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Glucose Oxidase (GO)

(ß-D-Glucose: oxygen 1-oxidoreductase; EC 1.1.3.4)

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

$220.00/MU

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

0.1 M Potassium phosphate buffer, pH 7.0.
1% O-Dianisidine in distilled water.
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.
10% D-Glucose in distilled water. Allow mutarotation to come to equilibrium by standing overnight.
Peroxidase (1 mg/ml) in distilled water.
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.

Set spectrophotometer (equipped with a strip chart recorder and temperature control) at 436 nm and 25°C.
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 25°C for 3-5 min. to achieve temperature equilibration.
Establish blank rate, if any, at 436 nm.
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.
Calculate the E436nm/min

Activity (U/mg) =	(ΔE_436nm/min)(Total Vol.)(Enz. Diln.)
	(8.3)(Enz. Vol.)(mg Enz./ml)

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