Enzymatic Assay of Alcohol Oxidase (EC 1.1.3.13)

Description

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 µmol of methanol to formaldehyde per minute at pH 7.5 at 25 °C.

Precautions

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

Reagents and Equipment Required

Potassium phosphate monobasic (Product No. P5379)
2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Product No. A9941)
Methanol (Product No. M1775)
Peroxidase (Product No. P8250)
Cuvettes and thermostatted spectrophotometer

Preparation (Storage/Stability)

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 (Product No. 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 (Product No.  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 (Product No. M1775) in ultrapure water.

Peroxidase Solution – Immediately before use, prepare a 250 units/mL solution of peroxidase (Product No. 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.

Procedure

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 (mL)	Blank (mL)
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 (mL)	Blank (mL)
Phosphate Buffer	–	0.10
Methanol Solution	0.10	0.10

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

Reagent	Test Sample (mL)	Blank (mL)
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.

Results

Calculations

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

Where:
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

2.

Units/mg solid =	units/mL enzyme
	mg solid/mLenzyme

3.

Units/mg protein =	units/mL enzyme
	mg protein/mL enzyme

References

1. Keesey J. 1987. Boehringer Mannheim Biochemicals. 58

2. Janssen FW, Ruelius HW. 1968. Alcohol oxidase, a flavoprotein from several basidiomycetes species. Biochimica et Biophysica Acta (BBA) - Enzymology. 151(2):330-342. https://doi.org/10.1016/0005-2744(68)90100-9

ABTS is a trademark of Roche.

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