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.
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.
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:
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.