Inhibition of Catechol Oxidase in Banana by Heavy Metals

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 / ====> / yellow pigment / ====> / brown MELANIN
catechol oxidase / oxygen in air

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

• 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.

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

• 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 cm3 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 toremove the very fine precipitate formed during the experiment.

11. The intensity of the yellow colour formed can be measured using a colorimeter.

Alternatively, if the SAPS colour chart below is used to estimate the results, put the test tubes to the side till next day toallow the brown melanins to form. If results are required sooner, mixing the test tube contents with air will speedup 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:

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.