Assay Procedure for Pyruvate Oxidase Bacterial

Principle

The appearance of quinoneimine dye is measured at 550 nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of hydrogen peroxide (half of a micromole of quinoneimine dye) per minute under the conditions described below.

Method

Reagents

Procedure

1. Prepare the following working solution in a brown bottle and store on ice.
   
   10 mL K-phosphate buffer, pH 5.9 (B)
   2 mL4-Aminoantipyrine solution (C)
   2 mLEHSPT solution (D)
   2 mLTPP solution (E)
   2 mLFAD solution (F)
   2 mLEDTA solution (G)
   2 mLMgSO₄ solution (H)
   3 mLPeroxidase (I)

2. Pipette 2.5 mL of working solution into a cuvette (d＝1.0 cm), add 0.5 mL of pyruvate solution (A), and equilibrate at 37 ℃ for 5 minutes.
   
3. Add 0.1 mL of the enzyme solution* and mix by gentle inversion.
   
4. Record the increase in optical density at 550 nm against water for 3-4 minutes in a spectrophotometer thermostated at 37 ℃, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).
   

At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (J), dilute to 0.1－0.5 U/mL with the same buffer and store on ice.

Calculation

Activity can be calculated by using the following formula：

Weight activity (U/mg)＝(U/mL)×1/C