Required practical 5: The effect of pH on amylase

Aim

To find how pH affects the time taken for amylase to digest starch, using a continuous sampling technique with iodine solution.

Background

Enzymes are biological catalysts. Amylase breaks down starch into sugars (maltose). Each enzyme has an active site with a shape that fits its substrate. The substrate must collide with the enzyme and fit into the active site for the reaction to happen.

Each enzyme works best at a particular pH, called its optimum pH. If the pH is too high or too low, the bonds that hold the enzyme in shape break and the active site changes shape. The substrate no longer fits and the enzyme is denatured.

To follow the reaction, you test the mixture for starch using iodine solution. Starch turns iodine from orange-brown to blue-black. When all of the starch has been digested, the iodine stays orange-brown. This is called a continuous sampling technique, because you take samples from the same reaction mixture repeatedly. The shorter the time, the faster the rate of reaction.

Hypothesis

Amylase will digest the starch fastest at about pH 7 and more slowly at higher and lower pH, because the active site of the enzyme is changed when the pH is not at the optimum.

Variables

Equipment

• 1% amylase solution
• 1% starch solution
• Buffer solutions of pH 4, 5, 6, 7 and 8
• Iodine solution in a dropping bottle
• Spotting tile (or a white tile with dimples)
• Test tubes, a test tube rack and a marker pen
• Water bath set to 35 °C and a thermometer
• Syringes (or graduated pipettes) for measuring 2 cm³, one for each solution
• Dropping pipettes or glass rods for transferring samples
• Stopwatch
• Eye protection

Risk assessment

Method

1. Set a water bath to 35 °C. Using a pipette, put one drop of iodine solution into each dimple of a spotting tile (as many dimples as the number of samples you will take).
2. Use a syringe to put 2 cm³ of starch solution into a test tube. Use a different syringe to add 2 cm³ of pH 4 buffer solution. Label the tube with the pH.
3. In a second test tube, put 2 cm³ of amylase solution.
4. Stand both test tubes in the water bath for 5 minutes so that the solutions reach 35 °C.
5. Pour the amylase into the starch and buffer tube, mix by gently swirling and start the stopwatch straight away. Keep the tube in the water bath.
6. After 30 seconds, use a clean dropping pipette to remove one drop of the mixture. Add it to a drop of iodine solution on the spotting tile and record the colour.
7. Take another sample every 30 seconds and add it to a fresh drop of iodine. Continue until the iodine stays orange-brown. This is the end point.
8. Record the time at which the iodine first stayed orange-brown. If it is still blue-black after 10 minutes, record '> 600'.
9. Repeat steps 2 to 8 using buffer solutions of pH 5, 6, 7 and 8. Use clean syringes and put fresh drops of iodine on a clean spotting tile each time.
10. Repeat the whole experiment two more times so that you have three results for each pH.
11. Calculate the mean time for each pH. Calculate the rate of reaction for each pH: rate = 1 ÷ mean time, in s⁻¹.
12. Plot a graph of rate of reaction against pH. Draw a smooth curve through the points and read off the optimum pH.

Results

Fill this table in as you go. Print the PDF for a copy to write on.

Drawing the graph

Plot a line graph. Put the pH of the buffer on the x-axis (4 to 8) and the rate of reaction (1 ÷ mean time, in s⁻¹) on the y-axis. Plot each point with a small cross and draw a smooth curve (not straight lines joining the points) that rises to a peak and falls again. Mark the optimum pH at the peak of the curve.