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HomeEnzyme Activity AssaysEnzymatic Assay of Alcohol Oxidase (EC 1.1.3.13)

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:

Enzymatic Assay of Alcohol Oxidase

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:

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

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

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

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.

3.

Materials
Loading

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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