Catechol oxidase

Safety

Results

Methods

Catechol oxidase is the enzyme responsible for the browning of fruit. It is easy to prepare from a number of different sources – bananas are particularly good – and the reaction is readily followed using a colorimeter.

Catechol oxidase has a number of alternative names, (Polyphenol oxidase, Diphenol oxidase, Tyrosinase, etc, EC 1.10.3.1 ). The reaction is also catalysed by EC 1.14.18.1 and it is not easy to distinguish between the enzymes.

The reaction catalysed is the oxidation of catechol to the yellow product 1,2-benzoquinone.

catechol + ½O₂ → 1,2-benzoquinone + H₂O

On exposure to air there is a further reaction in which the yellow benzoquinone is converted to dark brown melanin.

This reaction can be continuously monitored with the colorimeter and is particularly suitable for investigations into the effect of pH and the effects of competitive and non-competitive inhibitors.

Dopa oxidase is a similar phenol oxidase and a good alternative to this assay

The volumes and concentrations of catechol and its inhibitors, (if used), present minimal hazards. Good laboratory practices should be observed and the wearing of gloves is advised when preparing working solutions.

Absorbance vs Time

These results were obtained using 0.01M catechol (0.3cm³ of a 0.1M solution in a 3cm³ reaction mix) + 2.6cm³ of 0.2M phosphate buffer pH 6.8 added to 0.1cm³ of banana extract (see ‘Methods - Enzyme extraction’) then diluted 1:1 with water. The temperature was 25°C.

Effect of catechol concentration

Effect of pH (results of 2 experiments)

Effect of competitive and non-competitive inhibitors

Enzyme extraction

Catechol oxidase can be extracted from fruits that brown on exposure of their cut surfaces to air, potato and apple are commonly used. We have found banana to be easiest to use, and the extract retains its activity for weeks in the fridge.

To prepare the enzyme blend banana with two volumes of water or just squash the banana with a fork and crush it in a pestle and mortar with two volumes of water. 20g of banana with 40cm³ of water will give plenty of extract for most purposes.

Filter the extract through several layers of butter muslin and store refrigerated.

Reaction mixture

For continuous reading put 0.1cm³ of the enzyme extract in a cuvette and add 2.9cm³ of a mixture containing 0.5cm³ of catechol solution and 2.4cm³ of buffer.

The reaction can be performed at room temperature, (results reported here were obtained at 25°C).

The optimum pH is around 6.8 and catechol concentrations between 0.01M and 0.05M (final concentration in the cuvette) should give a good linear response when the concentration is plotted against the rate of reaction.

Catechol MWt = 110

Investigating the effect of pH

The effect of pH on the activity of catechol oxidase can be investigated using buffers ranging from pH4 to pH9. Citrate buffers are suitable up to pH8 – higher pHs can be obtained using a different buffer or, more easily, just by adding 2M NaOH drop by drop until the desired pH is reached.

A reaction mix containing 0.01M catechol should give a good response at 25°C. (0.3mls 0.1M catechol + 2.6mls buffer added to 0.1mls enzyme extract in a standard cuvette). The initial straight line portion of the graph of absorbance against time gives V_i – the initial rate of reaction.

Inhibitors

Competitive inhibition
para-hydroxy benzoic acid, (4-hydroxy benzoic acid, PHBA, p-salicylic acid), is a competitive inhibitor of catechol oxidase.
0.1cm³ of a 1M stock solution (dissolved in ethanol) added to a 3cm³ reaction mix is a suitable concentration to demonstrate inhibition.

Non-competitive inhibition
Phenylthiourea, (phenylthiocarbamide), is a non-competitive inhibitor. The solid is toxic but at the concentration used here the hazard is minimal.
0.1cm³ of a 0.02M stock solution added to a 3cm³ reaction mix is a suitable concentration.


PHBA stock solution	1M
phenylthiourea stock solution	0.02M

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