Cholesterol Oxidase Assay Procedure

Cholesterol oxidase from Streptomyces sp. has been used to assess the relationship between the micellar structure of model bile and the activity of esterase. Cholesterol oxidase has also been used to investigate the effects of sphingomyelin degradation on cell cholesterol oxidizability and steady-state distribution between the cell surface and the cell interior.

Native cholesterol oxidase (Product No. 228250) catalyzes oxidation of cholesterol to cholesterone and hydrogen peroxide (isoelectric points: 6.1 and 7.3). Cholesterol oxidase is suitable for determination of total cholesterol when coupled with cholinesterase and peroxidase.

Cholesterol Oxidase Assay Principle

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

Reagents

Procedure

1. Prepare the following working solution (20 tests volume), immediately before use and store on ice in a brown bottle.
   
   51.0 mL Buffer solution (A)
   4.0 mL Substrate solution (B)
   1.0 mL 4-AA solution (C)
   2.0 mL POD solution (E)

2. Pipette 2.9 mL of working solution into a cuvette (d＝1.0 cm) and equilibrate at 37 ℃ for about 3 minutes. Add 0.1 mL of phenol solution (D), mix and keep at 37 ℃ for another 2 minutes.
3. Add 0.1 mL of the enzyme solution* and mix with gentle inversion.
4. Record the increase in optical density at 500 nm 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 0.1－0.3 U/mL with the same buffer, and store on ice.

Calculation

Activity can be calculated by using the following formula：

Weight activity (U/mg)＝(U/mL)×1/C