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Assay Procedure for Choline Oxidase


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.




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.


  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).


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)




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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