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.

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