Xanthine Oxidase Assay

Method: The rate of formation of urate from hypoxanthine is determined by measuring increased absorbance at 290 nm.

A unit of activity is that forming one micromole of urate per minute at 25°C.


  • Buffer: 0.05 M Phosphate, pH 7.5
  • Substarte: 10 mg hypoxanthine in 500 ml reagent grade water


Dilute stock suspension with 0.05 M phosphate buffer, pH 7.5, to contain 0.1 to 0.2 units per ml.


Into cuvettes pipette the following:

  Test Control
Buffer 1.9 ml 1.9 ml
Reagent grade water ---- 1.0 ml
Enzyme 0.1 ml 0.1 ml
Substrate (at zero time) 1.0 ml ----

Record increase in absorbance and determine ΔA290 from the linear portion of the curve. The rate is proportional to enzyme concentration within limits of 0.01 to 0.02 units per test.



The molar absorbancy of uric acid = 1.22 X 104 cm-1 (Westerfeld et al. 1959)

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