Animal Tissues, Organs and Systems » Required Practical: pH and the Rate of Amylase Activity

The aim of the practical

You already know that amylase breaks down starch and that enzymes work best at a certain pH. In this practical you test that idea yourself. The question is: how does pH affect the rate at which amylase digests starch?

A good hypothesis is: amylase works fastest at one pH and slower at pHs above and below it, because a pH that is too high or too low changes the shape of the active site.

Key terms:

• Continuous sampling: taking small samples from one reaction mixture again and again, at regular times, to test it.
• Buffer solution: a liquid that keeps the pH steady at a set value.
• End point: the moment the test shows the reaction is finished. Here it is when the iodine no longer turns blue-black.

The variables

The spec says the temperature must be controlled using a water bath or an electric heater. Even a small change in temperature changes the rate, so a steady water bath (for example 35 °C) is important.

The method

1. Put a drop of iodine solution into each well of a spotting tile. Use one row for each pH.
2. Label a test tube for each pH. Add the same volume of starch solution and the same volume of buffer solution to each tube. Use buffers of known pH, and record the pH of each tube alongside its time.
3. Stand the tubes, and a separate tube of amylase solution, in the water bath. Leave them for a few minutes to reach the temperature of the bath.
4. Add a measured volume of amylase to the first tube. Start the stopwatch straight away and mix.
5. After 30 seconds, use a dropper or pipette to take a small sample and add it to the first drop of iodine on the tile. Note the colour.
6. Repeat every 30 seconds. Use a clean pipette each time, or rinse it, so you do not carry amylase from one pH to another.
7. Stop when the iodine stays orange-brown. This is the end point. Record the time.
8. Repeat the whole method for each pH, then repeat the investigation to check your results.

Reading the results

At the start the sample turns blue-black because starch is present. As amylase digests the starch, the colour gets weaker, and then the sample stays orange-brown. That means no starch is left.

The faster the colour disappears, the faster the enzyme is working. A tube at the optimum pH reaches the end point in the shortest time. A tube at a pH that is too high or too low takes longer, or may never reach the end point.

If the iodine is still blue-black when you stop, write 'more than' and the time (for example, more than 600 s). Do not guess a time.

Calculating the rate

Time and rate are opposites. A short time means a fast reaction. So the rate is worked out from the time:

rate = 1 ÷ time (units: s^-1, if the time is in seconds)

Drawing the graph

Plot a graph of amylase activity (rate) on the y-axis against pH on the x-axis. Choose a sensible scale, label both axes with units and plot each point carefully. Join the points with a smooth curve or straight lines drawn with a ruler, as the question asks.

You should see a peak. The pH at the top of the peak is the optimum pH for this amylase. The rate falls on both sides of the peak.

Safety and sources of error

• Wear eye protection. Iodine solution stains skin and clothes.
• Take care with the hot water bath and with electrical equipment near water.
• Wash your hands after handling enzyme solutions.

• Judging the end point: the colour change is gradual, so two people may disagree about when it stops. Use the same person and a white tile.
• Time gaps: samples every 30 seconds mean the true time could be up to 30 seconds earlier. A shorter gap gives a more accurate time.
• Temperature drift: the water bath may not stay exactly constant, so check it with a thermometer.
• Measuring volumes: use a syringe or pipette, not a beaker.

Exam-style question

A student investigates the effect of pH on the activity of amylase. She mixes starch solution, buffer solution and amylase at 35 °C. Every 30 seconds she tests a sample with iodine solution.

(a) What colour does the iodine turn when starch is present? [1 mark]

(b) How does the student know when all the starch has been digested? [1 mark]

(c) The starch was digested in 150 s at pH 6. Calculate the rate of reaction in s^-1. [2 marks]

(d) Give two variables the student must control. [2 marks]