Enzymes required practical

Aim: To investigate how pH affects the rate at which amylase breaks down starch, using iodine to detect when the starch has gone.

Variables

• Independent: pH of the buffer solution.
• Dependent: Time taken for the starch to be completely broken down (iodine stays orange-brown).
• Control: Temperature of the water bath; Concentration and volume of amylase; Concentration and volume of starch; Volume of buffer and the interval between samples

Equipment

• Spotting tile
• Iodine solution in a dropping bottle
• Amylase solution
• Starch solution
• Buffer solutions (e.g. pH 5, 6, 7, 8, 9)
• Water bath set at 35 °C
• Test tubes and rack
• Syringes or measuring cylinders
• Stopwatch
• Pipettes and a glass rod

Method

1. Put one drop of iodine solution into each well of a spotting tile.
2. Place a test tube containing amylase and a separate tube containing starch solution in the water bath for about five minutes so they reach 35 °C.
3. Add a measured volume of the first pH buffer to the starch tube.
4. Pour the amylase into the starch and buffer mixture, mix and start the stopwatch straight away.
5. Every 30 seconds, use a pipette to transfer a drop of the mixture to the next well of iodine.
6. Continue until the iodine no longer turns blue-black and stays orange-brown; record this time.
7. Repeat with each of the other pH buffers, using clean equipment each time.
8. Repeat the whole set to get at least three results at each pH and calculate means.

Safety

• Wear eye protection because iodine is an irritant and amylase can cause allergic reactions.
• Wash any enzyme off the skin immediately.
• Take care with hot water baths and avoid spilling water near electrical equipment.

Results and calculations

Record the time for starch to disappear at each pH and calculate a mean. Work out the rate of reaction as rate = 1000 ÷ time (units s^-1) and plot rate (or time) against pH to find the optimum pH, which gives the fastest rate.

Common mistakes and improvements

• Sampling only every 30 seconds limits resolution; take samples more often, such as every 10 seconds.
• Judging the end colour is subjective; compare against a control well containing starch and iodine only.
• Solutions not at the right temperature when mixed; allow them to equilibrate in the water bath first.
• Cross-contamination between samples; rinse the pipette between drops or use a new one.

Exam tips

• Explain the shape of the graph: rate rises to an optimum pH, then falls as the active site changes shape (denatures) and the substrate no longer fits.
• Explain why the solutions are kept at the same temperature and why a buffer is used: to control variables that also affect enzyme activity.
• Use rate = 1000 ÷ time or 1 ÷ time and give units correctly.
• Suggest improvements, such as testing more pH values close to the optimum or using a colorimeter for a more objective end point.
• Link the practical to the lock and key model and to the digestive system, where different enzymes work best at different pH values.