Enzymatic Assay of Glucose Oxidase

Document History

Derived from procedure SPGLUC01. Includes template updates to current SOP specification and incorporation of notes into the procedure. Refer to CR SOP-DEK ENZ 36.

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 @25°C

3.2    GOD = Glucose Oxidase

3.3.    POD = Peroxidase

3.4.    Unit definition of Glucose Oxidase = One unit will oxidize 1.0 μmole of β-D-glucose to D-gluconolactone and H2O2 per minute at pH 5.1 at 35ºC (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   GOD   > D-Glucono -1,5 – Lactone + H2O2
H2O2 + o-Dianisidine(reduced)   POD   > o-Dianisidine(oxidized)

5. RESPONSIBILITIES
It is the responsibility of all Analytical Services personnel to follow this protocol as written.

6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.

7. PROCEDURE

7.1.    CONDITIONS
T=35ºC, 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 35ºC (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, Sigma-Aldrich Product Number S8625 . Adjust to pH 5.1 at 35ºC with 1 M HCL.)

7.3.2.    0.21 mM o-Dianisidine Solution (o-DDH)
Dissolve 20mg of o-Dianisidine Dihydrochloride Sigma-Aldrich Product Number D9154 , or Sigma-Aldrich Product Number 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, Sigma-Aldrich Product Number 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 35ºC 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 (GOD)
For all Glucose Oxidase products (except for crude and liquid products) prepare an initial solution of 20-40 units/ml in cold buffer. Then immediately prior to use, further 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 min (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 (GOD) 0.1 -----
7.3.1 (Buffer) ----- 0.1

7.4.3    Immediately mix by inversion and record the increase in A500nm/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 A500nm 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 500nm
     0.1=Volume (in milliliters) of enzyme used

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

7.5.3 Units/mg protein = Units/mL enzyme
mg protein/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.)

8. REFERENCES & ATTACHMENTS
Bergmeyer, H.U.,Gawehn, K. and Grassl, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U. ed) Volume I, Second Edition, 457-458, Academic Press, Inc., New York, NY

9. APPROVAL
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