Lactic Oxidase

Unit definition

One unit is defined as the amount of enzyme which produces 1 μmol of H2O2 per minute under the conditions described below.

Reagents preparation

Reagent I：0.2 M pH 6.5 potassium phosphate buffer.

Reagent II： 1kU/mL peroxidase (POD) solution.

Reagent III：50 mM 4-AA solution.

Reagent IV： 0.5 M DL- Lactic acid solution, pH 6.5.

Reagent V：50 mM TOOS solution.

Enzyme diluent ： 10 mM pH 7.0 potassium phosphate solution with 10 μM FAD. to 0.05 – 0.2 U/mL.

Prepare reaction mixture as follows:

Reagent I                            10 ml

Reagent II                           0.25 mL

Reagent III                          1.5 mL

Reagent IV                         5 mL

Reagent V                          1.5 mL

Add ddH2O to                  50 ml

Procedure

1. Add 1 mL of the reaction mixture to 1 mL    cuvette.

2. Heat the reaction mixture at 37 °C for 5 min.
3. Add 20 μL of the enzyme solution to the cuvette and mix.

4. Record the ΔAs at 555 nm in 1 minute in a spectrophotometer thermo stated at 37 ℃.

* At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.

∆A = ∆As – ∆Ab