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3.3.3Exchange and Transport

Investigating Digestive Enzymes — AQA A Level Biology 7402 Revision Notes & Topical Questions

5 min read 9 parts Lesson 23 of 48 1 quiz

📚 AQA A Level Biology 7402 Hub / Topical Revision
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3.3.3 Investigating Digestive Enzymes — AQA A Level Biology 7402 notes: Plan, carry out and evaluate practical investigations of the factors that affect the activity of digestive enzymes, using suitable tests and controls.TestDetectsPositive resultIodine in potassium iodideStarch…

  • Protease on milk or gelatin
  • Lipase and pH indicator
  • Colorimetry
  • Agar plates
  • Exam-style question 1
  • Exam-style question 2

Section 1 of 9Biochemical tests used to follow digestion

TestDetectsPositive result
Iodine in potassium iodideStarchYellow-brown to blue-black
Benedict's reagent, heatedReducing sugarsBlue to green, yellow, orange, brick-red precipitate
BiuretPeptide bonds (protein)Blue to purple
Ethanol emulsion testLipidsCloudy white emulsion

For starch digestion by amylase, samples are taken at intervals and added to iodine; the time taken for the blue-black colour to disappear shows when all the starch has been hydrolysed. For sugar production, Benedict's reagent is used to give semi-quantitative results, or a colorimeter measures the colour more objectively.

Section 2 of 9Investigating the effect of pH or temperature on amylase

1
Place drops of iodine solution in a spotting tile.
2
Mix starch solution and a buffer of known pH in a test tube, and place in a water bath at a constant temperature to equilibrate.
3
Add amylase solution (also equilibrated) and start the stopwatch.
4
Every 30 seconds, remove a drop of the mixture and add to a well of iodine.
5
Record the time when iodine no longer turns blue-black.
6
Repeat for a range of pH values (for example 3, 5, 7, 9) or temperatures (for example 10, 20, 30, 40, 50, 60 °C), and repeat each at least three times.

Rate = 1 ÷ time taken. The independent variable is the pH or temperature; the dependent variable is the time taken (rate); the controlled variables are enzyme and substrate concentration and volume, and temperature (when pH is tested) or pH (when temperature is tested).

Section 3 of 9Other methods

1Protease on milk or gelatin
Protease on milk or gelatin cloudy milk clears or a photographic film becomes transparent as protein is digested. Time to clear gives rate.
2Lipase and pH indicator
Lipase and pH indicator milk with sodium carbonate and phenolphthalein is pink; as fatty acids form the pH falls and the colour disappears. Time taken gives rate.
3Colorimetry
Colorimetry transmission of light through a solution is measured; a calibration curve converts to concentration.
4Agar plates
Agar plates wells cut in starch agar are filled with enzyme; after incubation, iodine shows a clear zone of digestion whose diameter (or area) is measured.

Section 4 of 9Controls, reliability and safety

Control
A control tube with boiled (denatured) enzyme or with water instead of enzyme shows that digestion is caused by the enzyme.
Use a water bath or thermostatically controlled block to keep temperature constant, and use buffer solutions to hold pH constant.
Repeat to calculate a mean and identify anomalies; calculate range or standard deviation.
Safety: wear eye protection with iodine, Benedict's and acids/alkalis; avoid skin contact with enzymes (they can cause allergic reactions); take care with hot water baths.

Section 5 of 9Analysing and evaluating results

A graph of rate against pH or temperature gives a bell-shaped curve with a peak at the optimum.

1
At low temperature the enzyme is not denatured but molecules move slowly so the rate is low; above the optimum the rate falls rapidly as the enzyme is denatured.
2
For more precision, narrower intervals can be investigated around the optimum.
3
Sources of error include timing end-points subjectively, drops of mixture varying in volume, and heat transfer when mixing.
4
Improvements include using a colorimeter, equilibrating all solutions before mixing, and using a pH meter to check the buffers.

Section 6 of 9Worked example: planning to a mark scheme

Questions on required practicals usually reward the same few skills.

Plan
When asked to plan, state the independent variable and the range with units, the dependent variable and how you will measure it, the key controlled variables and how you will keep them constant, the number of repeats, and the safety precautions.
Evaluate
When asked to evaluate, identify a limitation, say how it affects the result, and suggest a specific improvement.

Examplea student used a stopwatch to find when the blue-black colour disappeared, but found that colour changes are gradual.

Worked example: planning to a mark scheme
Improvement: use a colorimeter with a red filter to measure absorbance at regular intervals and plot a graph, so that the end-point is not a matter of opinion.
Another limitation is that the temperature of the mixture could change after taking it out of the water bath, so the volume of reagent should be small and the mixture should be kept in the bath throughout.

Remember that the type of graph depends on the variables. A continuous independent variable, such as temperature, gives a line graph; categories, such as different foods, give a bar chart.

Section 7 of 9Exam-style questions

  1. Describe how you would investigate the effect of temperature on the rate of digestion of starch by amylase. [5]5 marks

  2. A student found that starch was digested in 120 s at 30 °C and in 60 s at 40 °C. Calculate the rate at each temperature and the percentage increase. [3]3 marks

  3. Explain the purpose of a control tube containing boiled enzyme. [2]2 marks

Section 8 of 9Key terms

TermMeaning
BufferSolution that resists changes in pH.
ControlTreatment in which the factor being tested is absent so that its effect can be compared.
EquilibrateAllow solutions to reach the experimental temperature before mixing.
ColorimeterInstrument that measures the absorbance or transmission of light through a solution.
Reducing sugarSugar that donates electrons to Benedict's reagent.
OptimumCondition giving the greatest enzyme activity.

Section 9 of 9Exam technique

1
Heating the iodine test; Benedict's reagent needs heating, iodine does not.
2
Failing to equilibrate the enzyme and substrate at the test temperature before mixing.
3
Not controlling pH while changing temperature.
4
Using a single repeat and calling the results reliable.
5
Stating that the rate is time taken, when rate is the reciprocal of time.

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