Amino Acid Oxidase, D- Assay

Method: The reaction velocity is determined in a peroxidase coupled system by measuring the increase in A₄₃₆ resulting from the oxidation of D-alanine. One unit oxidizes one micromole of D-alanine per min. at 37°C and pH 8.3 under the specified conditions.

Reagents

• 0.02 M Sodium pyrophosphate, pH 8.3
• 0.05 M D-Alanine in 0.02 M sodium pyrophosphate pH 8.3 (Substrate) and containing 0.0065% o-dianisidine
• 0.02 M Sodium pyrophosphate, pH 8.3 with 10 mg/ml peroxidase (Worthington code: HPOD) in reagent grade water

Enzyme

Dissolve purified enzyme at a concentration of 0.05-0.2 mg/ml in 0.02 M sodium pyrophosphate buffer, pH 8.3. Dissolve crude enzyme at 1-5 mg/ml in buffer.

Procedure

Adjust spectrophotometer to 436 nm and 37°C.

Pipette 2.8 ml of pyrophosphate/alanine/dianisidine mixture into cuvette. Add 0.1 ml of 1% peroxidase and allow temperature to equilibrate. Record blank rate for 5-10 minutes if present. Add 0.1 ml of appropriately diluted enzyme to yield a rate of 0.015-0.05 �A per minute. Record rate for 10 minutes. Calculate rate from linear portion of curve. Subtract blank rate if present.

Calculation