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.

Calculation

\frac{\text{Units}}{\text{mgP}}\text{ = } \frac{\frac{\Delta \text{A}}{\text{min}}\text{ x 1000}}{\text{1.22 x 10}^4\text{ x }\frac{\text{mg}}{\text{ml}}\text{ reaction mixture}}

\frac{\text{Units}}{\text{ml}}\text{ = } \frac{\frac{\Delta \text{A}}{\text{min}}\text{ x 1000 x 3 ml x dilution}}{\text{1.22 x 10}^4\text{ x 0.1 ml}}

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

	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	----

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Xanthine Oxidase - Assay

Xanthine Oxidase Assay