Testing the effect of pH on potato catalase: what the boiled control reveals
A student investigates the effect of pH on the enzyme catalase. 5 g of potato is ground with 20 cm³ of distilled water and filtered to make an extract. For each pH, 2 cm³ of extract is added to a test tube containing 10 cm³ of 1 % hydrogen peroxide and 5 cm³ of buffer solution at that pH. After exactly 2 minutes the height of the foam in the tube is measured with a ruler. The results are:
| pH | 3 | 5 | 7 | 9 | 11 |
|---|---|---|---|---|---|
| Foam height / cm | 0.4 | 3.1 | 6.8 | 2.6 | 0.9 |
The student then repeats the experiment at pH 7 using extract that has been boiled for 5 minutes and cooled, and records a foam height of 1.1 cm. She concludes that "catalase works best at pH 7 and is denatured at pH 11". A classmate says foam height is a poor measure because foam collapses when a lot of gas is produced.
1. What should the boiled extract have given, and what does the 1.1 cm result tell you about the reliability of the other readings?
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Boiling denatures catalase: the hydrogen bonds and other weak bonds holding the active site's shape break, the active site no longer fits hydrogen peroxide, and no enzyme-controlled reaction occurs. The boiled tube should therefore have shown no foam above the baseline.
Getting 1.1 cm means gas and foam appear even without working enzyme. Hydrogen peroxide breaks down slowly on its own, and adding and mixing the solutions creates foam. So roughly 1 cm is the floor of the method, not a measurement of enzyme activity.
The consequence is serious for the interpretation. The pH 3 reading (0.4 cm) and the pH 11 reading (0.9 cm) are at or below that floor, so the data cannot show that any enzyme activity occurred at those pH values - they are indistinguishable from zero. Only pH 5, 7 and 9 lie clearly above the baseline. Without the boiled control the student would have read the small foam heights as "a little activity", when the honest reading is "no detectable activity". A control is what allows that judgement to be made.
2. Is the claim that catalase is "denatured at pH 11" supported by this experiment? Explain, and describe a test that would settle it.
Read the worked answer
It is not supported as stated. The data show only that there is no detectable activity at pH 11. Extreme pH can alter the ionic bonds that hold the active site in shape, but that change may be reversible or irreversible - the experiment cannot tell them apart, and "denatured" specifically means irreversible loss of shape.
Test: hold extract at pH 11 for 5 minutes, then add buffer to bring it back to pH 7 and measure foam height in the usual way. If activity returns close to the 6.8 cm seen for fresh extract at pH 7, the change at pH 11 was reversible, so the enzyme was inhibited, not denatured. If it stays near the 1.1 cm baseline, the shape change was permanent and "denatured" is justified.
A second comparison helps: boiled extract at pH 7 should sit at the baseline. Anything above that is evidence of active enzyme. The word "denatured" should be used only when the loss of activity has been shown to be permanent.
3. How does the classmate's point about collapsing foam affect the shape of the pH curve, and what should be measured instead?
Read the worked answer
Foam is a gas trapped in liquid, and it breaks down over time. When gas is produced quickly - as at pH 7 - bubbles burst as fast as they form, so the height at 2 minutes under-records the true rate. Where the reaction is slow, the small amount of foam produced has time to stand and is recorded more faithfully.
The result is that the peak is flattened and the curve looks broader than it really is. The true optimum may be sharper and higher than the graph suggests, and the difference between pH 5, 7 and 9 is underestimated. The optimum pH value itself is probably still roughly right because pH 7 is clearly the highest, but the sizes of the differences cannot be trusted.
Better measure: collect the oxygen in a gas syringe and read the volume after 60 seconds, or time how long it takes to collect a fixed volume (say 20 cm³). Both measure gas produced rather than gas retained, and both give numbers that can be plotted and compared.