Cholesterol Esterase - Assay
Cholesterol Esterase Assay
The initial rate of hydrolysis of cholesterol ester is determined by following the increase in absorbance at 505 nm. One unit hydrolyzes 1 μmole of cholesterol ester per minute at 37°C and pH 7.0 under the specified conditions.
- 0.1 M Phosphate buffer, pH 7.0
- 0.7 mM 4-aminoantipyrine in 0.1 M Phosphate buffer, pH 7.0. Discard after 8 hours.
- 5.0 mM Phenol in 0.1 M Phosphate buffer, pH 7.0. Discard after 8 hours. Use only fresh, white phenol crystals.
- 5% Triton X-100. Store at 4°C.
- Sodium Cholate: Dissolve 3.51 g Sodium cholate in 10.0 ml reagent grade water. Discard after 8 hours.
- Cholesterol Oxidase: Dissolve 28.5 units cholesterol oxidase/ml in reagent grade water. Discard after 8 hours.
- Peroxidase: 8200 u/ml in reagent grade water. Discard after 8 hours.
- Cholesteryl Acetate. Dissolve 220 mg Cholesteryl Acetate (Eastman) in 50.0 ml Isopropanol (ACS Grade). Stir vigorously to dissolve. Store at 4° C.
Dissolve one mg/ml in 0.1 M Phosphate buffer and then prepare further dilutions (usually 2-3X) to achieve a rate of approximately 0.045/minute.
Adjust spectrophotometer to 505 nm and 37°C. Prepare master batch, sufficient for 5 tests, by pipetting into a beaker:
0.7 mM 4-aminoantipyrine 7.0 ml 5.0 mM Phenol 7.0 ml Sodium Cholate 0.5 ml 5% Triton X-100 0.5 ml Cholesterol Oxidase 0.25 ml Peroxidase 0.05 ml
Mix gently. Stored at 4°C, master batch is stable for 8 hours.
Pipette into test and blank cuvettes 3.0 ml master batch. Add 0.10 ml cholesterol acetate. Mixture should be used within 1 hour. Equilibrate temperature to 37°C. Add to test cuvettes, 0.1 ml enzyme dilution and 0.1 ml reagent grade water to the blank. If the blank rate exceeds 0.005 ΔA per minute, reincubate for 5 more minutes. Determine ΔA505 of test and blank cuvettes using the linear portion of the first 5 minutes of the reaction. Subtract blank rates, if any.