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.

Reagents

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

Enzyme

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

Procedure

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.

Calculations

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