Name: ______________________________ Biology block: ____________

Unit 3: Practical Assignment 5

You are provided with a solution, labelled E, containing an enzyme which coagulates (clots) milk. Enzyme
E hydrolyses (breaks) peptide bonds between certain amino acids in a protein found in milk and this results
in the coagulation of the milk. Calcium ions are required for this coagulation.

You are required to:

• carry out a trial test to think about sources of error
• investigate the effect of substrate concentration on this enzyme-catalysed coagulation.

When a mixture of milk, calcium chloride solution and E is gently rotated in a test-tube the coagulation
goes through the stages shown in the figure below. Stage 3 is the end-point of the enzyme-catalysed
coagulation.

You are provided with:

If C or E comes into contact with your skin, wash off immediately under cold water. It is recommended that
you wear suitable eye protection.

You are required to carry out a trial test (step 1 to step 16) before you start your investigation.
Read step 1 to step 16 before proceeding. Proceed as follows:

1. You are provided with a beaker labelled water-bath. Use the hot and cold water to set up a water-bath
in this beaker. The starting temperature of the water-bath should be between 35°C and 40°C. You will
not need to maintain this temperature during steps 2 to 15.
2. Put 10 cm3 of M into a test-tube.
3. Repeat step 2 so that you have three test-tubes containing M.
4. Put 1 cm3 of C into each test-tube.
5. Gently shake each of the test-tubes to mix M and C.
6. Take the temperature of the water-bath and record this temperature in (a)(ii).
7. Put the test-tubes into the water-bath and leave for at least 3 minutes.

a) i. Explain why the test-tubes are left in the water-bath for at least 3 minutes in step 7.
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8. Remove one of the test-tubes from the water-bath. The process of coagulation will start when E is
added to the test-tube.
9. Put 1cm3 of E into the test-tube, so that it runs down the side of the test-tube and forms a layer on the
surface of the mixture, as shown in the figure below.
10. Gently shake the test-tube to mix the solutions and start timing.
11. Hold the test-tube over a piece of black card on the table as shown in the figure below.
12. Gently rotate the test-tube to form a film of milk on the inside of the test-tube.

13. Observe the film until the end-point is reached (stage 3). Ignore any small bubbles on the inside of the
test-tube. Stop timing.
14. Record in (a)(iii) the time taken to reach the end-point. If the end-point has not been reached in 4
minutes, stop the experiment and record ‘more than 240’.
15. Repeat step 8 to step 14 with each of the other two test-tubes in the water-bath.
16. Take the temperature of the water-bath when the final test-tube has been removed and record this in
(a)(ii).

ii. Temperature may be a source of error in this investigation. State the temperatures of the water-bath.
temperature of water-bath taken in step 6 _______________ °C
temperature of water-bath taken in step 16 _______________ °C
Explain whether the temperature of the water-bath is a significant source of error in this investigation.

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iii. Record your results in an appropriate table.

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 iv. A significant source of error for this investigation is deciding when the end-point is reached. Suggest
one advantage of carrying out this trial test before investigating the effect of substrate concentration
on this enzyme-catalysed reaction.
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You are required to investigate the effect of substrate concentration on this enzyme-catalysed
coagulation.

v. Identify the dependent variable in this investigation.

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vi. You are required to prepare different concentrations of milk, using M. M is undiluted milk and is to
be referred to as 100% milk. You are required to make a serial dilution of M, which reduces the
concentration of M by half between each successive dilution. You will need to prepare 3 further
concentrations of milk. You will need to prepare 20 cm3 of each concentration. Complete the figure
below to show how you will dilute M by:
• stating, under each beaker, the volume and concentration of the milk available for use in the
investigation
• using one arrow, with a label above the beaker, to show the volume and concentration of milk
added to prepare the concentration
• using another arrow, with a label above the beaker, to show the volume of distilled water, W,
added to prepare the concentration.

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17. Prepare the concentrations of milk as decided in (a)(vi) and in the figure.
18. Adjust the temperature of the water-bath so that it is between 35°C and 40°C. You will not need to
maintain this temperature during step 19 to step 24.
19. Put 10 cm3 of the lowest concentration of milk into a test-tube.
20. Repeat step 19 with each of the other concentrations of milk that you have prepared and with 100%
milk.
21. Put 1 cm3 of C into each test-tube.
22. Gently shake each of the test-tubes to mix the milk and C.
23. Put the test-tubes in the water-bath and leave for at least 3 minutes.
While you are waiting read step 8 to step 13.
24. After 3 minutes remove one of the test-tubes from the water-bath. Add 1 cm3 of E as in step 9, then
repeat step 10 to step 13 and record in (a)(vii) the time taken to reach the end-point.
25. Repeat step 24 with each of the other test-tubes.

vii. Record your results in an appropriate table.

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viii. Describe a control that could be carried out as part of your investigation.
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ix. This procedure investigated the effect of substrate concentration on the activity of enzyme E, using
the time taken to reach the end-point. To modify this procedure for investigating another variable, the
substrate concentration would need to be standardised. Describe how the substrate concentration
could be standardised.
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Think about how you could modify this procedure to investigate the effect of temperature on the time
taken to reach the end-point. Describe the modifications needed to investigate the effect of
temperature.
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b) A scientist investigated the percentage mass of protein in milk produced by different animals. The
results are shown in the table below.

i. Draw a chart of the data shown in the table. Use a sharp pencil for drawing charts.

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ii. A scientist investigated enzyme-catalysed coagulation of milk using enzyme E. This enzyme
hydrolyses (breaks) peptide bonds between certain amino acids in a protein found in milk and this
results in the coagulation of the milk. The scientist recorded the time taken to reach the end-point with
each of the types of milk shown in the table. The shortest time to reach the end-point was recorded
when seal milk was investigated. Use your knowledge of enzymes and the data in the table to suggest
an explanation for this result.
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