UNIT DEFINITION

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

REAGENTS PREPARATION

Reagent I: 0.2 M Tris-HCl buffer, pH 8.0.

Reagent II: 1.0 M Sarcosine solution.

Reagent III: 1 kU/mL POD solution.

Reagent IV: 50 mM 4-AA solution.

Reagent V: 50 mM TOOS solution.

Enzyme diluent: 10 mM KH2PO4 – K2HPO4b uffer, pH 7.5.

Enzyme solution: Dilute the enzyme to 0.07 −0.17 U/ml with enzyme diluent.

REACTION MIXTURE (50 TESTS):

Reagent I                   5 mL

Reagent II                  10 mL

Reagent III                0.25 mL

Reagent IV                1.5 mL

Reagent V                  1.5 mL

Pure H2O                 31.75 mL

PROCEDURE

1. Pipette 1 ml reaction mixture to a cuvette.
2.  Preincubate the reaction mixture at 37℃ for 5 min.

3.  Add 20 ul the enzyme solution and mix to start the reaction, record ΔAs at 555 nm in 1 minute in a spectrophotometer.

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

CALCULATION