Core practical: the effect of pH on enzyme activity

Core practical: the effect of pH on enzyme activitySpec 1.10

A common version of this investigation uses amylase, an enzyme that breaks down starch, and iodine solution to show when the starch has gone.

1. Put drops of iodine solution into the wells of a spotting tile.
2. Add starch solution and a buffer solution of a known pH to a test tube. Put this tube, and a tube of amylase solution, in a water bath at a set temperature (for example 35 °C) for a few minutes.
3. Add the amylase solution to the starch and buffer, mix and start the stopwatch.
4. At regular intervals, for example every 30 seconds, use a dropping pipette to add a drop of the mixture to a well of iodine solution.
5. Record the time when the iodine solution stops turning blue-black and stays orange-brown. This shows all the starch has been broken down.
6. Repeat for buffer solutions of different pH values, and repeat each pH to calculate a mean.

Typical results: the shortest time is at the optimum pH, where the enzyme works fastest. Times get longer at pH values further from the optimum. Plot the rate (or time) against pH on a graph and draw a smooth curve through the points. Wear eye protection, because iodine solution and some buffers are irritants, and wipe up any spills.