Effects of Temperature and pH on Amylase and Pepsin Activity

Aim

This experiment investigates two related questions about enzyme activity: how temperature affects the rate at which amylase digests starch, and how pH affects the rate at which pepsin digests protein. Both parts test the idea that an enzyme's rate of reaction depends on the conditions around it, because heat and pH both affect the shape of the enzyme's active site.

Variables

• Manipulated variable (temperature test): temperature of the reaction mixture, set using water baths at about 10°C, 20°C, 37°C, 50°C and 70°C.
• Responding variable (temperature test): time taken for the iodine test to stop giving a blue-black colour, showing that the starch has been fully digested.
• Controlled variables (temperature test): pH of the mixture, volume and concentration of amylase and starch solution, and the interval between iodine tests.
• Manipulated variable (pH test): pH of the reaction mixture, set using buffer solutions such as pH 2, pH 4, pH 7 and pH 9.
• Responding variable (pH test): time taken for the egg albumin (protein) suspension to clear, showing that the protein has been digested.
• Controlled variables (pH test): temperature of the water bath, and the volume and concentration of pepsin solution and egg albumin suspension.

Materials and apparatus

• Amylase solution and starch solution
• Iodine solution and a white tile
• Pepsin solution and egg albumin (egg-white) suspension
• Buffer solutions at pH 2, pH 4, pH 7 and pH 9
• Water baths (or beakers of water) set at different temperatures, and a thermometer
• Test tubes, a test tube rack and a stopwatch
• Syringes, pipettes or droppers for measuring liquids
• A glass rod for transferring samples onto the tile

Procedure

1. Set up five water baths at about 10°C, 20°C, 37°C, 50°C and 70°C, and place a tube of starch solution and a tube of amylase solution into each to reach that temperature.
2. At the first temperature, mix the amylase and starch solution and start the stopwatch immediately.
3. At one-minute intervals, transfer a drop of the mixture onto a fresh spot of iodine solution on the tile.
4. Record the time the mixture stops turning blue-black with iodine, showing the starch is fully digested.
5. Repeat steps 2 to 4 at the other four temperatures, keeping the pH and the volume and concentration of amylase and starch constant.
6. Prepare four tubes of egg albumin suspension, adding an equal volume of a different buffer solution (pH 2, 4, 7 or 9) to each.
7. Add an equal volume of pepsin solution to each tube and place all four in a water bath at a constant temperature, such as 37°C.
8. Start the stopwatch and note the time taken for the cloudy albumin suspension to clear in each tube.
9. Repeat step 8 for each pH value, keeping the temperature and the volume and concentration of pepsin and albumin constant.
10. Record and tabulate the results of both the temperature test and the pH test.

Expected results

Starch is digested fastest at an optimum temperature and slowest, or not at all, at extreme temperatures, while protein is digested fastest at an optimum pH and slowest, or not at all, at pH values far from this optimum. The table below summarises the typical pattern seen in both parts of the experiment.

Conclusion

Both parts show that enzyme activity depends on the surrounding conditions. Amylase has an optimum temperature near normal body temperature, about 37°C, where enzyme and substrate molecules collide often enough for a fast reaction without the enzyme being damaged.

Pepsin has an optimum pH around 2, matching the acidic stomach conditions where it normally works. Away from these optima, digestion slows: at a low temperature or unsuitable pH the enzyme is simply less active, while at a high temperature or extreme pH its shape changes permanently, so the active site no longer fits the substrate and digestion stops.

Paper 3-style questions

Question 1, hypothesis. For the temperature part of this investigation, write a suitable hypothesis.

Model answer. As the temperature rises towards the optimum of about 37°C, the rate of starch digestion by amylase increases, so the time for the iodine test to stop turning blue-black gets shorter; above the optimum the rate falls again. A concise form is: the nearer the temperature is to the optimum, the faster the starch is digested.

Question 2, variables. State the manipulated, responding and one controlled variable for the pH part, and explain why that controlled variable must be fixed.

Model answer. Manipulated variable: pH of the reaction mixture. Responding variable: time for the egg-albumin suspension to clear.

Controlled variable: temperature of the water bath (or the volume and concentration of pepsin). It must be fixed so that any change in the clearing time is caused only by pH and not by a second factor, keeping the test fair.

Question 3, tabulation and graph. Describe how the temperature results should be tabulated and what graph should be plotted.

Model answer. Record temperature (°C) in the first column and time taken (s) in the next, with the unit in each heading and values to a consistent number of decimal places. Plot time taken on the y-axis against temperature on the x-axis, or plot rate (1 ÷ time) against temperature to give a clear peak at the optimum, and join the points with a smooth curve.

Question 4, inference. At 70°C the mixture stays blue-black throughout. Infer what has happened to the amylase and explain your reasoning.

Model answer. The amylase has been denatured: the high temperature has broken the bonds that hold the enzyme in shape, so its active site changes shape and no longer fits the starch. Starch is therefore not digested, so the iodine stays blue-black.

Denaturation is permanent, so activity does not return when the mixture is cooled.

Safety

• Move test tubes into and out of the hot water baths with a test-tube holder, and handle beakers of hot water with care to avoid scalds.
• Iodine solution stains skin, clothing and benches and irritates the eyes; wipe up spills at once and keep it away from your face.
• Wash your hands after handling egg albumin and pepsin, and clear the bench of spills.
• Report and clear broken glass straight away, and never pick up glass with bare hands.

Common mistakes

Source:SRC-DSKP-EN

Frequently asked questions