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

A. Pyruvate solution	：0.3 M ［378 mg of Pyruvate・K salt (MW＝126.15)/10 mL of H2O］
B. K-phosphate buffer, pH 5.9	：0.15 M
C. 4-Aminoantipyrine solution	：0.15% (150 mg of 4-Aminoantipyrine/100 mL of H2O)
D. EHSPT (TOOS) solution	：0.3% ［300 mg of EHSPT (N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine) /100 mL H2O］
E. TPP solution	：3 mM ［13.8 mg of TPP (Thiamine pyrophosphate)(MW＝460.77)/10 mL H2O］
F . FAD solution	：0.15 mM ［1.3 mg of FAD・2Na salt (MW＝865.55)/10 mL H2O］
G. EDTA solution	：15 mM ［590 mg of EDTA・2Na salt (MW＝394.22)/100 mL H2O］
H. MgSO4 solution	：0.15 M ［3.4 g of MgSO4・7H2O (246.48)/100 mL H2O］
I . Peroxidase solution	：50 U/mL ［45 mg of peroxidase (110 purpurogallin units/mg)/100 mL H2O］
J . Enzyme diluent	：50 mM K-phosphate buffer, pH 5.7

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)

Concentration in Assay Mixture
Pyruvate	48 mM
K-Phosphate buffer	50 mM
4-Aminoantipyrine	0.48 mM
EHSPT	0.58 mM
TPP	0.19 mM
FAD	0.01 mM
EDTA	0.97 mM
MgSO4	9.7 mM
Peroxidase	ca.4.8 U/mL

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

Vt	：Total volume (3.10 mL)
Vs	：Sample volume (0.10 mL)
36.88	：Millimolar extinction coefficient of quinoneimine dye under the assay condition (F/micromole)
1/2	：Factor based on the fact that one mole of H2O2 produces half of a mole of quinoneimine dye.
1.0	：Light path length (cm)
df	：Dilution factor
C	：Enzyme concentration in dissolution (c mg/mL)