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Assay Procedure for Glycerol 3-phosphate Oxidase

Principle

The appearance of quinoneimine dye is measured at 500nm 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. D,L-α-Glycerophosphate solution
:0.2M[Weigh 6.48g of D, L-α-glycerophosphate (disodium salt, MW=324.17),
dissolve in 80ml of 0.125M Tris-HCl buffer, pH 8.0 contg. 0.125% of Triton X-100 and after adjusting the pH to 8.1 at 25℃ with 2.0N HCl, fill up to 100ml with H2O.] (Stable for two weeks if stored at 0-5℃)
B. 4-AA solution :0.1% (100mg of 4-aminoantipyrine /100ml of H2O)(Store at 4℃ in an amber bottle)
C. Phenol solution :0.1% (100mg phenol/100ml of H2O)(Store at 4℃ in an amber bottle)
D. Peroxidase solution :0.025%[25mg peroxidase (110 purpurogallin units/mg)/100ml of H2O]
(Should be prepared fresh)
E. SDS solution :0.25% (1.25g sodium dodecyl sulfate/500ml of H2O)
F. Enzyme diluent :20mM Tris-HCl buffer, pH 7.5 contg. 0.2% of BSA

Procedure

  1. Prepare the following working solution (100 tests) in a brownish bottle and store on ice.

    50ml Substrate solution    (A)
    10ml 4-AA solution              (B)
    20ml Phenol solution         (C)
    20ml Peroxidase solution (D)

 

Concentration in assay mixture
Tris-HCl buffer 50 mM
Glycerophosphate 98mM
4-Aminoantipyrine 0.48mM
Phenol 2.1 mM
Triton X-100 0.049 %
POD ca.5.4 U/ml

 

  1. Pipette 1.0ml of working solution into a test tube and equilibrate at 37℃ for about 5 minutes.
  2. Add 0.02ml of the enzyme solution* and mix.
  3. After exactly 10 minutes at 37℃, add 2.0ml of SDS solution (E) to stop the reaction and measure the optical density at 500nm against water (OD test).

At the same time, prepare the blank by first mixing 1.0ml of working solution with 2.0ml of SDS solution after 10min-incubation at 37℃, followed by addition of the enzyme solution (OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (1.0mg/ml or more) and dilute to 0.15-0.35U/ml with the same buffer, immediately before assay.

Calculations

Activity can be calculated by using the following formula:

 

Weight activity (U/mg)=(U/ml)×1/C

 

Vt :Total volume (3.02ml)
Vs :Sample volume (0.02ml)
13.3 :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.
t Reaction time (10 minutes)
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

     
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