Assay Procedure for Cholesterol Esterase

PRINCIPLE

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

Method

Reagents

Reagents
A. 0.2M K-Phosphate buffer, pH 7.0
B. Cholesterol linoleate solution	：To 39mg of cholesterol linoleate, add 2.0ml of isopropanol and dissolve completely by heating slightly. Mix with about 80ml of 1.0% (v/v) hot Triton X-100 solution (preheated at 72～74 ℃) to the cholesterol linoleate solution and keep the solution in a hot water bath (72～74 ℃), stirring for 30 minutes. The solution will turn clear and then cloudy. Cool under running water with gentle agitation until temperature of the solution goes down to room temperature. Add 600mg of Na-cholate and dissove. Fill up the solution to 100ml with 1.0% Triton X-100 solution. This solution is stable at 4 ℃ for at least 5 days.
C. 4-AA solution	：1.76% (1.76g 4-aminoantipyrine/100ml of H2O)(Store at 4 ℃ in a brownish bottle)
D. Phenol solution	：6.0% (6.0g phenol/100ml of H2O)(Store at 4 ℃ in a brownish bottle)
E. POD solution	：Horseradish peroxidase 7,500 purpurogallin units/50ml of 0.1M K-phosphate buffer, pH 7.0 (150PU/ml)(Prepare freshly)
F. COD solution	：Streptomyces sp. cholesterol oxidase 1,500U/5.0ml of ice-cold H2O (300 U/ml)(Should be prepared fresh)
G. Enzyme diluent	：20mM K-phosphate buffer, pH 7.5 containing 2mM MgCl2, 0.5mM EDTA-Na3 and 0.2% BSA

Procedure

1. Prepare the following working solution (50 tests) in a brownish bottle.
   75 ml Buffer solution (A)
   50 ml Substrate solution (B)
   2.5ml 4-AA solution (C)
   5.0ml Phenol solution (D)
   5.0ml POD solution (E)

Concentration in assay mixture

K-Phosphate buffer	0.11 M
Cholesterol linoleate	0.20mM
4-Aminoantipyrine	1.5 mM
Phenol	22 mM
EDTA	17 μM
Isopropanol	0.68 %
COD	ca.10 U/ml
POD	ca. 5.1 U/ml

1. Pipette 2.75ml of working solution into a cuvette (d＝1.0cm) and equilibrate at 37 ℃ for about 5 minutes. Add 0.1ml of COD solution (F), mix and keep at 37 ℃ for another 2 minutes.
   
2. Add 0.1ml of the enzyme solution* and mix with gentle inversion.
   
3. Record the increase in optical density at 500nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37℃, and calculate theΔOD per minute from the initial linear portion of the curve (ΔOD test).
   

At the same time, measure the blank rate (ΔOD blank) using the same method as the test except that the enzyme diluent (G) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (G), and dilute to 0.08－0.22U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula：

Vt	：Total volume (2.95ml)
Vs	：Sample volume (0.1ml)
13.78	：Millimolar extinction coefficient of quinoneimine dye under the assay conditions (F/micromole)
1/2	：Factor based on the fact that one mole of H2O2 produces half a mole of quinoneimine dye
1.0	：Light path length (cm)
df	：Dilution factor
C	：Enzyme concentration in dissolution (c mg/ml)

This procedure is for informational purposes. For a current copy of our quality control procedure, please contact our Technical Service Department.