Uricase Assay

Method: The reaction velocity is determined by measuring the decrease in absorbance at 290 nm resulting from the oxidation of uric acid to allantoin. One unit oxidizes one micromole of uric acid per minute at 25°C and pH 8.5 under the specified conditions.

Reagents

• 0.1 M Sodium borate buffer, pH 8.5
• 0.12 mM Uric acid. Dissolve 60 mg lithium carbonate in 15 ml water and filter. Prepare fresh solution by dissolving 100 mg uric acid in filtrate. Heating to 50-60°C may be necessary to effect solution. Cool and bring to a volume of 100 ml with reagent grade water. Dilute 1/100 with 0.1 M borate, pH 8.5.

Note: Prior to use, the 0.1 M sodium borate buffer and the 0.12 mM uric acid solution should be oxygenated by bubbling O₂ through the solutions for 10-15 minutes. Reoxygenate every 20 minutes.

Enzyme

Dissolve at one mg/ml in cold (5°C) 0.1 M sodium borate buffer, pH 8.5.

Immediately prior to assay, dilute further to a concentration of 0.01-0.1 units/ml.

Procedure

Adjust the spectrophotometer to 290 nm and 25°C.

Pipette into a cuvette as follows:

Borate buffer	0.5 ml
0.12 mM Uric acid	2.0 ml

Incubate in spectrophotometer for 4-5 minutes to achieve temperature equilibration and to establish blank rate, if any. At zero time add 0.5 ml of enzyme and record decrease in A₂₉₀ for 6-7 minutes. Calculate ΔA₂₉₀ from initial linear portion of curve.

Calculation