UNIT DEFINITION

One unit is defined as the amount of enzyme which consumes 1 μmol of glycerol per minute at 37 ℃ under the conditions specified in the assay procedure.

REAGENTS PREPARATION

Reagent I: 0.3 M glycerol.

Reagent II: 1 kU/mL POD.

Reagent III: 50 mM TOOS.

Reagent IV: 50 mM 4-AA.

Reagent V: 20 U/mL L-α-glycerophosphate oxidase.

Reagent VI: 200 mM HEPES, contains 20 mM MgCl2 and 40 mM ATP (pH 7.9).

Reaction mixture:

Reagent I                            1.67 mL

Reagent II                            400 μL

Reagent III                           3 mL

Reagent IV                           3 mL

Reagent V                           40 mL

Pure water                          Set to 100 mL

Enzyme dilution buffer: 20 mM, pH 7.5 potassium phosphate buffer.

Sample: Dilute the sample to 0.2 – 0.4 U/mL by enzyme dilution buffer.

PROCEDURE

1. Add 1mL reaction mixture to the 1 mL cuvette.
2. Preincubate at 37 ℃ for 2 min.
3. Add 0.02 mL 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