Cholesterol oxidase (EC 126.96.36.199, from Microorganism)
Cholesterol + O2 ⇒ △4-Cholesten-3-one + H2O2
One unit is defined as the amount of enzyme which generates 1 μmol of H2O2 per minute at 37℃ under the conditions described below.
Reagent I: 0.1 M pH 6.5 potassium phosphate buffer.
Reagent II: 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. Then add 90 mL pure water and boiled for 30-60s, the substrate solution become cloudy. Cool under running water with gentle agitation, the solution will turn clear. Then add 0.4 g sodium cholate and fill up the solution to 100 mL by pure water. This solution is stabilized at room temperature for one week. e. If it becomes cloudy, warm slightly while stirring until it clears.
Reagent III: 100 U/mL POD.
Reagent IV: 50 mM 4-AA solution.
Reagent V: 50 mM TOOS solution.
Enzyme dilution buffer: 20 mM potassium phosphate buffer, pH 7.0, contains 0.2% BSA.
Sample: The enzyme was diluted to 0.05-0.2 U/mL with enzyme dilution buffer.
REACTION MIX :
- Preincubate the reaction mixture at 37℃ for 5 min.
- Add 0.9 mL reaction mix to the 1 mL cuvette.
- Add 0.1 mL enzyme solution to the 1mL cuvette and mix.
- Record the ΔAs at 555 nm in 1 minute in a spectrophotometer thermostated at 37℃.
At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution
Vt: Total volume (1mL)
Vs: enzyme volume (0.1mL)
df: dilution factor
C: Enzyme concentration (mg/mL)
1.0: Light path length (cm)
1/2: 1mol H2O2 will react to 1/2 mol Quinoneimine dye
39.2: Millimolar extinction coefficient of quinoneimine dye under 555 nm (cm2/μmol)
- Alain, C.C. et. al. (1973) Clin. Chem., 20,
- Tarbutton, P. N. and Gunter, C. R. (1974) Clin. Chem., 20, 724.
- Nomoto, S. (1976) Rinsho Kensa, 20, 688.
- Kameno, K., Nakano, N. and Baba, S. (1976) Jap. J. Clin. Path., 24, 650.