Enzymes

1 Plan and risk assessment

Amylase is an enzyme that breaks starch down into the sugar maltose. Iodine solution turns blue-black while starch is there and stays orange-brown once it has all gone. Each enzyme works fastest at its optimum pH; away from it the shape of the active site changes, so starch fits less well and the reaction slows.

mean rate of reaction = amount of starch broken down ÷ time taken

Prediction

2 Apparatus

• ✓Spotting tiles (two for each pH) Hold the drops of iodine solution that each sample is tested in.
• ✓Iodine solution in a dropping bottle Turns blue-black when starch is present; stays orange-brown when it is not.
• ✓1 % starch solution The substrate.
• ✓1 % amylase solution The enzyme.
• ✓Buffer solutions: pH 4, 5, 6, 7, 8 and 9 Keep each mixture at one pH: the independent variable.
• ✓Three syringes: 5 cm³ for starch, 1 cm³ for buffer, 1 cm³ for amylase (resolution 0.2 cm³ and 0.01 cm³) Measure the same volumes for every run.
• ✓Test tubes in a rack Hold the starch with buffer, and the amylase.
• ✓Water bath at 37 °C Keeps the temperature the same for every run.
• ✓Thermometer (resolution 1 °C) Checks the water bath temperature.
• ✓Glass rod Stirs the mixture and carries each drop to a well.
• ✓Beaker of water For rinsing the glass rod between drops.
• ✓Stopclock (resolution 1 s) Times each run from the moment you mix.
• ✓Safety goggles Protect your eyes from iodine solution.

3 Method: your choices

• Which version are you doing?: AQA required practical 5: the effect of pH on amylaseSix pH buffers in a water bath at 37 °C: find the optimum pH.
• Choose how often to test a drop: Every 30 secondsEach time you record is at most 30 s after the real end point. Neighbouring pH values usually give different times.
• Warm them in the water bath first: DoneTemperature changes the rate of an enzyme reaction. Both solutions must already be at the bath temperature when they meet, so the only thing changing between runs is the pH.
• Mix the amylase in: DoneThe amylase must be spread evenly through the starch, so every drop you test is the same mixture.
• Rinse the rod between drops: DoneMixture left on the rod from the last drop still contains starch. Carried into the next well it can turn that drop blue-black.
• When do you stop?: At the first drop that stays orange-brown: the iodine does not turn blue-black at allYou record the time when the starch has all been broken down.

• 0.0 Struck through: the anomaly, retaken; the new reading is beside it.

Anomaly: Time at pH 8, trial 2, retaken. Likely cause: an air bubble in the amylase syringe: less amylase went in, so the starch took much longer to break down.

6 Analysis

1. Mean rate of reaction

   mean rate of reaction = amount of starch broken down ÷ time taken

   Each run started with 50 mg of starch, and it was all broken down by the end point. Calculate the mean rate of starch breakdown at pH 7, in mg per minute.

   33.3 mg/min Right first time

2. Shape of the graph

   Describe the shape of your curve.

   It rises to a highest rate (the optimum pH) and then falls on the other side. Right first time

3. The optimum pH

   Read the pH below the highest point of your curve.

   Use your curve. At what pH does amylase work fastest? Read it to 1 decimal place.

   6.5 Right first time

4. What your testing interval means

   Your first run at pH 7 was recorded as 90 s: the first drop that stayed orange-brown, tested every 30 s. When did the starch really run out?

   Some time between 60 s and 90 s. Right first time

7 Conclusion and evaluation

Conclusion

Sources of error

Improvement

Repeatable or reproducible

9 Exam questions on your data

On your own readings, self-marked against the mark scheme, with error carried forward where it applies. Total 30/30.

1. 01.1[1 mark]

   A student investigated the effect of pH on the time amylase takes to break down starch. They mixed starch solution, a pH buffer and amylase solution in a water bath at 37 °C. Every 30 seconds they put a drop of the mixture into iodine solution on a spotting tile.

   What was the independent variable in this investigation?

   Your answer

   The pH of the buffer solution

   1/1 self-marked

   • pH (of the buffer / solution / mixture) (ignore acidity or alkalinity)

   Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

   Model answer: The pH of the buffer solution.

2. 01.2[2 marks]

   A student investigated the effect of pH on the activity of amylase. Starch solution, amylase and a buffer were mixed in a water bath, and a drop of the mixture was tested with iodine solution at regular intervals.

   Give two control variables in this investigation.

   Your answer

   The temperature of the water bath and the volume of starch solution.

   2/2 self-marked

   • Temperature (of the water bath)
   • Volume of starch (solution)
   • Volume of amylase (solution)
   • Volume of buffer
   • Concentration of starch or of amylase
   • Number of drops (volume) of iodine; size of the drop of mixture
   • Time between tests; time in the water bath before mixing
   • "Amount" of starch, amylase or buffer (allow "amount" once only)

   Self-marked: 2 of 2 marks. This is your own judgement against the mark scheme.

   Model answer: The temperature of the water bath; the volume of starch solution (also allowed: volume of amylase or of buffer, concentrations, drops of iodine, time between tests).

3. 01.3[1 mark]

   You stood the starch and amylase tubes in the water bath for 5 minutes before mixing them.

   Suggest why both tubes should be left in the water bath for 5 minutes before they are mixed.

   Your answer

   So that both solutions reach 37 °C, the temperature of the water bath, before they are mixed.

   1/1 self-marked

   • So both solutions reach the temperature of the water bath / 37 °C (the same temperature) before mixing; allow: to equilibrate

   Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

   Model answer: So that both solutions reach 37 °C, the temperature of the water bath, before they are mixed.

4. 01.4[2 marks]

   The student used iodine solution from a dropping bottle.

   Give one harm iodine solution could cause, and one way to reduce the risk.

   Your answer

   It could splash into your eyes and irritate them, so wear safety goggles.

   2/2 self-marked

   • Harm: irritates (stings, damages) the eyes; stains or irritates the skin (ignore "harmful" or "dangerous" on its own; do not accept burns)
   • Control: wear safety goggles (eye protection); wipe up spills; wash hands (skin); wear gloves

   Self-marked: 2 of 2 marks. This is your own judgement against the mark scheme.

   Model answer: It could irritate your eyes if it splashes. Wear safety goggles.

5. 02.1[1 mark]

   How did the student know that all the starch had been broken down?

   Your answer

   The iodine solution stayed orange-brown and did not turn blue-black.

   1/1 self-marked

   • The iodine (solution) stayed orange-brown / did not turn blue-black (both the reagent and the colour are needed; ignore "did not turn blue" or "purple")

   Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

   Model answer: A drop of the mixture no longer turned the iodine solution blue-black: the iodine stayed orange-brown.

6. 02.2[1 mark]

   Table 1 shows your three times at pH 8.

   Which trial gave an anomalous result?

   Select one row.

   Table 1

   Trial	Time taken for the starch to be broken down at pH 8 in s
   1	120
   2	510
   3	150

   Your answer

   trial 2

   1/1

   • Trial 2: 510 s is far from the other two, which agree

   Full marks. Well done. Trial 2 (510 s) is far from the other two times, which agree with each other.

   Model answer: Trial 2. Its time, 510 s, is far longer than the other two, which agree with each other.

7. 02.3[2 marks]

   Look again at Table 1.

   Calculate the mean time at pH 8.

   Do not use the anomalous result.

   Your answer

   135

   Working: mean = (120 + 150) ÷ 2 = 135 s

   2/2

   • (120 + 150) ÷ 2 (the anomaly left out)
   • 135 (s)

   Full marks. Clear and precise.

   Model answer: mean = (120 + 150) ÷ 2 = 135 s

8. 02.4[3 marks]

   Each run started with 50 mg of starch. All of it was broken down by the end point.

   Use your answer to Question 02.3 to calculate the mean rate of starch breakdown at pH 8.

   Give your answer in mg per minute.

   Your answer

   22

   Working: time = 135 s ÷ 60 = 2.25 min rate = 50 mg ÷ 2.25 min = 22 mg/min

   3/3

   • time in minutes: 135 ÷ 60 = 2.25 (or × 60 at the end)
   • rate = 50 ÷ time (amount of starch ÷ time)
   • 22 (mg/min); allow error carried forward from 02.3

   Full marks. That is exactly what the mark scheme is looking for.

   Model answer: time = 135 s ÷ 60 = 2.25 min rate = 50 mg ÷ 2.25 min = 22 mg/min

9. 02.5[1 mark]

   Figure 1 shows your mean rate at each pH, with a curve of best fit.

   Use Figure 1 to determine the optimum pH of the amylase.

   Your answer

   6.5

   1/1

   • 6.5 (the pH at the top of the curve); allow 6.3 to 6.8

   Full marks. Well done.

   Model answer: About pH 6.5: the pH at the top of the curve.

10. 02.6[1 mark]

    You tested a drop every 30 seconds. Another student tested a drop only once every minute.

    Suggest why the other student’s times would be less accurate.

    Your answer

    The starch could have been broken down at any time in the minute between tests, so the time could be up to a minute late.

    1/1 self-marked

    • The starch could have been broken down at any time during the minute between tests / the end point could be up to a minute before it is seen; allow: the time can only be a whole number of minutes

    Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

    Model answer: The starch could have all been broken down at any time in the minute between two tests, so the recorded time could be up to a minute after the real end point.

11. 02.7[1 mark]

    Your fastest was pH 6 and pH 7 (the same mean time).

    How could you find the optimum pH of amylase more precisely?

    Tick (✓) one box.

    • ✓ Test more pH values between pH 6 and pH 7. (your choice)
    • Repeat every pH five more times.
    • Test pH 1, 2 and 3 as well.
    • Use a more concentrated amylase solution.

    Your answer

    Test more pH values between pH 6 and pH 7.

    1/1

    • Test more pH values between pH 6 and pH 7.

    Full marks. Well done.

    Model answer: Test more pH values between pH 6 and pH 7, for example every 0.2.

12. 03.1[4 marks]

    Your mean time was 430 s at pH 4 and 90 s at pH 6.

    Explain why the starch was broken down much more slowly at pH 4 than at pH 6.

    Your answer

    At pH 6 the active site is complementary to starch, so the starch fits. At pH 4 the shape of the active site changes and the amylase is denatured, so starch no longer fits. Fewer enzyme-substrate complexes form, so the rate of reaction is slower.

    4/4 self-marked

    • At pH 6 starch fits the active site (the active site is complementary to starch)
    • At pH 4 the shape of the active site changes / the amylase is denatured (allow: damaged; do not accept killed or died)
    • So starch no longer fits (is not complementary to) the active site, or fits less well
    • Fewer enzyme–substrate complexes form (per second), so the rate of reaction is slower

    Self-marked: 4 of 4 marks. This is your own judgement against the mark scheme.

    Model answer: At pH 6 the active site of amylase is complementary to starch, so starch fits and enzyme–substrate complexes form quickly. At pH 4 the shape of the active site changes (the amylase is denatured), so starch no longer fits as well. Fewer enzyme–substrate complexes form, so the rate of reaction is slower.

13. 03.2[1 mark]

    Another student said: "At pH 7 the iodine stayed orange-brown after 90 seconds, so the amylase had stopped working."

    Which is the correct explanation of the orange-brown colour?

    Tick (✓) one box.

    • ✓ All the starch had been broken down, so none was left to turn the iodine blue-black. (your choice)
    • The amylase had been denatured, so it stopped working.
    • Iodine turns orange-brown when sugar is present.
    • All the iodine had been used up.

    Your answer

    All the starch had been broken down, so none was left to turn the iodine blue-black.

    1/1

    • All the starch had been broken down, so none was left to turn the iodine blue-black.

    Full marks. Your answer meets all the points for this question.

    Model answer: All the starch had been broken down, so none was left to turn the iodine blue-black.

14. 04.1[2 marks]

    Another student mixed the solutions straight from the bench, without warming them in the water bath first.

    Explain how this would affect their times.

    Your answer

    The solutions started at room temperature, cooler than 37 °C, so the amylase worked slowly at first and the times were longer.

    2/2 self-marked

    • The solutions (amylase) started below 37 °C / at room temperature, so the reaction was slower at first (less kinetic energy, fewer collisions)
    • So the times were longer (the rates lower)

    Self-marked: 2 of 2 marks. This is your own judgement against the mark scheme.

    Model answer: The solutions started at room temperature, below 37 °C, so the amylase molecules had less kinetic energy and worked more slowly at first. Their times would be longer, the short ones most.

15. 04.2[1 mark]

    The student did three runs at each pH.

    Give one reason for doing three runs at each pH.

    Your answer

    So that a mean can be calculated, which reduces the effect of random errors.

    1/1 self-marked

    • To calculate a mean (which reduces the effect of random errors); or to identify anomalies; or to check the results are repeatable

    Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

    Model answer: So a mean can be calculated, which reduces the effect of random errors (or so an anomaly can be identified).

16. 05[6 marks]

    A student wants to investigate the effect of pH on the time taken for amylase to break down starch.

    The student has: starch solution, amylase solution, buffer solutions of different pH, iodine solution, a spotting tile, syringes, test tubes, a glass rod, a water bath and a stopclock.

    Describe a method the student could use.

    Your answer

    1. Put one drop of iodine solution into each well of a spotting tile. 2. Measure 5 cm³ of starch solution and 1 cm³ of pH 5 buffer into a test tube with a syringe, and 1 cm³ of amylase into another tube. 3. Stand both tubes in a water bath at 37 °C for 5 minutes so the temperature is controlled. 4. Add the amylase to the starch and buffer, stir, and start the stopclock. 5. Every 30 seconds, use a glass rod to put a drop of the mixture into the next well of iodine. 6. Blue-black means starch is still there; record the time when the iodine stays orange-brown. 7. Repeat with buffers of pH 6, 7, 8 and 9, using the same volumes and concentrations. 8. Repeat each pH three times and calculate a mean time.

    6/6 self-marked

    • Put (drops of) iodine solution into the wells of a spotting tile
    • Measure volumes of starch, amylase and buffer (with a syringe or measuring cylinder)
    • Mix the amylase with the starch and the buffer (pH solution) and start the timer
    • Put a drop of the mixture into the iodine at regular intervals (every 30 seconds)
    • Blue-black shows starch is present; record the time when the iodine stays orange-brown
    • Repeat with buffers of different pH (at least three)
    • Control the temperature with a water bath (warm both solutions first)
    • Keep the volumes and concentrations of starch and amylase the same
    • Repeat each pH and calculate a mean

    Self-marked: Level 3, 6 of 6 marks. You ticked 9 of 9 indicative points. This is your own judgement against the mark scheme.

    Model answer: 1. Put one drop of iodine solution into each well of a spotting tile. 2. Measure 5 cm³ of starch solution and 1 cm³ of pH 5 buffer into a test tube with a syringe, and 1 cm³ of amylase into another tube. 3. Stand both tubes in a water bath at 37 °C for 5 minutes so the temperature is controlled. 4. Add the amylase to the starch and buffer, stir, and start the stopclock. 5. Every 30 seconds, use a glass rod to put a drop of the mixture into the next well of iodine. 6. Blue-black means starch is still there; record the time when the iodine stays orange-brown. 7. Repeat with buffers of pH 6, 7, 8 and 9, using the same volumes and concentrations. 8. Repeat each pH three times and calculate a mean time.

Unseen data: the same skills, new apparatus

1. 01[1 mark]

   Pepsin is a protease enzyme in the stomach. A student measured the mean rate at which pepsin digested egg white at different pH values.

   Table 1

   pH	Mean rate of digestion in mg/min
   1	5.4
   2	12.1
   3	8.0
   4	1.6
   5	0.1
   6	0.0

   The student wants to find the optimum pH of pepsin more precisely.

   Which pH values should the student test next?

   Tick (✓) one box.

   • ✓ pH 1, 1.25, 1.5, 1.75, 2 … 3 (your choice)
   • pH 2 again, ten more times
   • pH 7, 8, 9 and 10
   • pH 1, 2, 3, 4, 5 and 6 at a higher temperature

   Your answer

   pH 1, 1.25, 1.5, 1.75, 2 … 3

   1/1

   • pH 1, 1.25, 1.5, 1.75, 2 … 3

   Full marks. Your answer meets all the points for this question.

   Model answer: pH 1, 1.25, 1.5, 1.75, 2 … 3 The highest rate is at pH 2, so the optimum lies between pH 1 and pH 3: test values close together in that range.

2. 02[2 marks]

   A protease solution digested 80 mg of gelatine in 4 minutes.

   Calculate the mean rate of digestion in mg per minute.

   Your answer

   20

   Working: 240 / 60 = 4 80 / 4 = 20

   2/2

   • Time in minutes (4 min), or mass ÷ time in seconds
   • Mean rate = 20 mg/min

   Full marks. Clear and precise.

   Model answer: time = 240 s ÷ 60 = 4 min mean rate = 80 ÷ 4 = 20 mg/min

3. 03[2 marks]

   A student added potato extract, which contains the enzyme catalase, to hydrogen peroxide. They collected the oxygen made in 1 minute. They did this four times.

   Table 3

   Repeat	Volume of oxygen in cm³
   1	9
   2	21
   3	21
   4	22

   Calculate the mean volume of oxygen.

   Do not include the anomalous result. Give your answer to 3 significant figures.

   Your answer

   21.3

   Working: 21 + 21 + 22 = 64 64 / 3 = 21.3

   2/2

   • Leaves out repeat 1 and adds the other three
   • Mean = 21.3 cm³

   Full marks. Your answer meets all the points for this question.

   Model answer: Repeat 1 (9 cm³) is anomalous. mean = (21 + 21 + 22) ÷ 3 = 21.3 cm³