Alcohol oxidase is used for qualitative and quantitative determination of methanol or ethanol (blood alcohol) by colorimetric procedures. Unlike present ADH based assays for ethanol in biological specimens, no labile cofactors or trapping agents are required and considerably less enzyme is used per assay. Quantitative determination of alcohol using an oxygen electrode (enzyme electrode) is also possible. With or without catalase, alcohol oxidase can be used to remove alcohol or scavenge oxygen. Using dilute methanol, sensitive samples can be sterilized through release of the products formaldehyde and hydrogen peroxide. The enzyme can also be used to generate H₂O₂ in radio-iodine labelling procedures.
Biochemical Physiological Actions:
Alcohol oxidase catalyzes the oxidation of short-chain, primary, aliphatic alcohols to their respective aldehydes. It has the highest affinity for methanol. Alcohol oxidase is involved in the metabolism of methanol, which is important for yeasts that utilize methanol as their sole energy source. Alcohol oxidase is primarily found in the peroxisome but may be present in the cytoplasm as well.
Reaction: Alcohol + O₂ ⟶ Aldehyde + H₂O₂
Specificity:
The enzyme is specific for short-chain, linear aliphatic alcohols and oxidizes the following substrates with a relative activity: methanol = ethanol > n-propanol > n-butanol. It also catalyzes the oxidation of allyl and propargyl alcohol, and ethyl mercaptan and formaldehyde at slower rates. Branched chain alcohols, C₂-aldehydes or higher, ketones and organic acids are not substrates. However, some of these reagents can give false positive responses due to trace contamination with ethanol. Oxygen is the only known hydrogen acceptor. At air saturation, the apparent Kₘ for alcohol oxidase is 0.7 mM using methanol and 9.0 mM using ethanol as the substrate. In oxygen saturated solutions the Kₘ values are higher. The turnover number is 2000 sec⁻¹.
Inactivation and Stability:
Freezing does not inactivate the enzyme. Compared to other oxidases, it is relatively resistant to p-chloromercuribenzoate, heavy metals and hydrogen peroxide. Hydrogen peroxide does not inactivate the enzyme at concentrations many times higher than those reported to inactivate previously-described yeast alcohol oxidases (Hansenula, Candida). The temperature optimum is 45°C and pH optimum is 7.2 (optimal working range pH 5.5 to 9).
Suggested Applications:
The qualitative and quantitative determination of methanol and/or ethanol (blood alcohol) by colorimetric procedures. Unlike present ABTS based assays for ethanol in biological specimens, no colloid factors or trapping agents are required and considerably less enzyme is used per assay. Quantitative determination of alcohol using an oxygen electrode (enzyme electrode) is also possible. With or without catalase, alcohol oxidase can be used to remove residual or scavenge oxygen.
Using dilute methanol, sensitive samples can be sterilized through removal of the products formaldehyde and hydrogen peroxide. The enzyme can also be used to generate H₂O₂ in radio-iodine labeling procedures.
Determining Alcohol Oxidase Activity by ABTS Assay:
This method is provided in order to establish a standard method for the determination of alcohol oxidase enzyme activity in highly concentrated and viscous solutions. Because of the high viscosity of alcohol oxidase products, extra care must be taken in pipetting and mixing the dilutions used in the ABTS activity assay.
Materials:
1. Concentrated alcohol oxidase (500 to 1500 EU/ml) in sucrose, warmed to room temperature.
2. ABTS stock solution:
16.0 mg ABTS (2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid))
2 ml absolute ethanol
1 ml of 1 mg/ml horseradish peroxidase in distilled water
0.1 M potassium phosphate buffer, pH 7.5
Completely dissolve the ABTS in a portion of the buffer (about 75 ml), then add the ethanol and peroxidase and make up to a final volume of 100 ml.
3. 0.1 M Potassium phosphate buffer, pH 7.5
4. Positive displacement 100 μl pipette or Hamilton syringe.
5. Recording spectrophotometer suitable for enzyme kinetics.
Dilution Procedure:
The following procedure is for preparing dilutions of highly viscous materials for the ABTS assay. Non-viscous solutions may be assayed directly.
1. Duplicate dilutions should be tested.
2. Pipet 9.9 ml of 0.1 M potassium phosphate buffer into two test tubes.
3. To the first test tube, add 100 μl alcohol oxidase solution using a 100 μl positive displacement pipette (1:100 dilution).
a. Slowly draw sample into pipette, discharge sample, and immediately draw sample into pipette.
b. Wipe off pipette tip taking care not to remove any sample from tip because of the absorbancy of the blotting tissue.
c. Pipet sample into the first test tube, dipping the pipette tip into the second.
d. Cover test tube with parafilm, manually shake vigorously for 5 seconds, vortex for 5 seconds, and then shake vigorously again for 5 seconds.
4. To the second test tube, add 100 μl of the 1:100 dilution using a positive displacement pipette (1:10,000 dilution), repeating steps 3a through 3d.
Determine the alcohol oxidase activity of the 1:10,000 dilution using the standard ABTS assay below.
ABTS Assay:
- Pipet 2.5 ml of ABTS stock solution (at room temperature and saturated with air) into a 3 ml cuvette.
- Pipet 25 μl of a 1:10,000 dilution of alcohol oxidase into the cuvette, cover with parafilm and mix by inverting 3 times. Insert cuvette into spectrophotometer.
- Record absorbance change for 3 minutes at 390 nanometers.
Calculations:
- Calculate the slope (change in absorbance/minute) of the resultant curve from 30 to 180 seconds.
- Multiply the slope by 3.06 (extinction coefficient plus cuvette dilution factor) and by the initial dilution factor (10,000).
Notes:
- Precisely weigh the ABTS. Slight variations in the amount can result in errors in the assay. ABTS solution should be made immediately before use.
- The major errors associated with obtaining reproducible results with this alcohol oxidase assay are in accurately pipetting and mixing the highly viscous alcohol oxidase products. Therefore, extreme care should be taken in pipetting and mixing the initial dilutions.
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