In order to determine catechol oxidase of a crude extract prepared from plant you can used catechol as substrate at two pH value (pH 5.0 and pH 7).

1- Preparation of catechol solution at 0.4 M in H3PO4 (orthophosphoric acid) at 0.5 mM (The acidic medium avoid chemical oxidation of catechol).

2- In a cuvette (1 mL) add 0.2 mL ofcatechol (at 0.4 M) and 0.780 mL of phosphate buffer (pH 7.0) (or acetate buffer pH 5.0-0.05 M).

3- After mixing the medium, add 20 µl enzyme extract without dilution and started the time.

4- After each 10 seconds, the absorbance was noted during 5 min (at 410 nm).

This protocol can be modified (enzyme volume : 10, 20...100 µl)in order to otain an increase of 0.3 Abs/min

If your enzyme extract is less actif you can concentrate the enzyme by precipitation with ammonium sulphate sel or acetone.

You can also tested if your enzyme is fully active or exist in a latent form (adding SDS detergent (40 µl sds at 0.1 M in 1 mL of reactionnel medium).

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