Galactose Oxidase Assay

Method: The reaction velocity is measured in a peroxidase/o-tolidine coupled system as an increase in A₄₂₅ resulting from the oxidation of galactose. One unit results in a change in A₄₂₅ of 1.0 per minute at 25°C and pH 6.0 under the defined conditions.

Reagents

• 0.1 M Potassium phosphate buffer, pH 6.0
• 0.5% o-tolidine. Note: o-tolidine has been reported to be carcinogenic. Handle with care.
• Peroxidase. Dissolve Worthington peroxidase (Code: HPOD) at a concentration of approximately 60 u/ml in reagent grade water.
• 10% galactose. Allow to come to equilibrium of mutarotation by allowing to stand overnight.

Enzyme

Dissolve at a concentration of 1 mg/ml in reagent grade water. Dilute further for assay to a concentration of 0.2 - 0.5 units/ml.

Procedure

Adjust spectrophotometer to 425 nm and 25°C.

Prepare tolidine-buffer mixture by adding 0.1 ml tolidine to 12 ml 0.1 M potassium phosphate buffer pH 6.0.

Pipette into each cuvette as follows:

Tolidine-buffer solution	1.7 ml
10% Galactose	1.5 ml
Peroxidase	0.1 ml

Incubate in spectrophotometer at 25°C for 3 - 4 mintues to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml of appropriately diluted enzyme and record increase in A₄₂₅/min. from initial linear portion of the curve.

Calculation