To standardize a procedure for the determination of the enzymatic assay of cholesterol oxidase.
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).
Cholesterol + O2 Cholesterol Oxidase > H2O2 + 4-Cholesten-3-one
H2O2 + o-Dianisidine (reduced) POD > 2 H2O + o-Dianisidine (Oxidized)
Analytical Services laboratory personnel should follow this procedure as written.
Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.
T = 25 °C, pH = 7.5, A500nm, Light path = 1 cm
Continuous Spectrophotometric Rate Determination
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:
4. Mix by inversion and equilibrate to 25 °C using a suitable thermostatted spectrophotometer. Monitor the A500nM until constant. Then add:
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.
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.
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.
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