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.


  • 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.


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:

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.



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