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 Product No.  C7274) and from Brevibacterium sp. (discontinued Product No. C8153).

3. Definitions

• ODA - o-Dianisidine
• POD - Peroxidase
• Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 °C
• 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

Analytical Services laboratory personnel should follow this procedure as written.

6. Safety

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

7. Procedure

7.1 Conditions:

T = 25 °C, pH = 7.5, A_500nm, Light path = 1 cm

7.2 Method:

Continuous Spectrophotometric Rate Determination

7.3 Reagents:

• 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, Product Number such as P5379, in purified water. Adjust pH to 7.5 at 25 °C with 1 M KOH.
• 7.3.2 1% (w/v) o-Dianisidine Solution (ODA)
  Prepare a 10.0 mg/mL solution of o-Dianisidine, such as Product No. D9154, in Reagent (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, such as Product No.  C8503, in 10 mL of Triton X-100.  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, Product No. 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

7.4 Test Method

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

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.

3. Pipette the following reagents (milliliters) 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

4. Mix by inversion and equilibrate to 25 °C using a suitable thermostatted spectrophotometer. Monitor the A_500nM 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 A_500nm for approximately 5 minutes. Obtain the ΔA_500nm / minute using the maximum linear rate for both the Test and Blank.

7.5 Calculations

	Units/mL enzyme =	(ΔA500nm/min Test - ΔA500nm/min Blank)(3.0)(df)
		(7.5)(0.10)
Where:     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

	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 Unit Definition

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

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

7.7 Final Assay Concentration:

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.