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Assay Procedure for Cholesterol Oxidase


The appearance of quinoneimine dye formed when coupled with 4-aminoantipyrine and phenol is measured at 500nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per minute under the conditions described below.




A. 0.1M K-Phosphate buffer, pH 7.0  
B. Cholesterol solution :To 5.0ml of Triton X-100 on a hot plate or in a water bath, add 500mg of cholesterol and mix with a stirring bar until cholesterol dissolves. Add 90ml of distilled water to the hot cholesterol-Triton X-100 solution by slowly pouring along a stirring bar. Stir and allow to boil for 30 to 60 seconds. The solution will be cloudy. Cool under running water with gentle agitation, the solution will turn clear. Add 4.0g of sodium cholate and dissolve. Fill up the solution to 100ml with distilled water. This solution is stable for about one week at room temperature. If it becomes cloudy, warm slightly while stirring until it clears.
C. 4-AA solution :1.76% (1.76g 4-aminoantipyrine/100ml of H2O)
D. Phenol solution :6.0% (6.0g phenol/100ml of H2O)
E. POD solution :Horseradish peroxidase 15,000 purpurogallin units/100ml of buffer (A)
F. Enzyme diluent :20mM K-Phosphate buffer, pH 7.0 contg. 0.2% bovine serum albumin


  1. Prepare the following working solution (20 tests volume), immediately before use and store on ice in a brownish bottle.

    51.0ml Buffer solution (A)
    4.0ml Substrate solution (B)
    1.0ml 4-AA solution (C)
    2.0ml POD solution (E)
Concentration in assay mixture
K-Phosphate buffer 87 mM
Cholesterol 0.89mM
4-Aminoantipyrine 1.4 mM
Phenol 21 mM
Triton X-100 0.34 %
Sodium cholate 64 mM
BSA 33μg/ml
POD 5 U/ml
  1. Pipette 2.9ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37℃ for about 3 minutes. Add 0.1ml of Phenol solution (D), mix and keep at 37℃ for another 2 minutes.
  2. Add 0.1ml of the enzyme solution* and mix with gentle inversion.
  3. Record the increase in optical density at 500nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37℃, and calculate the ΔOD per minute from the linear portion of the curve (ΔOD test).

* At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution. Dissolve the enzyme preparation in ice-cold enzyme diluent (F), and dilute to 0.1-0.3U/ml with the same buffer, and store on ice.



Activity can be calculated by using the following formula:


Weight activity (U/mg)=(U/ml)×1/C


Vt :Total volume (3.1ml)
Vs :Sample volume (0.1ml)
13.78 :Millimolar extinction coefficient of quinoneimine dye under the assay conditions (F/micromole)
1/2 :Factor based on the fact that one mole of H2O2 produces half a mole of quinoneimine dye.
1.0 :Light path length (cm)
df :Dilution factor
C :Enzyme concentration in dissolution (c mg/ml)


This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.


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