D-Lactate Dehydrogenase

UNIT DEFINITION

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

REAGENTS PREPARATION

Reagent I: Add 0.04 g sodium pyruvate in 0.1 M, pH 7.7 Tris-HCl buffer and set to 100 mL.

Reagent II: Add 2.11 g CAPS in 80 mL pure water and adjust to pH 9.7, then add 0.12 g NADH and set to 100 mL.

Enzyme diluent: 10 mM, pH 7.0 potassium phosphate, contains 0.1% BSA.

Sample: The enzyme was diluted to 1-2 U/mL with Enzyme diluent.

PROCEDURE

1. Add 1mL Reagent I and 0.02mL enzyme solution to the cuvette.

2. Preincubate the reaction mixture at 37 ℃ for 5 min.

3. Add 0.2 mL Reagent II and mix, record the ΔAs at 340 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 in stead of the enzyme solution.

∆A = ∆As – ∆Ab