Certain features of will be down for maintenance the evening of Friday August 18th starting at 8:00 pm CDT until Saturday August 19th at 12:01 pm CDT.   Please note that you still have telephone and email access to our local offices. We apologize for any inconvenience.

Enzymatic Assay of Alcohol Oxidase


This procedure may be used for all Alcohol Oxidase products.

The continuous spectrophotometric rate determination (A405, Light path = 1 cm) is based on the following reactions:

ABTS™ - 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)
POD - peroxidase

Unit Definition: One unit of Alcohol Oxidase will oxidize 1.0 µmole of methanol to formaldehyde per minute at pH 7.5 at 25 °C.


Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents and Equipment Required

Potassium phosphate monobasic (Catalog Number P5379)
2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Catalog Number A9941)
Methanol (Catalog Number M1775)
Peroxidase (Catalog Number P8250)
Cuvettes and thermostatted spectrophotometer

Preparation Instructions

Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.

Phosphate Buffer (100 mM Potassium Phosphate Buffer, pH 7.5 at 25 °C) – Prepare a 13.6 mg/ml solution of potassium phosphate monobasic (Catalog Number P5379) in ultrapure water. Adjust pH to 7.5 at 25 °C with 1 M KOH.

ABTS Solution (2 mM) – Prepare a 1.0 mg/ml solution of ABTS (Catalog Number A9941) in Phosphate Buffer. Adjust pH to 7.5 at 25 °C with 1M KOH, if necessary. PREPARE FRESH. Immediately before use, bubble O2 gas through the solution for ~5 minutes.

Methanol Solution [1.0% (v/v)] – Prepare a 0.01 ml/ml solution of methanol (Catalog Number M1775) in ultrapure water.

Peroxidase Solution – Immediately before use, prepare a 250 units/ml solution of peroxidase (Catalog Number P8250) in ultrapure water.
Note: Unit Definition: One unit of peroxidase will form 1.0 mg of purpurogallin from pyrogallol in 20 seconds at pH 6.0 at 20 °C. In general, peroxidase will use ABTS as an electron donor. 

Alcohol Oxidase Solution – Immediately before use, prepare a solution containing 0.1 unit/ml of Alcohol Oxidase in cold Phosphate Buffer.


Final Assay Concentrations – In a 3.01 ml reaction mix, the final concentrations are 96 mM potassium phosphate, 2 mM ABTS, 0.033% (v/v) methanol, 2.5 units POD, and 0.01 unit alcohol oxidase.

1. Pipette the following reagents into suitable cuvettes:

Reagent Test Sample
ABTS Solution
2.80 2.80
Peroxidase Solution 0.01 0.01

2. Mix by inversion and equilibrate to 25 °C using a suitable thermostatted spectrophotometer. Monitor the A405 until constant. Then add:

Reagent Test Sample
Phosphate Buffer
Methanol Solution 0.10 0.10

3. Mix by inversion and monitor the A405 until constant. Then add:

Reagent Test Sample
Alcohol Oxidase Solution 0.10

Immediately mix by inversion and record the increase in A405 for ~5 minutes. Obtain the ΔA405/minute using the maximum linear rate for both the Test Sample and the Blank.




Units/ml enzyme =
(ΔA405/minute Test Sample – ΔA405/minute Blank) (3.01) (df)
(36.8) (0.10)

3.01 = Total volume (in milliliters) of assay
df = Dilution Factor
36.8 = Millimolar extinction coefficient of ABTS at 405 nm
0.10 = Volume (in milliliters) of enzyme used


Units/mg solid = units/ml enzyme
mg solid/ml enzyme



Units/mg protein = units/ml enzyme
mg protein/ml enzyme




  1. Keesey, J., Biochemica Information, 1st ed., Boehringer Mannheim Biochemicals, (Indianapolis, IN: 1987) p. 58.
  2. Jassen, F.W., and Ruelius, H.W., Biochemica et Biophysica Acta, 151, 330-342 (1968).


ABTS is a trademark of Roche.

07/13 - 1

Related Links