Diaphorase​

UNIT DEFINITION

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

REAGENTS PREPARATION

Reagent I: 200mM pH7.0 Tris-HCl buffer.

Reagent II: 36 mM NADH solution.

Reagent III: 2.4 mM DCPIP solution.

Enzyme diluent: 200 mM Tris-HCl, pH7.5, contains 0.5% Tween20.

Sample: dilute the enzyme to 0.1-0.25 U/ml with enzyme diluent.

PROCEDURE

1. Add 2.4ml pure water, 0.3ml Reagent I and 0.1ml Reagent II into a 3ml cuvette and equilibrate at 37 ℃ for about 4 minutes.

2. Add 0.1 mL of samples and 0.1 mL DCPIP to mix.

3. Record the ΔAs at 600 nm in 1 minute in a spectrophotometer thermostated 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