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Glucose Oxidase (GO)

(ß-D-Glucose: oxygen 1-oxidoreductase; EC

Glucose oxidase catalyzes the following reaction:

beta-D-Glucose + Enzyme-FAD ------> Enzyme-FADH2 + p-D-Gluconolactone
          Enzyme-FADH2 + O2 ------> Enzyme-FAD + H2O2 
Glucose oxidase is a flavoenzyme and it has been used clinically in glucose determination (Williams D.C., Huff, G.F. and W.R. Seitz, Clin Chem., 22, 372, 1976). The enzyme has a molecular weight of 160,000.

1.Source: Aspergillus brasiliensis
   Form: Freeze-dried powder 
   Solubility: Distilled water or dilute buffer 
   Stability: Store at -20 C (-4 F) 
   Activity (approx.): 200-250 U/mg 
   Protein (approx.): 90-95% 
   Catalog No.: 077A0250 

That amount of enzyme which causes the oxidation of one micromole of glucose per minute at 25C and pH 7.0.

The reaction velocity of the enzyme-catalyzed reaction is determined by an increase in absorbancy at 436 nm resulting from the oxidation of O-dianisidine through a peroxidase coupled system.

  1. 0.1 M Potassium phosphate buffer, pH 7.0.
  2. 1% O-Dianisidine in distilled water.
  3. Dianisidine - buffer mixture: prepared by mixing 1.0 ml of O-Dianisidine (10 mg/ml) with 100 ml of 0.1 M phosphate buffer, pH 7.0. The solution is then oxygenated for 5 min.
  4. 10% D-Glucose in distilled water. Allow mutarotation to come to equilibrium by standing overnight.
  5. Peroxidase (1 mg/ml) in distilled water.
  6. Glucose oxidase (test enzyme) solution prepared in 0.1 M phosphate buffer, pH 7.0 to yield a final concentration of 0.15 U/ml.

  1. Set spectrophotometer (equipped with a strip chart recorder and temperature control) at 436 nm and 25C.
  2. Into a cuvette place the following reagents:

    Dianisidine - buffer mixture (oxygenated) 2.5 ml
    10% D-Glucose 0.3 ml
    Peroxidase 0.1 ml

    Incubate in spectrophotometer at 25C for 3-5 min. to achieve temperature equilibration.

  3. Establish blank rate, if any, at 436 nm.
  4. Initiate the reaction by adding 0.1 ml of glucose oxidase (enzyme) solution. Record the increase in absorbance at 436 nm for 4-6 min.
  5. Calculate the (delta)E436nm/min

Activity (U/mg) = (ΔE436nm/min)(Total Vol.)(Enz. Diln.)
(8.3)(Enz. Vol.)(mg Enz./ml)

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