Required practical - The effect of pH on the rate of an enzyme controlled reaction
Laura Armstrong & Joe Wolfensohn
Teachers
Contents
Recall Questions
This topic requires prior knowledge of enzyme structure and function, including the role of the active site and the effect of temperature and pH on enzyme activity.
What is an enzyme and what does it do?
An enzyme is a biological catalyst that speeds up chemical reactions without being used up.
What happens to an enzyme if the temperature is too high?
The enzyme may become denatured, changing the shape of its active site so it no longer binds to the substrate.
What does the active site of an enzyme do?
It is complementary to, and binds to, the specific substrate to form an enzyme-substrate complex to cause a reaction.
Topic Explainer Video
Check out this @JoeDoesBiology video that explains the effect of pH on the rate of an enzyme controlled reaction, then read the study notes. Once you’ve gone through them, don’t forget to try the practice questions!
Method Overview
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Set up a water bath to keep the temperature constant (typically 37°C).
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Add 2 drops of iodine solution to each well in a spotting tile.
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Mix amylase enzyme and a buffer solution of known pH in a test tube.
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Put starch solution into another test tube.
The enzyme mixture and the starch solution should be put in the water bath separately so they can equilibrate to the same temperature before being mixed together.
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Add the starch solution to the enzyme and buffer test tube.
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Start a stopwatch and every 10 seconds, use a pipette to transfer a drop of the mixture into a well of iodine solution.
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Observe the colour change:
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Blue-black = starch still present.
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Orange / brown = starch digested.
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Record the time it takes for iodine solution to stop turning blue-black. When the iodine solution stops turning blue-black (and remains orange / brown) this tells us all the starch has been broken down into sugar.
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You can then repeat the investigation using different pH buffers- for example, pH 5, 9 and 14
Variables
|
Type |
Variable |
|
Independent |
pH of buffer solution |
|
Dependent |
Time taken for starch to be digested (time taken for iodine to stop going blue-black) |
|
Control |
Temperature, volume of all solutions, enzyme and substrate concentration. |
The experimental set up could also be changed to investigate the effect of temperature. Different water baths would be used at different temperatures and the pH would have to be controlled by using the same buffer solution in each test tube.
Improving precision and accuracy
To improve the precision of the rate of reaction repeat the experiment at least 3 times at each pH value and calculate the mean. Any anomalies can also be removed before calculating the mean.
To improve the accuracy of the optimum pH use smaller intervals between pH buffers, for example, use pH buffers of 2, 3, 4, 5, 6 etc.
Rate of Reaction
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The rate of reaction can be calculated by 1 ÷ time
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Identify the optimum pH (the fastest rate of reaction).
No answer provided.
Key Term
- Buffer solution - maintains a constant pH.
No answer provided.
Exam Tips
- Know all the variables for the experiment, how they are changed, measured and controlled.
- Include these when planning an investigation.
- Try to give at least 3 controlled variables. In this experiment you should control the volume of iodine in each well, the volume and concentration of enzyme and substrate as well as the temperature.
- Make sure to read the study note in the Exam Skills section on ‘Planning an Investigation’.
No answer provided.
Practice Question
Describe how you would investigate the effect of pH on the activity of amylase. (6 marks)
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Set up a spotting tile with iodine solution in each well.
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Add all solutions to a water bath to let the temperature equilibrate.
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Add amylase and starch to a test tube with a buffer solution of known pH.
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Start a stopwatch and transfer a sample to the iodine every 10 / 20 / 30 seconds.
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Repeat until the iodine no longer turns blue-black, recording the time.
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Repeat with different pH values using different buffer solutions.
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Repeat at least 3 times at each pH value and calculate the mean time.
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Compare the time taken to digest starch to determine which pH gives the fastest rate.
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Note- to get 6 marks, you have to include at least 3 control variables in your answer. For example - put the same volume of iodine into each well of the spotting tile, use the same volume of starch and amylase in each test tube, have the water bath set at the same temperature throughout.
More Practice
Try to answer the practice question from the TikTok video on your own, then watch the video to see how well you did!