AffiZYME® Ascorbate oxidase
Abbreviation: ASOD
Preparation and Specification:
- Appearance: Light blue amorphous powder, lyophilized
- Activity: Grade III 200U/mg-solid or more (containing approx. 70% of stabilizers)
- Contaminants: Catalase ≤1.0×10⁻¹ %, Phosphatase ≤2.0×10⁻² %
- Stabilizers: BSA, borax, basic amino acids.
Properties:
- Stability: Stable at -20°C for at least one year
- Molecular Weight: 132,000, 140,000
- Isoelectric Point: Between 6.0 and 7.8, 8.2
- Michaelis Constant: 2.5×10⁻⁴M (Ascorbate)
- Structure: 8 copper atoms per mol of enzyme
- Inhibitors: Cyanide, Na₂S, diethyldithiocarbamate (Na)
- Optimum pH: 5.6
- Optimum Temperature: Approx. 30°C
- pH Stability: pH 7.0−10.0 (25°C, 17hr)
- Thermal Stability: Below 40°C (pH 8.0, 30min)
- Substrate Specificity: Oxidizes ascorbic acid and several ascorbic derivatives
Applications:
- Useful for enzymatic determination of ascorbic acid and eliminating interference in clinical analysis.
Assay:
- Principle: Ascorbate oxidase catalyzes the oxidation of ascorbic acid to dehydroascorbic acid.
- Unit Definition: One unit causes the decrease of one micromole of ascorbic acid per minute.
- Method: Spectrophotometry at 245nm.
- Reagents: Ascorbic acid solution, Na₂HPO₄ solution, HCl solution, Enzyme diluent.
- Procedure: Reaction mixture preparation, enzyme addition, stop reaction with HCl, measure optical density.
- Calculation: Activity calculation based on optical density changes.