Practical 3: pH and enzyme activity

Aim

To investigate how pH affects the time taken for amylase to break down starch.

Background

Amylase is an enzyme that breaks down starch. Like all enzymes it has an active site whose shape is held by bonds between the amino acids of the protein. The substrate fits into the active site, so the shape must be right for the enzyme to work.

Each enzyme works best at its optimum pH. As the pH moves away from the optimum, the extra hydrogen ions or hydroxide ions interfere with the bonds that hold the enzyme in shape, so the active site changes shape and the substrate fits less well. At extreme pH values the enzyme is denatured and the substrate no longer fits.

A buffer solution keeps its pH almost constant when the enzyme and starch are added. The time for all the starch to be broken down is found by adding samples of the mixture to iodine solution, which is blue-black with starch and stays orange-brown when no starch is left.

Hypothesis

Amylase will break down starch in the shortest time at about pH 7, and will take longer at lower and higher pH values because the active site changes shape and the enzyme is denatured.

Variables

Equipment

• Starch solution (1%), 2 cm³ per tube
• Amylase solution (1%), 2 cm³ per tube
• Buffer solutions at pH 3, 5, 7, 9 and 11, 1 cm³ per tube
• Iodine solution in a dropping bottle
• Spotting tile with 6 or more wells
• Water bath at 35 °C (a 250 cm³ beaker of water on a thermostatic heater, or hot and cold water) and a thermometer
• Test tubes, test tube rack and marker pen
• Syringes or graduated pipettes (2 cm³ and 5 cm³)
• Dropping pipettes or glass rods for sampling
• Stopwatch or timer
• Eye protection

Risk assessment

Method

1. Put on eye protection. Set up a water bath at 35 °C and check the temperature with a thermometer.
2. Put one drop of iodine solution into each well of a spotting tile (at least 6 wells for each pH).
3. Label a test tube for each pH. Put 2 cm³ of starch solution into each tube, then add 1 cm³ of the matching buffer solution.
4. Label a second set of tubes and put 2 cm³ of amylase solution in each.
5. Stand the starch and buffer tube for the first pH and its tube of amylase in the water bath for 5 minutes.
6. Pour the amylase into the starch and buffer tube, mix, return it to the water bath and start the stopwatch at once.
7. After 30 seconds, use a clean dropping pipette to transfer one drop of the mixture into the first well of iodine solution. Record the colour.
8. Take a further drop every 30 seconds and add it to the next well. Continue until the iodine stays orange-brown.
9. Record the time when the iodine first stays orange-brown. If it is still blue-black after 20 minutes, record '>20 min'.
10. Repeat steps 5–9 for each of the other pH values, using fresh tubes and a clean pipette each time.
11. Repeat the whole investigation at least twice more and calculate the mean time at each pH.
12. Calculate the rate at each pH as 1 ÷ mean time (min⁻¹) and plot a graph of rate against pH.

Results

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

Drawing the graph

Plot rate (1 ÷ mean time, min⁻¹) on the y-axis against pH on the x-axis. Mark each point with a cross and draw a smooth curve of best fit through them. Read off the pH with the highest rate: this is the optimum pH for the amylase to the accuracy of the pH values tested.