Investigating Enzymes and pH

Explained

Changing the pH and watching the rate

This practical is the temperature investigation with one variable swapped. Instead of changing the temperature and holding the pH steady, you change the pH and hold the temperature steady.

The method

Put drops of iodine solution in the wells of a spotting tile. In a test tube, mix amylase, starch and a buffer solution at your chosen pH, and start a stopwatch.

A buffer is a solution that holds the pH at a set value even as the reaction proceeds. Without one, the pH would drift and you would not know what you had actually tested.

Every thirty seconds, take a drop of the mixture and add it to a fresh well. While starch remains the iodine turns blue black; when it stays orange brown, the starch has all been digested. Record that time, then repeat the whole thing with buffers at pH 3, 5, 7, 9 and 11.

Turning time into rate

A shorter time means a faster reaction, so calculate the rate as one divided by the time taken. The numbers then rise as the reaction gets faster, and the graph peaks at the optimum instead of dipping, which is much easier to read.

The results

Rate is low at low pH, rises to a maximum, and falls away again at high pH. The peak is the optimum pH, which for amylase is about 7.

The explanation is the same mechanism as for temperature, but with a different cause. An enzyme's active site has a specific shape held together by bonds within the protein. Extremes of pH break those bonds, so the active site changes shape, the substrate no longer fits, and no enzyme substrate complexes form. The enzyme has been denatured, and the change cannot be undone by returning the pH to normal.

Note what is different from the temperature curve. There, the rise before the optimum was caused by increasing kinetic energy and more frequent collisions. Here, moving from pH 3 towards pH 7 does not make anything move faster; it simply un-distorts the active site, so more enzyme molecules are working properly. Using the kinetic energy explanation on a pH graph is a common way to lose the mark.

Making it fair

Only the pH should change. Keep the volume and concentration of amylase and starch the same, keep the temperature the same by using a water bath, and use the same volume of buffer each time.

Bring the solutions to the test temperature before mixing them, and keep the sampling interval the same throughout, since the interval limits how precisely you can locate the end point. Repeat each pH and take a mean.

Why different enzymes differ

Optimum pH depends on where the enzyme normally works. Amylase in the mouth and small intestine works best near neutral. Protease in the stomach has an optimum around pH 2, because the stomach produces hydrochloric acid.

So if a question gives you an enzyme with an unusual optimum, the location is the explanation, and saying which organ it comes from is often the mark.