Practical skill 4: Effect of temperature and pH on enzyme activity (amylase)

Aim

To investigate how temperature and pH affect the activity of the enzyme amylase by timing how long it takes to break down starch.

Background

Enzymes are proteins that are involved in all metabolic reactions, where they function as biological catalysts. Amylase is an enzyme that breaks down starch (the substrate) to maltose. Each enzyme has an optimum temperature and an optimum pH at which its activity is highest.

Starch gives a blue-black colour with iodine solution. When all the starch has been broken down, a drop of the mixture stays orange-brown when it is added to iodine solution. The time taken to reach this end point is a measure of enzyme activity: the shorter the time, the faster the reaction. The rate of reaction can be calculated as 1000 ÷ time.

Below the optimum temperature the enzyme and substrate molecules have less kinetic energy, so there are fewer effective collisions and the rate is low. At temperatures above the optimum the shape of the active site changes and the substrate no longer fits: the enzyme is denatured. A pH that is too high or too low also changes the shape of the active site and can denature the enzyme.

Hypothesis

The rate of starch breakdown will increase with temperature up to an optimum temperature of about 40 °C and then decrease, because the enzyme is denatured above the optimum. The rate will be highest at about pH 7 and lower at more acidic and more alkaline pH values.

Variables

Equipment

• Starch solution, 1%, 100 cm³; amylase solution, 1%, 40 cm³
• Buffer solutions at pH 4, 5, 6, 7, 8 and 9 (20 cm³ of each)
• Iodine in potassium iodide solution (iodine solution) in a dropper bottle
• Spotting tile (or white tile with drops of iodine); dropping pipettes
• Large test-tubes (150 mm × 25 mm) for the starch and buffer, standard test-tubes (125 mm × 15 mm) for the amylase, and a rack; syringes, 5 cm³ and 10 cm³
• Beakers, 250 cm³, to use as water baths; kettle or hot water, and ice; thermometer, –10 °C to +110 °C
• Stop-clock reading to 1 s; glass rod or stirring rod; marker pen; eye protection

Risk assessment

Method

1. Put on eye protection. Put a drop of iodine solution in each well of a spotting tile (have a second tile ready, because up to 20 samples may be needed).
2. Use a syringe to put 5 cm³ of starch solution and 5 cm³ of pH 7 buffer into a large test-tube. In a standard test-tube put 2 cm³ of amylase solution.
3. Part A (temperature): set up a water bath in a 250 cm³ beaker at the first temperature (10 °C, using ice). Stand both tubes in the water bath for 5 minutes so they reach the temperature of the bath. Check the temperature with a thermometer.
4. Pour the amylase into the starch and buffer, mix with a clean glass rod and start the stop-clock. Keep the tube in the water bath.
5. After 30 seconds, use a clean dropping pipette to take one drop of the mixture and add it to a drop of iodine solution on the tile. Record the colour.
6. Repeat every 30 seconds with a clean pipette and a new drop of iodine each time until the iodine stays orange-brown. Record this time as the time for the starch to be broken down.
7. If the iodine is still blue-black after 600 s (10 minutes), stop and record the time as '> 600'.
8. Repeat steps 2 to 7 at 20, 30, 40, 50 and 60 °C, using fresh solutions each time. Keep the water bath within 1 °C of the temperature by adding hot or cold water.
9. Part B (pH): use a water bath at 35 °C and repeat steps 2 to 7, using buffers at pH 4, 5, 6, 7, 8 and 9 instead of the pH 7 buffer.
10. Calculate the rate of reaction for each value, using rate = 1000 ÷ time (use a rate of 0 if the end point was not reached).
11. Plot graphs of rate of reaction against temperature and against pH. Use each graph to find the optimum.
12. Repeat the investigation at least twice more and calculate mean times for each value.

Results

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

Drawing the graph

Plot rate of reaction / arbitrary units (y-axis) against temperature / °C (x-axis). Mark points with crosses (×), use more than half of the grid in both directions and draw a single smooth best-fit curve (not straight lines between points). Read the optimum temperature from the highest point of the curve. Do the same for rate of reaction against pH in Part B.