Practical: temperature, pH and enzyme activitySpec 2.12, 2.14B
In short
The effect of temperature or pH on enzyme activity can be investigated using amylase and starch. Drops of the mixture are added to iodine solution every 30 seconds, and the time is recorded when the iodine stays orange-brown, showing all the starch is broken down. The shortest time, and so the fastest rate, is at the optimum.
A common version of these investigations uses amylase, an enzyme that breaks down starch, and iodine solution to show when the starch has gone. Iodine stays orange-brown when there is no starch left.
Effect of temperature on enzyme activity
Independent variable: temperature; dependent variable: time for the starch to be broken down. Keep pH, volumes and concentrations the same each time.
- Put drops of iodine solution into the wells of a spotting tile.
- Set up water baths at different temperatures, for example 10, 20, 30, 40 and 50 °C.
- Put starch solution and a buffer solution of one fixed pH in a test tube, and amylase solution in a second tube. Leave both in the water bath for a few minutes to reach the temperature.
- Add the amylase to the starch and buffer, mix and start the stopwatch.
- Every 30 seconds, use a dropping pipette to add a drop of the mixture to a well of iodine solution.
- Record the time when the iodine stays orange-brown. This shows all the starch has been broken down.
- Repeat at each temperature and calculate a mean time.
Typical results: the shortest time is at the optimum temperature. The time is longer at low temperatures because there are fewer collisions, and at high temperatures the enzyme is denatured so the starch may never be broken down. Calculate the rate as 1 ÷ time and plot it against temperature. Take care with hot water, and wear eye protection because iodine solution is an irritant.
| Variable | How it is controlled |
|---|---|
| pH | Add the same buffer solution to every tube |
| Volume and concentration of enzyme | Use the same amount of the same solution each time |
| Volume and concentration of starch | Use the same amount of the same solution each time |
Biology only (what this means)
Biology only: only in International GCSE Biology. Double Award students can skip it. What the labels meanEffect of pH on enzyme activity
Biology only (what this means)
Biology only: only in International GCSE Biology. Double Award students can skip it. What the labels meanIndependent variable: pH, using buffer solutions; dependent variable: time for the starch to be broken down. Keep temperature, volumes and concentrations the same each time.
Biology only (what this means)
Biology only: only in International GCSE Biology. Double Award students can skip it. What the labels meanUse the same method, but keep the temperature fixed by putting all the tubes in one water bath (for example at 35 °C), and use buffer solutions of different pH values, such as pH 3, 5, 7, 9 and 11. Repeat each pH and calculate a mean.
Biology only (what this means)
Biology only: only in International GCSE Biology. Double Award students can skip it. What the labels meanTypical results for the pH practical: the shortest time is at the optimum pH, where the enzyme works fastest. Times get longer at pH values further from the optimum. Plot the rate (1 ÷ time) against pH and draw a smooth curve through the points.
Quick check
Which elements are present in proteins but not in carbohydrates or lipids?
Show answer
Nitrogen (and sometimes sulfur).
What are the smaller units that make up a protein?
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Amino acids.
What colour does Benedict's solution turn with glucose when heated?
Show answer
From blue to green, yellow, orange or brick-red.
What is meant by a biological catalyst?
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An enzyme that speeds up a reaction in a living thing without being used up.
Why does a very high temperature stop an enzyme working?
Show answer
The shape of the active site changes, so the substrate no longer fits and the enzyme is denatured.
Written and checked against the Edexcel IGCSE Science Double Award (4SD0) specification · Updated October 2026