3. 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.

  1. Set the spectrophotometer (equipped with strip chart recorder and temperature control) at 290 nm and 37°C.
  2. 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.
  3. Record blank rate at 290 nm, if any.
  4. Add 0.1 ml enzyme solution to the cuvette. Mix and record the increase in absorbance at 290 nm for 5 minutes.
  5. Calculate µE290 nm/min

Activity (U/mg) = (ΔE290nm/min)(Total Vol.)(Enz. Diln.)
(12.3)(Enz. Vol.)(mg Enz./ml)

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