Cholesterol Esterase is an enzyme that hydrolyzes cholesterol ester to cholesterol and free fatty acid in the intestinal lumen. Cholesterol synthesized in the acinar cells and is stored in zymogen granules. Cholesterol esterase is also known as bile salt-stimulated lipase and carboxy ester lipasea, acts function for acceleration of cholesterol absorption.
APPLICATIONS
This enzyme is used to catalyze the hydrolysis of cholesterol esters into cholesterol and fatty acids, and is applied in in vitro research.
Assay procedure
Principle
The formation of quinoneimine dye by reaction between 4-aminoantipyrine and phenol is measured at 500 nm by spectrophotometry.
Unit Definition
One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 micromole of hydrogen peroxide (equivalent to 1/2 micromole of quinoneimine dye) per minute under the following reaction conditions.
Reagents
A. Sample Diluent: Dissolve 2.72 g of KH₂PO₄ in 800 mL of deionized water. Roughly adjust the pH to 7.5 using NaOH solution. Add and dissolve 0.19 g of MgCl₂, 0.186 g of EDTA·2Na, 2 g of BSA, and 1 g of NaN₃ sequentially. Precisely adjust the pH to 7.0 at 25°C using KOH solution. Dilute the solution to a final volume of 1000 mL with deionized water.
B. Reagent 1: Dissolve 14 g of KH₂PO₄ in 800 mL of deionized water. Roughly adjust the pH to 7.0 using KOH solution. Add and dissolve 0.961 g of 4-aminoantipyrine, 15 KU of peroxidase, 30 KU of cholesterol oxidase, and 1 g of NaN₃ sequentially. Precisely adjust the pH to 7.0 at 25°C using KOH solution. Dilute the solution to a final volume of 1000 mL with deionized water.
C. Reagent 2a: Dissolve 14 g of KH₂PO₄ in 800 mL of deionized water. Roughly adjust the pH to 7.0 using KOH solution. Add and dissolve 6 g of phenol and 2 g of NaN₃. Precisely adjust the pH to 7.0 at 25°C using KOH solution. Dilute the solution to a final volume of 1000 mL with deionized water.
D. Reagent 2b: Dissolve 1 g of Triton X-100 in 80 mL of deionized water, and heat the solution in a water bath at 70°C. Dissolve 39 mg of cholesterol linoleate in 2 mL of isopropanol, then slowly add this solution to the above Triton X-100 aqueous solution (maintained in the 70°C water bath). Rinse the 10 mL beaker (used for dissolving cholesterol linoleate) 2-3 times with a small amount of the 70°C Triton X-100 aqueous solution, and combine all rinses into the main solution. Stir the solution in the 70°C water bath for 3-5 minutes until it becomes clear, then stop the water bath heating. When the solution temperature drops to 60°C, add and dissolve 0.6 g of sodium cholate. Cool the solution to room temperature, then dilute it to a final volume of 1000 mL with deionized water.
Procedure
1. Weigh the required amount of enzyme using a weighing boat. To ensure weighing accuracy, the enzyme mass should be at least 10 mg. Dissolve the enzyme in the sample diluent (Reagent A) and dilute to the desired volume.
2. Add the following reagents in sequence according to the specified volume ratio: 0.3 mL of Reagent 1, 0.45 mL of Reagent 2a, and 0.45 mL of Reagent 2b. Preheat the mixture at 37°C for 180 seconds.
3. Add 0.04 mL of the enzyme solution to the preheated mixture, mix well, and allow the reaction to proceed for 2 minutes.
4. Measure the rate of absorbance change within 1 minute at a wavelength of 505 nm using a spectrophotometer. The linear range of enzyme activity for this method is 100-300 U/L.
5. For the blank control: Replace the enzyme solution with the sample diluent (Reagent A), and repeat steps 2-4 to measure the blank absorbance change rate.
Calculation
The enzyme activity is calculated using the following formula:
Vt：Total volume of the reaction mixture（ml）
ε：Molar extinction coefficient, with a value of 13.78（cm²/μmol）
Vs：Volume of the enzyme solution to be tested（ml）
d：Light path length of the cuvette（cm）
D：Dilution factor of the enzyme solution
0.5：Reaction coefficient, representing that 1 mole of H₂O₂ produces 0.5 moles of quinoneimin dye
ΔA/min：ΔA/min（test sample）－ΔA/min（blank）（ABS/min）
