Inhibition of Catechol Oxidase in banana by heavy
metals
Students’ Sheet
Introduction
When some fruit and vegetables are damaged you no doubt will have noticed they turn brown.
This is due to an enzyme called catechol oxidase. This enzyme acts on its substrate catechol to
form a yellow compound which then reacts with oxygen in the air to form brown melanin
pigments. The more active the enzyme, the stronger the brown colouration.
Catechol
====>
catechol
oxidase
yellow pigment
====>
oxygen in
air
brown MELANIN
You are going to extract the catechol oxidase from banana and mix it with varying concentrations
of lead which is thought to inhibit the enzyme. A control tube will also be present. The experiment
is carried out in the presence of a buffer solution. Such solutions are used to try and prevent
changes in pH. Measuring the intensity of the brown colour formed at the end of the experiment
will indicate how active the enzyme is and thus how much the volume of lead added has inhibited
the enzyme.
Instructions
Part 1 - Extracting the enzyme
You are going to extract the catechol oxidase from banana and mix it with varying concentrations
of lead which is thought to inhibit the enzyme. A control tube will also be present. The experiment
is carried out in the presence of a buffer solution. Such solutions are used to try and prevent
changes in pH. Measuring the intensity of the brown colour formed at the end of the experiment
will indicate how active the enzyme is and thus how much the volume of lead added has inhibited
the enzyme.
Collect
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8 g banana (a slice of banana about 1cm thick)
a little sand
25 cm3 distilled water
mortar and pestle
piece of muslin
small beaker
centrifuge tube
1. Mix the banana, sand and water together thoroughly using a mortar and pestle.
2. Strain the mixture through muslin into a beaker.
Science & Plants for Schools: www.saps.org.uk
Inhibition of catechol oxidase in banana by heavy metals: p. 1
This document may be photocopied for educational use in any institution taking part in the SAPS programme.
It may not be photocopied for any other purpose. Revised 2010.
3. The enzyme, catechol oxidase, is present in the clear liquid in the beaker.
4. Very occasionally it may be necessary to pour the beaker contents into a centrifuge tube and
centrifuge for about 3 minutes until all small fragments of fruit have settled at the bottom of the
tube.
Part 2 – The experiment
Collect
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marker pen
buffer solution pH 7
0.09 M (1%) catechol solution
0.13 M (5%) lead ethanoate (lead (II) acetate) solution
2 x 10 cm3 syringes
5 boiling tubes
5 test tubes
2 x 1 cm3 pipettes
colour chart
test tube rack
5 filter funnels
5 filter papers (No. 6)
goggles
gloves
Label 5 boiling tubes A, B, C, D and E.
With reference to the table below, add varying volumes of buffer solution and lead ethanoate to
each tube using the 10 cm 3 syringe
Test tubes
A
B
C
D
E
Volume of
buffer (cm3)
10
9
8
5
2
Volume of
Lead
Ethanoate
(cm3)
0
1
2
5
8
Take great care while using the lead ethanoate solution. It is TOXIC!
8. Add 1 cm3 of catechol solution to each boiling tube. The catechol is a substrate for the enzyme
to act on.
9. Finally, add 1 cm3 of the enzyme, catechol oxidase, extracted from the banana, to each boiling
tube.
10. Filter the contents of each test tube with the No. 6 filter papers. These high quality filter
papers are required to remove the very fine precipitate formed during the experiment.
11. The intensity of the yellow colour formed can be measured using a colorimeter.
Science & Plants for Schools: www.saps.org.uk
Inhibition of catechol oxidase in banana by heavy metals: p. 2
This document may be photocopied for educational use in any institution taking part in the SAPS programme.
It may not be photocopied for any other purpose. Revised 2010.
Alternatively, if the SAPS colour chart below is used to estimate the results, put the test tubes to
the side till next day to allow the brown melanins to form. If results are required sooner, mixing
the test tube contents with air will speed up melanin formation.
12. Draw up a table of results using correct headings and appropriate units.
13. Compare your results with other groups and if necessary calculate an average for each tube.
14. Present these final results as a graph with suitable scales and axes labelled with quantities
and units. The volume of lead ethanoate added will form the horizontal x-axis, % transmission will
be on the vertical y-axis.
Questions
1. What is the aim of this experiment?
2. What causes cut fruit to turn brown?
3. Write down the chemical reaction that is occurring in such fruit
4. Why is a buffer solution included in each test tube?
5. Give a reason why it is important for the total volume of liquid in each test tube in the
experiment to be the same?
6. How can you tell how active the enzyme is?
7. In what way is lead affecting the activity of the enzyme?
8. Why would scientists repeat the entire experiment even when they obtained 'satisfactory'
results the first time?
Science & Plants for Schools: www.saps.org.uk
Inhibition of catechol oxidase in banana by heavy metals: p. 3
This document may be photocopied for educational use in any institution taking part in the SAPS programme.
It may not be photocopied for any other purpose. Revised 2010.