Enzymatic Assay of Glucose Oxidase

1. Objective

The objective of this procedure is to standardize a procedure for the enzymatic assay of glucose oxidase.

2. Scope

This procedure applies to all products that have a specification for glucose oxidase activity.

3. Definitions

3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm at 25 °C

3.2 GO = Glucose Oxidase

3.3. POD = Peroxidase

3.4. Unit definition of glucose oxidase = One unit will oxidize 1.0 μMol of β-D-glucose to D-gluconolactone and H₂O₂ per minute at pH 5.1 at 35 °C (equivalent to an O₂ uptake of 22.4 μL per minute). If the reaction mix is saturated with oxygen, the activity may increase by up to 100%.

4. Discussion

β -D-Glucose + O2 + H₂O ^GO > D-Glucono -1,5 – Lactone + H₂O₂
H₂O₂ + o-Dianisidine(reduced) ^POD > o-Dianisidine(oxidized)

5. Responsibilities

Analytical Services personnel should follow this protocol as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1. CONDITIONS
T=35 °C, pH= 5.1, A , Light path=1 cm

7.2. METHOD: Continuous Spectrophotometric Rate Determination

7.3. REAGENTS

7.3.1. 50 mM Sodium Acetate Buffer, pH 5.1 at 35 °C (Buffer)
Prepare 500 mL in purified water saturated with oxygen (saturate the water for five minutes and let it stand overnight before running the assay) using sodium acetate, trihydrate, (Product No. S8625). Adjust to pH 5.1 at 35 °C with 1 M HCL.)

7.3.2. 0.21 mM o-dianisidine Solution (o-DDH)
Dissolve 20 mg of o-dianisidine dihydrochloride (Product No. D9154) , or (Product No. F5803) , in 8 mL purified water saturated in oxygen using an amber vial or other vial protected from light. Then dilute 5.35 mL to 200 mL with Reagent 7.3.1

7.3.3. 10% (w/v) β-D-(+) Glucose Substrate Solution
Prepare 60 mL in purified water using β-D-(+) Glucose (Product No. G5250).

7.3.4. 0.17 mM o-dianisidine and 1.72% (w/v) glucose solution (Reaction Cocktail)
Immediately before use, prepare 232 mL by combining 192 mL of reagent 7.3.2 with 40 mL of reagent 7.3.3. Equilibrate to 35 °C and adjust to pH 5.1 if necessary with 1 M HCL. Prepare fresh.

7.3.5. Peroxidase Enzyme solution (POD)
Immediately before use, prepare a solution containing 60 purpurogallin units/mL of POD Type II in cold purified water.

7.3.6. Glucose Oxidase Enzyme Solution (GO)
For all Glucose Oxidase products (except for crude and liquid products) prepare an initial solution of 20-40 units/mL in cold buffer, and immediately before use, dilute to 0.4-0.8 units/mL in cold buffer (this is zero time). Keep the initial solution on ice. Make a fresh second dilution in cold buffer and run again after 60 minutes (this is 60 min time). For all crude and liquid products see table below.

Crude products and liquids	First Dilution	Second Dilution
G1262	0.2 mg/mL	No further dilution required
G6766	0.2 mg/mL	No further dilution required
G6125	2.0 mg/mL	0.1 mL+6.4 mL Buffer
G6641	1.0 mg/mL	0.1 mL+5.0 mL Buffer
G9010	0.1 mL+5.0 mL Buffer	0.1 ml+3.0 mL Buffer
G2133	0.1 mg/mL	0.1 mL+5.0 mL Buffer
G7016	0.2 mg/mL	0.1 mL+5.0 mL Buffer
G7141	0.1 mg/mL	0.1 mL+5.0 mL Buffer
G7779 (1.0 mg/mL)	0.1 mL+0.5 mL Buffer	0.1 mL+5.0 mL Buffer
G7779 (0.01 mg /mL)	Neat	1.0 mL+3.0 mL Buffer

7.4 TEST METHOD

7.4.1 Pipette the following reagents into suitable cuvettes:

	Test	Blank
7.3.4 (Reaction Cocktail)	2.9	2.9
7.3.5 (POD)	0.1	0.1

7.4.2 Then add:

7.3.6 (GO)	0.1	-----
7.3.1 (Buffer)	-----	0.1

7.4.3 Immediately mix by inversion and record the increase in A_500nm/min for six minutes. Obtain the maximum linear rate for both the Test and the Blank using a minimum of a 1.0 minute period and a minimum of four A_500nm points.

7.5 CALCULATIONS

7.5.1	Units/mL enzyme =	(ΔA500nm/minTest - ΔA500nm/ min Blank) * (3.1) * (df)
		(7.5) * (0.1)

3.1=volume (in milliliters) of assay
df=Dilution factor
7.5=Millimolar extinction coefficient of oxidized o-dianisidine at 500 nM
0.1=Volume (in milliliters) of enzyme used

7.5.2	Units/mg solid =	Units/mL enzyme
		mg solid/mL enzyme

7.6 FINAL ASSAY CONCENTRATION:
In a 3.10 mL reaction mix, the final concentrations are 48 mM sodium acetate, 0.16 mM o-dianisidine, 1.61% (w/v) glucose, and 6 units peroxidase (concentration of glucose oxidase will vary as to which product is used.)