These practical protocols are designed for students doing an Extended Project Qualification, Advanced Higher investigation, or IB investigation in biology.
They are linked to our Food waste – bruising and browning project starter.
Possible areas for investigation
- How important a role does post-harvest browning play in wastage of fruit and vegetables worldwide, and what steps can be taken to prevent it?
- What is the catechol oxidase content of a given fruit at different stages of ripeness?
- What is the catechol oxidase content of different varieties of the same fruit?
- What are the catechol oxidase content of different fruits?
A student who does a project to investigate catechol oxidase activity in plant tissues is likely to need the following equipment and materials
- samples of fruit or vegetable (about 8 g)
- 10 cm3 distilled water
- a little sand
- mortar and pestle
- piece of muslin
- small beaker
- centrifuge (optional)
- centrifuge tubes (optional)
- marker pen
- buffer solution, pH 7
- 0.09 M (1%) catechol solution
- 1 x 10 cm3 syringe
- 2 x 1 cm3 syringes
- colorimeter with 440 nm filter
Note – catechol oxidase has a shelf life of only 2-3 days and should be stored in the dark.
The enzyme catechol oxidase can be extracted from plant tissues by simply grinding plant tissue with a little sand and distilled water, then filtering or centrifuging. Enzyme is present in the liquid extract. Catechol oxidase activity can then be estimated by measuring the rate of colour formation on adding the enzyme to its substrate, catechol in buffer. A colorimeter is used to measure absorbance at 440 nm.
Enzyme activity starts immediately on addition of the enzyme so you must be prepared to start measuring the absorbance.
o samples of fruit or vegetable (about 8 g)
o 10 cm3 distilled water
o a little sand
o mortar and pestle
o piece of muslin
o small beaker
o centrifuge tubes
o marker pen
o buffer solution, pH 7
o 0.09 M (1%) catechol solution
o 1 x 10 cm3 syringe
o 2 x 1 cm3 syringes
o Colorimeter with 440 nm filter
1. Mash the fruit, sand and water together thoroughly using a mortar and pestle.
2. Strain the mixture through muslin into a beaker (moisten the muslin first to speed up the filtering process).
Note: the enzyme, catechol oxidase is present in the clear liquid in the beaker. With some fruits and vegetables, it may be necessary to centrifuge the strained liquid to remove the solids. In this instance, pour the strained liquid into a centrifuge tube and centrifuge for about three minutes.
3 Use a syringe to add 10 cm3 buffer solution to a boiling tube.
4 Add 1 cm3 catechol solution to the boiling tube.
5 Prepare a table to record your results showing time in seconds and absorbance at 440nm.
6 Fill a cuvette with water and place in the colorimeter. Calibrate it to zero.
7 Add 1 cm3 enzyme solution (extracted from the fruit or vegetable) to the boiling tube and start the stop-clock.
8 Immediately transfer the solution to a cuvette and record the absorbance at 440 nm every 30 s for five minutes.