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Assay Procedure for Choline Oxidase

Principle

The appearance of quinoneimine dye 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

 

A. Choline chloride solution :2.1%[2.1g choline chloride/100ml of Tris-HCl buffer (D)](Should be prepared fresh)
B. 4-AA solution :1.0% (1.0g 4-aminoantipyrine/100ml of H2O)(Store at 4℃ in a brownish bottle)
C. Phenol solution :1.0% (1.0g phenol/100ml of H2O)(Store at 4℃ in a brownish bottle)
D. Tris-HCl buffer :0.1M Tris-HCl buffer, pH 8.0[Dissolve 12.1g of Tris (MW=121.14) in ca.800ml of H2O and, after adjusting the pH to 8.0 at 25℃ with 2.0 N HCl, fill up to 1,000ml with H2O.]
E. Enzyme diluent :10mM Tris-HCl buffer, pH 8.0 contg. 2mM EDTA and 1.0% KCl.

Procedure

  1. 1. Prepare the following working solution (100ml) in a brownish bottle and store on ice.

    97 ml Substrate solution (A)
    1.0ml 4-AA solution          (B)
    2.0ml Phenol solution     (C)
    5.0mg Peroxidase from horseradish (110 purpurogallin units/mg)(Toyobo GradeⅢ)
Concentration in assay mixture
Tris buffer 97 mM
Choline chloride 0.14 M
EDTA 33 μM
KCI 2.2 mM
4-Aminoantipyrine 0.48 mM
Phenol 2.1 mM
POD ca.4.92U/ml
  1. Pipette 3.0ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.
  2. Add 0.05ml of the enzyme solution* and mix by gentle inversion.
  3. Record the increase in optical density at 500nm against the working solution for 3 to 4 minutes in a spectrophotometer thermostated at 37℃, and calculate theΔOD per minute from the initial linear portion of the curve.

* Dissolve the enzyme preparation in ice-cold Tris-HCl buffer (D) and dilute to 0.1-0.5U/ml with enzyme
diluent (E).

Calculation

Activity can be calculated by using the following formula:

 

Weight activity (U/mg)=(U/ml)×1/C

 

Vt :Total volume (3.05ml)
Vs :Sample volume (0.05ml)
12.0 :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)

 

Materials

     

This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

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