Acyl-CoA oxidase is an FAD-dependent enzyme that catalyzes the oxidation of acyl-CoA to produce α,β-enoyl-CoA and H₂O₂.
Application: For in vitro research.
Assay Procedure
Principle
Unit Definition
One unit of enzyme activity equals the amount of enzyme required to produce 1 micromole of H₂O₂ (equivalent to 1/2 micromole of quinone dye) within 1 minute under specified reaction conditions.
Reagents
Dilution solution: Add 2.3 g potassium dihydrogen phosphate and 22.8 g potassium hydrogen phosphate to 800 mL deionized water, stir to dissolve, adjust pH to 7.60 with potassium hydroxide, and make up to 1 L.
Reagent A: Dissolve 24.23 grams of Tris in 800 mL of water, stir until fully dissolved, then adjust the pH to 8.00 with hydrochloric acid at 25°C.
Reagent B: 15 mM 4-AA solution (0.305 g of 4-AA dissolved in 100 mL water and thoroughly mixed).
Reagent C: 0.2% (W/V) phenol solution (2 grams of phenol dissolved in 1 liter of water);
Reagent D: A 50 U/ml POD solution (diluted by 100 mL of Reagent A for 5kU enzyme);
Reagent E: 1% Triton X-100 solution (dissolve 10 g Triton X-100 in 1 L water);
Reagent F: Dissolve 10 mg of palmitoyl-CoA in 2 mL of diluent;
Reagent G (Acetyl-CoA Oxidase Dilution): Before use, prepare the dilution solution from 3.1 by adding FAD monosodium salt to achieve 8 mg/L and ATP to 1.52 g/L. This solution is the Acetyl-CoA Oxidase Dilution.
Test sample: Weigh the required enzyme with a Weighing tube before testing. When weighing the enzyme, at least 10mg of enzyme should be weighed to ensure the accuracy of weighing. Dissolve the enzyme with enzyme dilution solution and adjust the volume to the preparation volume.
Procedure
Add the following reagents to the cuvette in the specified proportions: 0.2 mL of Reagent A, 0.1 mL of Reagent B, 0.1 mL of Reagent C, 0.1 mL of Reagent D, 0.1 mL of Reagent E, 0.1 mL of Reagent F, and 0.3 mL of deionized water. After preheating at 37°C for 300 seconds, add 0.02 mL of the enzyme solution to be tested. Measure the absorbance change rate within 1 minute at a wavelength of 500 nm. The linear range of this method is 0.2-0.5ku/L.
Calculation
The enzyme activity is calculated using the following formula:
Vt: Total volume of the reaction mixture (ml)
ε: Molar extinction coefficient, with a value of 12 (cm²/μmol)
Vs: Volume of the enzyme solution to be tested (ml)
d: Light path length of the cuvette (cm)
D: Dilution factor of the enzyme solution
△A/min: △A/min(test sample)-△A/min(blank) (ABS/min)
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