Core practical 2: The effect of pH on enzyme activity

Aim

To investigate how pH affects the time taken for amylase to break down starch, and to find the optimum pH for the enzyme.

Background

Enzymes are biological catalysts. Each enzyme has an active site with a specific shape that fits its substrate. Amylase is an enzyme that breaks down the substrate starch into the sugar maltose.

The pH of the solution affects the bonds that hold the enzyme in shape. At an extreme pH the shape of the active site changes, the substrate no longer fits and the enzyme is denatured. Each enzyme works fastest at its optimum pH.

Starch turns iodine solution from orange-brown to blue-black. When all of the starch has been broken down, a drop of the mixture no longer turns iodine blue-black. The time taken for this to happen is a measure of the enzyme activity: the shorter the time, the faster the reaction.

A buffer solution keeps the pH of the mixture constant, so pH is the only thing that changes. The rate of reaction can be calculated from the time: rate = 1 ÷ time.

Hypothesis

The starch will be broken down fastest at about pH 7, because amylase from saliva has an optimum pH close to neutral. At pH values further from this, the shape of the active site will change and the enzyme will work more slowly.

Variables

Equipment

• Amylase solution, 1% (2 cm³ per test)
• Starch solution, 1% (2 cm³ per test)
• Buffer solutions at pH 4, 5, 6, 7, 8 and 9 (2 cm³ per test)
• Iodine solution in a dropper bottle
• Spotting tile
• Water bath set at 35 °C
• Thermometer
• Test tubes and test tube rack
• Three 5 cm³ syringes, or graduated pipettes, for measuring volumes
• Dropping pipettes or glass rods
• Stopwatch
• Marker pen for labelling tubes
• Eye protection

Risk assessment

Method

1. Put on eye protection. Set the water bath to 35 °C and check with a thermometer.
2. Label six test tubes with the pH values 4 to 9. Put 2 cm³ of starch solution in each tube and add 2 cm³ of the matching buffer solution.
3. Label six more test tubes with the pH values and put 2 cm³ of amylase solution in each.
4. Put drops of iodine solution into the wells of a spotting tile. Use one row of wells for each pH, with a drop in every well.
5. Stand the tubes in the water bath for 5 minutes, so that the contents reach 35 °C.
6. Start with pH 7. Add the 2 cm³ of amylase to the starch and buffer mixture, mix with a clean dropping pipette and start the stopwatch at once.
7. After 10 s, use the dropping pipette to put one drop of the mixture into the first iodine well. Record the colour.
8. Repeat every 10 s with a new well each time, until the drop of the mixture no longer turns blue-black (it stays orange-brown).
9. Record the time when the iodine first stays orange-brown. This is the time taken for all the starch to be broken down.
10. Rinse the pipette. Repeat steps 6 to 9 for each of the other pH values, using the correct tubes and a fresh row of iodine wells each time.
11. Repeat the whole experiment at least once more and calculate the mean time for each pH.
12. Calculate the rate of reaction for each pH: rate = 1 ÷ mean time, in s⁻¹. To avoid very small numbers, multiply by 1000 and record the rate in ×10⁻³ s⁻¹ (for example, 1 ÷ 40 s = 0.025 s⁻¹ = 25 × 10⁻³ s⁻¹).
13. Plot a graph of rate against pH and draw a smooth curve through the points.

Results

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

Drawing the graph

Plot a line graph with pH on the x-axis (4 to 9) and rate of reaction (×10⁻³ s⁻¹) on the y-axis, starting from zero. Plot each mean point with a small cross and join them with a smooth curve (not ruled lines between points). The peak of the curve shows the optimum pH.