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Polyphenoloxidase assay

Polyphenoloxidase (catechol oxidases) assay

Browning of the cut surface of some fruits and vegetables is due the presence of a group of enzymes called polyphenoloxidases. These enzymes are released by the broken cells and they catalyse the reaction between colourless molecules called polyphenols and molecular oxygen. This reaction creates coloured compounds and these new compunds can spontaenously cross react with one another to form black-brown complexes called melanins.

One example of a substrate for these enzymes is catechol, hence the alternative name ‘catechol oxidases’ for these enzymes. Catechol is oxidised initially to the orange compound benzoquinone which is then converted to melanins.The conversion to melanin is spontaneous but slow.

catechol + oxygen
benzoquinone + water

Food processing and cooking often involve procedures which are intended to inhibit the action of polyphenoloxidases. Why do you think a cook immediately places freshly peeled potatoes into a pan of water? Or why do people squeeze a few drops of lemon juice on to a freshly cut avocado? Mushrooms contain high levels of polyphenoloxidases, so how do you think pre-sliced packaged ones can be prevented from going brown?

The assay

In this technique, the change from a colourless solution of catechol to coloured benzoquinone is followed with a colorimeter. A fruit extract is added to a solution of catechol and the rate of formation of coloured benzoquinone is measured. The faster the rate of increase in absorbance of the reaction mixture, the greater the polyphenoloxidase activity of the fruit extract.

Ideas for investigations into the activity of these enzymes in plant tissues

Alternative techniques for monitoring the progress of the reaction include a ‘low-tech’ method, such as following the reaction by allowing the formation of melanins overnight, or a ‘high-tech’ method, by following the uptake of dissolved oxygen from a reaction mixture using an oxygen electrode.

For more information, you will find suggestions for experiments with potato polyphenoloxidases at Typing the search term polyphenoloxidase on WWW search engines will provide plenty of hits.

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