Enzymatic Assay of Cholesterol Oxidase

1. Objective

To standardize a procedure for the determination of the enzymatic assay of Cholesterol Oxidase.

2. Scope

This procedure applies to products that have a specification for the enzymatic activity of cholesterol oxidase. This assay is NOT to be used to assay Cholesterol Oxidase from Schizophyllum commune (Discontinued Sigma-Aldrich Product Number C7274) and from Brevibacterium sp. (Discontinued Sigma-Aldrich Product Number C8153).

3. Definitions

3.1 ODA - o-Dianisidine

3.2 POD - Peroxidase

3.3 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC

3.4 Peroxidase Unit Definition - One unit will form 1.0 mg purpuroallin in 20 seconds at pH 6.0 at 20°C.

4. Discussion

Cholesterol + O2    Cholesterol Oxidase   > H2O2 + 4-Cholesten-3-one
H2O2 + o-Dianisidine (reduced)    POD    > 2 H2O + o-Dianisidine (Oxidized)

5. Responsibilities

It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. Safety

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

7. Procedure

T = 25°C, pH = 7.5, A500nm, Light path = 1 cm

Continuous Spectrophotometric Rate Determination


7.3.1    50 mM Potassium Phosphate Buffer, pH 7.5 at 25°C (Buffer)
            Prepare a 6.8 mg/ml solution of Potassium Phosphate, Sigma-Aldrich Product Number such as P5379, in purified water. Adjust pH to 7.5 at 25°C with 1M KOH.

7.3.2    1% (w/v) o-Dianisidine Solution (ODA)
            Prepare a 10.0 mg/ml solution of o-Dianisidine, Sigma-Aldrich Product Number such as D9154, in Reagent 7.3.1 (Buffer). (Prepare Fresh)

7.3.3    0.5% (w/v) Cholesterol with 10% (w/v) Trition X-100 Solution (Cholesterol)
            Initially dissolve 500 mg of Cholesterol, Sigma-Aldrich Product Number such as C8503, in 10 ml of Triton X-100, Sigma-Aldrich Product Number such as X-100. (Triton is a registered trademark of Union Carbide Chemicals and Plastics Co., Inc.) Heat while stirring until the solution clarifies. Then slowly add 90 ml of purified water warmed to 70-80°C. Continue stirring the solution at 70-80°C for 10 min. Solution may become cloudy upon addition of water but can still be used for the assay.

7.3.4    Peroxidase Enzyme Solution (POD)
            Immediately before use, prepare a 100 pyrogallol unit/ml solution of Peroxidase, Sigma-Aldrich Product Number P8250, in purified water.

7.3.5    Cholesterol Oxidase Enzyme Solution (Enzyme)
            Immediately before use, prepare a solution containing 0.1 – 0.2 unit/mL of cholesterol oxidase in cold Reagent 7.3.1 (Buffer).


7.4.1    Prepare a reaction cocktail by pipetting (in milliliters) the following reagents into a suitable container:

Reagent 7.3.1 (Buffer) 40.00
Reagent 7.3.2 (ODA) 0.50


7.4.2    Mix thoroughly by swirling and adjust to pH 7.5 at 25°C with 0.1 M HCl or KOH if necessary. Add Reagent 7.3.1 to a final volume of 50 ml, mix by swirling thoroughly and oxygenate for approximately 10 minutes before use.

7.4.3    Pipette ( in milliliters) the following reagents into suitable cuvettes:

  Test Blank
Reaction Cocktail 2.70 2.70
Reagent 7.3.3 (Cholesterol) 0.10 0.10
Reagent 7.3.4 (POD) 0.10 0.10


7.4.4    Mix by inversion and equilibrate to 25 °C using a suitable thermostatted spectrophotometer. Monitor the A500nm until constant. Then add:

Reagent 7.3.1 (Buffer) ----- 0.10
Reagent 7.3.5 (Enzyme) 0.10 -----


     Immediately mix by inversion and record the increase in A500nm for approximately 5 minutes. Obtain the ΔA500nm / minute using the maximum linear rate for both the Test and Blank.


7.5.1 Units/ml enzyme = (ΔA500nm/min Test - ΔA500nm/min Blank)(3.0)(df)


    3.0 = Total volume (in milliliters) of assay
    df = Dilution Factor
    7.5 = Millimolar extinction coefficient of o-Dianisidine (Oxidized) at 500nm
    0.10 = 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 solid/mg s enzyme



7.6.1    One unit will convert 1.0 μmole of cholesterol to 4-cholesten-3-one per minute at pH 7.5 at 25°C.

7.6.2    Note: 4-cholesten-3-one may undergo isomerization.

In a 3.0 ml reaction mix, the final concentrations are 46 mM potassium phosphate, 0.009% o-dianisidine, 0.017% (w/v) cholesterol, 0.33% (v/v) Triton X-100, 10 units peroxidase, and 0.01 – 0.02 unit cholesterol oxidase.

8. References & Attachments

8.1 Replaces SPCHOL04.

8.2 Masurekar, P.S. and Goodhue, C.T. (1978) United States Patent No. 4,093,717, Eastman Kodak Company, Rochester, NY.

9. Approval

Review, approvals and signatures for this document will be generated electronically using EDMS. Print a “For Use” copy if hardcopy with signature verification is required.


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