Cholesterol esterase (EC 188.8.131.52, from Microorganism)
Cholesterol ester + H2O ⇒ Cholesterol + Fatty acid
One unit of cholesterol esterase is defined as the amount of enzyme producing 1 μmol H2O2 per minute under standard determination conditions.
Reagent I：0.2 M K3PO4 solution, pH 6.0.
Reagent II: Add 39 mg cholesterol linoleic acid ester to 2.0 ml isopropanol, heat by water bath for a complete dissolution. Add about 80 ml of 1% Genapol X-80 preheated at 72-74 ℃, water bath at 72-74 ℃ and stir for 30 min, the solution will become clear first and then turbid. Cool the solution to room temperature with water. Add 0.6 g sodium cholate, after dissolution, dilute to 100 ml with 1% Genapol X-80 solution.
Reagent III：1 kU/mL POD.
Reagent IV：50 mM TOOS solution.
Reagent V：50 mM 4-AA solution.
Reagent VI：300 U/mL COX enzyme solution.
Enzyme diluent：20 mM K3PO4 solution, pH 6.0. 2 mM MgCl2, 0.5 mM EDTA-Na2 and 0.2% BSA. Add the following components in the order specified
- Add 2.75 mL of the reaction mixture to a 3ml cuvette.
- Add 0.1 mL of reagent VI, water bath at 37℃ for 2 min.
- Add 0.1 mL of enzyme samples and mix well
- Measure absorbance change (As) in 1 minute at 555nm at 37 °C.
*Set a blank control with enzyme diluent ， measure the blank absorbance change (∆Ab)
2.95: Total volume of reaction solution (mL)
0.10: Volume of enzyme solution (mL)
1.0: Optical Path (cm)
df: Dilution multiple
1/2: 1 mol of H2O2 yields 1/2 mol of quinoneimine dye
C: Enzyme concentration (mg/mL)
39.2: Millimolar extinction coefficient of quinoneimine dye under 555 nm (cm2/μmol)