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Ascorbate Oxidase (AO)

(L-Ascorbate:Oxygen oxidoreductase; EC 1.10.3.3) Ascorbate oxidase catalyzes the following reaction:

                     AO
Ascorbic acid +  O2 ----->  Dehydroascorbic acid + H2O

Ascorbate oxidase is a copper-containing enzyme. It has been isolated from a wide variety of fruits and vegetables, including Valencia oranges (Baker, R.A.; and Bruemmer, J.H., Proc. Fla. State Hort. Soc., 81, 269, 1968).

1.Source:	Zucchini Squash
Form:	Freeze-dried powder
Solubility:	Dilute buffer
Stability:	Store at -20° C (-4° F)
Activity (approx.):	200-575 U/mg protein
Protein (approx.):	20% (biuret)
Stabilizer (approx.):	80%
Contaminants (approx.):	Catalase ² 1.0 x 10-1% Phosphatase ² 2.0 x 10-2%
Catalog No.:	061A0500

$5.00/KU

That amount of enzyme required to catalyze the oxidation of one micromole ascorbic acid per minute at 25°C and pH 5.6.

The reaction velocity of the enzyme-catalyzed reaction is based on determining the rate of decrease in the absorbance at 265 nm due to the oxidation of ascorbic acid to dehydroascorbic acid.

0.1 M Phosphate buffer, pH 5.6 (containing 0.186 g EDTA ● 2H₂O sodium salt per L).
0.005 M Ascorbic acid in phosphate buffer.
Ascorbic oxidase (enzyme) solution. Prepared in cold buffer to yield a final concentration of 0.1 U/ml.

Set spectrophotometer (equipped with a strip chart recorder and temperature control) at 265 nm and 25°C.

Into quartz cuvettes, pipette the following reagents in the amounts indicated:

                        TEST                BLANK
Phosphate buffer        2.9 ml              3.0 ml 
Ascorbic acid           0.1 ml              0.1 ml

Incubate the cuvettes in the spectrophotometer at 25°C and record blank rate, if any, at 265 nm.

Initiate the reaction by adding 0.1 ml enzyme solution to the TEST cuvette. Record the decrease in absorbance at 265 nm for 5 min.
Calculate E_265nm/min

Activity (U/mg) =	(ΔE_265nm/min)(Total Vol.)(Enz. Diln.)
	(13.386)(Enz. Vol.)(mg Enz./ml)

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