Glucose Oxidase Assay

Method: The reaction velocity is determined by an increase in absorbance at 460 nm resulting from the oxidation of o-dianisidine through a peroxidase coupled system. One unit causes the oxidation of one micromole of o-dianisidine per minute at 25°C and pH 6.0 under the conditions specified.

Reagents

• 0.1 M Potassium phosphate buffer, pH 6.0
• 1% o-Dianisidine: Note: o-dianisidine has been reported to be carcinogenic in the solid form. Handle with care.
• Peroxidase: Dissolve peroxidase (Worthington product code HPOD) at a concentration of 200 micrograms per ml in reagent grade water.
• 18% Glucose: Allow mutarotation to come to equilibrium by standing overnight at room temperature.
• Dianisidine-buffer mixture: Prepare by diluting 0.1 ml of 1% o-dianisidine in 12 ml of 0.1 M potassium phosphate buffer pH 6.0. Saturate with oxygen for 10 minutes within 30 minutes of use.

Enzyme

Dissolve at one mg/ml in reagent grade water. Dilute further to 0.02 - 0.06 ΔA/min. in reagent grade water for assay.

Procedure

Set spectrophotometer at 460 nm and 25°C. Pipette into cuvette as follows:

Dianisidine-buffer mixture, pH 6.0 (oxygenated)	2.5 ml
18% Glucose	0.3 ml
Peroxidase	0.1 ml

Incubate in spectrophotometer for 3 - 5 minutes to achieve temperature equilibration and establish blank rate if any. Add 0.1 ml of appropriately diluted enzyme and record increase in A₄₆₀ for 4 - 5 minutes. Calculate ΔA₄₆₀ from the initial linear portion of the curve.

Calculation