Specialists in the Isolation and Purification of Horseradish Peroxidase and a Select Range of Enzymes

Assay Procedure for Glucose Oxidase  (Ref. No. FGAP029)

Glucose Oxidase [GO] catalyses the oxidation of Glucose to Gluconic Acid.

The generation of H₂O₂ is indirectly measured by oxidation of O-dianisidine in the presence of peroxidase. [POD]

	Glucose Oxidase
b - D - Glucose + O2 + H2O		D-gluconic acid + H2O2
	POD
H2O2 + O-dianisidine(reduced)		O-dianisidine (oxidised) + H2O

Unit Definition

One unit of activity is defined as the amount of enzyme that will catalyse the oxidation of 1 micro mole of Glucose per minute at 25^°C under the assay conditions.

Note

O-dianisidine is a known carcinogen. Handle reagent with gloves. Clean spillages. Observe ALL safety precautions when handling this substance.

Precautions

1. O-dianisidine is a known carcinogen. Ensure that gloves and face mask are worn when handling this substance. Spillages must be disposed of safely.

2. Allow one hour for Glucose to mutarotate before using.

3. The Glucose Oxidase trace is non-linear. It is important to measure the first two minutes of the curve.

4. Do not allow cuvettes containing the equilibrated assay cocktail to stand in the spectrophotometer. This will cause de-oxygenation of the buffer and lower assay results. Use immediately.

Reagents

A	1M KOH Dissolve 5.61 g KOH [MM56.11] in 80 ml distilled H2O. Adjust volume to 100 ml.
B	0.1M KH2PO4 pH 7.0 Dissolve 13.6 g KH2PO4 [MM136.09] in ± 800 ml distilled H2O. Adjust pH to 7.0 with 1M KOH[A]. Transfer to a volumetric flask and adjust volume to 1000 ml with distilled H2O. [Stable for one month at 2°C to 8°C.
C	10% [m/v] b-D-glucose Dissolve 1 g D-glucose [MM180.16] in 10 ml distilled H2O. ALLOW ONE HOUR TO MUTAROTATE BEFORE USING. (Prepare fresh daily)
D	O-Dianisidine dihydrochloride Solution Dissolve 6.6 mg O-dianisidine dihydrochloride [MM 317.2] in 1 ml distilled H2O. Adjust volume to 100 ml with 0.1M KH2PO4 pH7.0 [B]. Prepare fresh daily. Equilibrate to 25°C in a water bath and bubble pure oxygen into the solution for at least 5 minutes. Note When a series of assays are being performed, leave the oxygen bubbling into the solution continuously. WEAR GLOVES AND FACEMASK WHEN HANDLING O-DIANISIDINE DIHYDROCHLORIDE. CLEAN UP ANY SPILLAGES IMMEDIATELY.
E	Peroxidase Solution Dissolve POD at a concentration of 1 mg POD / ml distilled H2O. Prepare fresh daily. [POD solution to contain 200 - 250 Purpurogallin units/ml] [20 - 25 Purpurogallin units required per determination]
F	Enzyme Solution For freeze-dried preparations, dissolve enzyme at a concentration of 10 mg GO / ml distilled H2O [AVOID FOAMING. DO NOT USE VORTEX MIXER. DISSOLVE ON ICE BY GENTLE AGITATION PERIODICALLY]. Test at least two weighings for each batch. Prepare test samples fresh daily. Immediately prior to assay dilute approximately to yield {0.15 £ units £ 0.3 u/ml [0.04 £ DA436 £ 0.08]} in 0.1M KH2PO4 pH 7.0 buffer.

Procedure

l = 436nm
Temperature = 25^°C
Path length = 10 mm
Cuvette volume =  3.1 ml
Pipette the following into a cuvette:

0-dianisidine [D]	2.40 ml
Glucose [C]	0.50 ml
Peroxidase [E]	0.10 ml

Mix and allow to equilibrate to 25^°C. Monitor background rate and measure DOD₁/min.

Add enzyme [F]	0.10 ml	and mix well
TOTAL	3.10 ml

Monitor the OD rate over the first two minutes. Measure DOD₂/minute over the linear portion of the reaction. Repeat above procedure in triplicate for each weighing / sample.

Caclulation

where:

Corrected DOD/min = DOD₂/min - DOD₁/min
TV = Total Volume [3.1 ml]
8.3 = millimolar extraction coefficient for O-dianisidine (oxidised)
SV = Sample Volume

For Freeze-dried Powders

Activity [u/mg] =	u/ml
	EC

where EC = enzyme concentration in mg/ml original solution. Determine the mean value for each batch being tested.

Note

1. O-dianisidine is a known carcinogen. HANDLE WITH CARE.

2. Allow glucose to mutarotate for one hour before using same.

3. Dilute enzyme to 0.15 to 0.3 u/ml in 0.1M KH₂PO₄ pH 7.0 buffer, just prior to assay.

Unit Conversion

It should be noted that the Glucose Oxidase assay procedure described above involves the oxygenation of the chromogen solution, o-dianisidine. This procedure gives a higher activity than without oxygenation.

One unit as described above x 0.6 = units without oxygenation.