Ascorbate oxidase catalyzes the following reaction:
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).
Store at -20° C (-4° F)
250-575 U/mg protein
Catalase ² 1.0 x 10-1% Phosphatase ² 2.0 x 10-2%
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
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.