UNIT DEFINITION

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

REAGENTS PREPARATION

Reagent I: Add 0.0372 g EDTA·2Na, 0.07 g sodium cholate, 1mL 4-AA (50 mM) in 40 mL of 0.1 M pH 7.5 potassium phosphate buffer, adjust to pH 7.5, then add 200 U xanthine oxidase and set to 50 mL.

Reagent II: Add 0.161 g inosine, 9.5 mL TOOS (50 mM), 0.5 mL POD (1 kU/mL) to 0.1 M pH 7.7 potassium phosphate buffer, adjust to pH 7.7, then set to 50 mL.

Enzyme diluent: Add 0.0372 g EDTA·2Na and 0.07 g sodium cholate in 50 mL of 0.1M pH 7.5 potassium phosphate.

Sample: Dilute the sample to 0.2-0.3 U/mL by enzyme diluent.

PROCEDURE

1. Add 0.75 mL of Reagent I, 0.15 mL of Reagent II to a cuvette and preincubate the reaction mixture at 37 ℃ for 5 min.

2. Add 0.015 mL enzyme solution to the cuvette and mix.

3. 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