Catalase and the pH conclusion that outran the data
A student investigates the effect of pH on catalase activity in potato tissue. She cuts 1 g of potato into discs, adds them to 5 cm³ of 3% hydrogen peroxide adjusted to pH 4, 6, 7, 8 or 10 with buffer solution, and records the volume of oxygen collected in a gas syringe after 60 seconds. Each pH is repeated three times. At pH 7 the volumes were 40, 42 and 61 cm³; at pH 10 they were 2, 3 and 4 cm³. She used potato discs cut from three different potatoes, and some discs were bruised. The timer was started before the syringe was sealed. No control tube without buffer was included. She concludes that the optimum pH for catalase is 7 and that the enzyme is denatured at pH 10. She writes that pH 7 is 'the best pH' without further comment.
1. Identify two variables that were not adequately controlled in this investigation and explain how each could have changed the measured oxygen volume.
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Any two of the following, with a mechanism:
- Potato mass and surface area. Catalase is in the tissue; more tissue or more cut surface means more enzyme available, so more hydrogen peroxide breaks down and more oxygen is collected. The discs came from three potatoes and were not standardised.
- Temperature. Enzyme-catalysed reactions rise with temperature up to the optimum. If tubes warmed differently, the rate could change even at the same pH. The setup gives no evidence that all tubes were at the same temperature.
- Timing. Starting the timer before sealing the syringe means some oxygen escaped or was not counted; the 60-second period is therefore not exactly the same reaction time for each tube.
- Potato condition. Bruised tissue may have damaged cells and different enzyme activity, adding variation.
A controlled variable must be held constant because it could otherwise explain the result; simply naming it is not enough.
2. Evaluate the conclusion that pH 7 is the optimum and that catalase is denatured at pH 10.
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The pH 7 results are 40, 42 and 61 cm³; the mean is 47.7 cm³, but the 61 cm³ repeat is far above the other two. Without it the mean is 41 cm³, still the highest of the five pH values, so pH 7 is the best-supported optimum among those tested. However, only five pH values were used, so the true optimum could lie between 6 and 8; 'the best pH' is not a scientific statement because it does not define the variable or the condition. The pH 10 results (2, 3, 4 cm³) are low but not zero. A low rate does not prove denaturation: the enzyme may be reversibly inhibited or only partly active. Denaturation would need evidence such as no recovery after restoring pH, or a structural test. The conclusion overstates the data.
3. Suggest two improvements and state what each would improve.
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- Use one potato and cut equal-mass, equal-thickness discs with a cork borer. This standardises enzyme quantity and surface area, reducing variation between repeats.
- Start timing only after the syringe is sealed and use a water bath at a fixed temperature (e.g. 25 °C). This makes reaction time and temperature consistent, so pH is the only intended variable.
Additional improvements: use a dissolved-oxygen probe or pressure sensor rather than reading a syringe, and repeat at more pH values (e.g. every 0.5 unit between 5 and 9) to locate the optimum more precisely. A control without buffer or with boiled potato would show whether the oxygen is enzyme-derived.