Enzymatic Assay of Alcohol Oxidase

1. OBJECTIVE
To standardize a procedure for the determination of the enzymatic assay of Alcohol Oxidase.

2. SCOPE
This procedure applies to products that have a specification for the enzymatic activity of alcohol oxidase.

3. DEFINITIONS

3.1 ABTS - 2,2’-Azino-bis-(3-Ethylbenzothiazoline-6-Sulfonic Acid)

3.2 POD - Peroxidase

3.3 Purified water - water from a deionizing system, resistivity ~18MΩ•cm @ 25°C

3.4 Peroxidase Unit Definition - One unit will form 1.0 mg purpurogallin from pyrogallol in 20 seconds at pH 6.0 at 20°C. In general, Peroxidase will use ABTS as an electron donor.

4. DISCUSSION
Methanol + O₂    ^{Alcohol Oxidase}   > Formaldehyde + H₂O₂
         H₂O₂ + ABTS    ^POD   >2 H₂O + Oxidized ABTS

5. RESPONSIBILITIES
It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. SAFETY
Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.
Formaldehyde is produced during this reaction. The amount produced is less than SOP 1.25 states (greater than 0.1% by volume), but every precaution should be taken to minimize exposure.

7. PROCEDURE

7.1 CONDITIONS:
T = 25°C, pH = 7.5, A_405nm, Light path = 1 cm

7.2 METHOD:
Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1    100 mM Potassium Phosphate Buffer, pH 7.5 at 25°C (Buffer)
             Prepare a 13.6 mg /ml solution of Potassium Phosphate, Sigma-Aldrich Product Number such as P5379, in purified water. Adjust pH to 7.5 at 25°C with 1M KOH.

7.3.2    2 mM ABTS Solution (ABTS)
            Prepare a 1.0 mg/ml solution of ABTS, Sigma-Aldrich Product Number such as A9941, in Reagent 7.3.1 (Buffer). Adjust pH to 7.5 25°C with 1M KOH if necessary. PREPARE FRESH. Immediately before use, bubble O₂ gas through the solution for approximately 5 min.

7.3.3    1.0% (v/v) Methanol Solution (Methanol)
            Prepare a 0.01 ml/ml solution of Methanol, Sigma-Aldrich Product Number such as M1775, in purified water.

7.3.4    Peroxidase Enzyme Solution (POD)
            Immediately before use, prepare a 250 units/ml solution of Peroxidase, Sigma-Aldrich Product Number such as P8250, in purified water.

7.3.5    Alcohol Oxidase Enzyme Solution (Enzyme)
            Immediately before use, prepare a solution containing 0.1 unit/ml of Alcohol Oxidase in cold Reagent 7.3.1 (Buffer).

7.4 TEST METHOD

7.4.1    Pipette ( in milliliters) the following reagents into suitable cuvettes:

7.4.2    Mix by inversion and equilibrate to 25°C using a suitable thermostatted spectrophotometer. Monitor the A_405nm until constant. Then add:

7.4.3    Mix by inversion and monitor the A_405nm until constant. Then add:

    Immediately mix by inversion and record the increase in A_405nm for approximately 5 minutes. Obtain the ΔA_405nm / minute using the maximum linear rate for both the Test and the Blank.

7.5 CALCULATIONS

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

7.6 UNIT DEFINITION
One unit will oxidize 1.0 μmole of methanol to formaldehyde per minute at pH 7.5 at 25°C.

7.7 FINAL ASSAY CONCENTRATION :
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.

8. REFERENCES & ATTACHMENTS

8.1 Replaces SPMETH01.

8.2 Keesey, J. (1987) Biochemica Information, 1^st ed., p. 58, Boehringer Mannheim Biochemicals, Indianapolis, IN.

8.3 Jassen, F.W. and Ruelius, H.W. (1968) Biochemica et Biophysica Acta 151, 330-342.

8.4 ABTS is a registered trademark of Boehringer Mannheim GmbH.

9. APPROVAL

Review, approvals and signatures for this document will be generated electronically using EDMS. Print a “For Use” copy if hardcopy with signature verification is required.

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Materials