In 1909 the Danish chemist Søren Sørensen was studying enzymes at the Carlsberg Laboratory in Copenhagen. He found that how well they worked depended on how acidic the solution was, and to describe that he introduced the pH scale. What the "p" originally stood for is still disputed. The pH values on the buffer labels in this practical use his scale.
This lesson covers the enzymes required practical for AQA GCSE Biology Topic 2 (Organisation): the effect of pH on the rate of reaction of amylase. It's also useful for Combined Science.
What amylase does
Amylase is a carbohydrase: it breaks starch down into sugars. You can't see this happen, so you use iodine solution. Iodine turns from orange-brown to blue-black when starch is present, and stays orange-brown when the starch has gone.
The method
- Put one drop of iodine solution in each well of a spotting tile. Add a drop of starch solution to the first well: this blue-black "zero time" drop is your colour reference.
- Put starch solution, amylase solution and a buffer of known pH in separate tubes in a water bath (AQA's method uses 35°C). Let them reach that temperature.
- Add the buffer to the starch, then add the amylase and start the stopclock straight away. Stir.
- At regular intervals, take a drop of the mixture with a glass rod and put it on the next drop of iodine. Rinse the rod each time. The specification says to test every 30 seconds; AQA's student sheet samples every 10 seconds.
- Stop when the iodine stays orange-brown. Record the time.
- Repeat with buffers of other pH values, such as pH 5, 6, 7 and 8.
The variables
- Independent variable: pH, set with a buffer, which holds the mixture at a fixed pH.
- Dependent variable: the time taken for the starch to be completely broken down.
- Control variables: temperature, the volumes and concentrations of starch and amylase, the volume of buffer, and the sampling interval.
The water bath matters because temperature also affects enzymes. If it changed between tests, you couldn't tell whether pH caused the difference.
Turning time into rate
A short time means a fast reaction, so rate is the inverse of time:
rate = 1 ÷ time
If the starch is gone after 40 seconds, the rate is 1 ÷ 40 = 0.025 s⁻¹. Some revision guides use 1000 ÷ time instead, which gives larger, easier numbers to plot. Either way, the pattern on the graph is the same. If the starch never disappears in the time you allow, record that the reaction didn't finish rather than inventing a time.
The results graph
Plot pH on the x-axis and rate on the y-axis, and draw a smooth curve. The rate rises to a peak, then falls. The peak is the optimum pH, where amylase works fastest. AQA's teacher notes give pH 6 as the optimum for amylase, although AQA's own trial results in the same notes were fastest at pH 5. Different amylases have different optimums, so use the one your own results show.
Why pH matters: the active site
An enzyme works because its active site has a shape that fits its substrate, the lock and key model. Changing the pH away from the optimum changes the shape of the active site, so starch fits less well and the rate drops. At extremes of pH the shape changes so much that the substrate no longer fits. The enzyme is then denatured.
Safety
Wear eye protection, and remember that iodine solution stains skin and clothes. If you heat the water bath with a Bunsen burner, take care with the flame and the hot water.
Common exam mistakes
- "The enzyme is killed." Enzymes are protein molecules, not living things. Say the active site changes shape, or the enzyme is denatured.
- Mixing up time and rate. The longest time is the slowest rate.
- Saying the optimum is always pH 7. It depends on the enzyme. Read it from the graph.
- Calling temperature the independent variable. Here it's a control variable.
- Forgetting the limit of sampling. You only know the end point to within one interval.
Frequently asked questions
What is the independent variable in the enzyme pH practical?
The pH of the buffer solution. The dependent variable is the time for the starch to be broken down.
Why do you use iodine in the amylase practical?
Iodine shows whether starch is still there: blue-black means starch remains, and orange-brown means it has all been broken down.
How do you calculate the rate of reaction from time?
Divide 1 by the time in seconds. Some guides use 1000 ÷ time to give bigger numbers.
What happens to an enzyme at the wrong pH?
Its active site changes shape, so the substrate fits less well and the rate falls. At extreme pH the enzyme is denatured.
Try it
Say each answer before you flip the card. Then put yourself in Sørensen's place in 1909: you've found that acidity changes how fast an enzyme works. What would you have tested next?
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