2.3.7Structure and Functions in Living Organisms: Part 1
Practical: Investigating pH and Enzyme Activity
5 min read 6 parts Lesson 13 of 36 Unit quiz
Section 1 of 6The Idea Behind the Investigation
Enzyme and substrate
- Amylase is an enzyme that digests the substrate starch into the sugar maltose.
Independent variable
- The pH is changed by using buffer solutions, each of which keeps the mixture at a fixed pH.
Dependent variable
- The time taken for all the starch to be broken down is measured with a stopwatch.
Iodine test
- Iodine solution is yellow-brown and turns blue-black when starch is present, so it shows when digestion is finished.
Section 2 of 6Method
1
Put a drop of iodine solution into each well of a spotting tile.
2
Place amylase solution and a buffer of known pH, such as pH 3, into a test tube, and add starch solution while starting the stopwatch.
3
Every 10 seconds, use a pipette to transfer a drop of the mixture to a fresh well of iodine.
4
Continue until the iodine stays yellow-brown, which shows that no starch remains, and record that time.
5
Repeat with buffers at other values, such as pH 5, 7, 9 and 11, using fresh solutions each time.
Section 3 of 6Control Variables and Safety
Control Variables and Safety
Temperature
- Keep every mixture at the same temperature, for example in a water bath, because temperature also changes enzyme activity.
Volumes and concentrations
- Use the same volume and concentration of amylase and starch in every test so only pH differs.
Repeats
- Repeat each pH at least three times and calculate a mean to improve reliability.
Safety
- Wear eye protection because iodine and amylase solutions can irritate the eyes, and wear gloves if skin is sensitive.
Section 4 of 6Interpreting Results
Optimum pH
- The pH with the shortest time for the iodine to stay yellow-brown is the optimum, where the enzyme is most active.
Rate of reaction
- Rate can be calculated as 1 ÷ time, so a shorter time means a faster rate.
Away from the optimum
- At higher or lower pH values digestion is slower because the shape of the active site changes.
Denaturation
- Extreme pH values denature the enzyme permanently, so the substrate can no longer fit and starch is not digested.
| pH | Time for starch to disappear | Rate (1 ÷ time) |
|---|---|---|
| 3 | 200 s | 0.005 s⁻¹ |
| 7 | 40 s | 0.025 s⁻¹ |
| 11 | No change in 300 s | Almost 0 |
Section 5 of 6Worked Example
Question
- In an amylase investigation the iodine stayed blue-black for 200 s at pH 3 but turned yellow-brown after 40 s at pH 7.
- Calculate the rate at each pH and state which is closer to the optimum.
- Rate = 1 ÷ time.
- At pH 3: rate = 1 ÷ 200 s = 0.005 s⁻¹.
- At pH 7: rate = 1 ÷ 40 s = 0.025 s⁻¹.
- Compare the twothe rate at pH 7 is 0.025 ÷ 0.005 = 5 times faster.
Answer pH 7 is closer to the optimum because the rate is five times greater than at pH 3.
Section 6 of 6Exam Checklist & Tips
2.3.7 – Key Points for the Exam
- Enzyme A biological catalyst that speeds up a reaction without being used up.
- Optimum pH The pH at which an enzyme works at its fastest rate.
- Buffer solution A solution that keeps the pH of a mixture at a set value.
- Denaturation Permanent change in the shape of an enzyme's active site, so it can no longer work.
- Substrate The molecule on which an enzyme acts.
Common Mistakes
- Saying the enzyme is killed by extreme pH; enzymes are not alive, they are denatured.
- Failing to keep temperature and volumes the same, so the test is not fair.
- Reading the iodine result the wrong way round: blue-black means starch is still present.
- Using a single reading at each pH and calling the result reliable without any repeats.