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:
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.
References
ABTS is a trademark of Roche.
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