Assay Procedure for Pyruvate Oxidase Bacterial

Principle

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

Unit definition

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

Method

Reagents

 

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

Procedure

  1. Prepare the following working solution in a brownish bottle and store on ice.

    10ml K-phosphate buffer, pH 5.9 (B)
    2ml 4-Aminoantipyrine solution   (C)
    2ml EHSPT solution                       (D)
    2ml TPP solutionn                          (E)
    2ml FAD solution                             (F)
    2ml EDTA solution                           (G)
    2ml MgSO4 solution                       (H)
    3ml Peroxidase                               ( I )
Concentration in assay mixture
Pyruvate 48 mM
K-Phosphate buffer 50 mM
4-Aminoantipyrine 0.48mM
EHSPT 0.58mM
TPP 0.19mM
FAD 0.01mM
EDTA 0.97mM
MgSO4 9.7 mM
Peroxidase ca.4.8 U/ml
  1. Pipette 2.5ml of working solution into a cuvette (d=1.0cm), add 0.5ml of pyruvate solution (A), and equilibrate at 37℃ for about 5minutes.
  2. Add 0.1ml of the enzyme solution* and mix by gentle inversion.
  3. Record the increase in optical density at 550nm against water for 3 to 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 0.1-0.5U/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 (3.10ml)
Vs :Sample volume (0.10ml)
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 a mole of quinoneimine dye.
1.0 :Light path length (cm)
df :Dilution factor
C :Enzyme concentration in dissolution (c mg/ml)

 

This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

Materials

     
Related Links