Catalase in Potato Extract: Denaturation or Just Slowing?
A student grinds 20 g of fresh potato, filters the extract through cheesecloth, and keeps it on ice. For each trial she adds 1 mL of extract to a tube containing 5 mL of 3% hydrogen peroxide held at the stated temperature and counts the oxygen bubbles collected in an inverted graduated tube over two minutes.
Results:
- 4 °C: 8 bubbles in the first 15 s, then a steady 10-12 per 15 s for the full two minutes.
- 22 °C: 30 bubbles in the first 15 s, falling to about 12 per 15 s by minute two.
- 37 °C: 60 bubbles in the first 15 s, about 40 per 15 s after that; the highest two-minute total of the series.
- 60 °C: 50 bubbles in the first 15 s, 4 in the next 15 s, then none for the rest of the run.
- 80 °C: 2 bubbles in two minutes.
- 37 °C buffered to pH 2: fewer than 3 bubbles in two minutes.
The student must explain what each result shows about the enzyme.
1. Which treatment is evidence of denaturation rather than simple slowing, and what single follow-up experiment would confirm your interpretation?
Read the worked answer
The 60 °C tube. Its activity does not merely decline, it collapses: 50 bubbles in the first 15 s - faster than the 22 °C tube ever managed - then 4, then nothing, and it does not recover while held at 60 °C. Cold slowing looks different: the 4 °C tube starts slowly (8 bubbles) but holds a steady 10-12 per 15 s for two minutes.
To confirm, cool the 60 °C tube back to 37 °C, add fresh 3% hydrogen peroxide (the original substrate is largely spent), and measure again. A denatured enzyme stays inactive; the 4 °C tube rewarmed to 37 °C should recover the full burst. Substrate replenishment matters, otherwise a dead tube and a used-up tube look identical.
2. Why did the 37 °C tube outpace the 22 °C tube, and why did pH 2 at the same 37 °C nearly abolish activity? Would you expect the pH effect to reverse?
Read the worked answer
Heat raises molecular motion, so enzyme and substrate collide more often and more collisions carry enough energy to react; 37 °C is closer to this enzyme's optimum, which the data show.
Extreme pH changes which R groups are protonated, breaking the ionic and hydrogen bonds that hold the folded tertiary structure. The active site changes shape and hydrogen peroxide no longer fits. Whether it reverses depends on how long the enzyme sat at pH 2: short exposure is often partly reversible, because restoring pH 7 lets the polypeptide refold, while prolonged exposure tends to be irreversible, like prolonged heat. That is exactly why assays control pH with a buffer and not only temperature.
3. The lab partner concludes that the 80 °C result means the potato extract contained no catalase. Evaluate that conclusion.
Read the worked answer
Reject it. The same extract, from the same batch, produced 60 bubbles per 15 s at 37 °C and steady activity at 4 °C, so catalase was present; the treatment changed, not the extract.
Two weaknesses remain. The 80 °C tube sits at the extreme of the series and gives 2 bubbles, near the limit of the counting method, so it is a poor test of presence. And there is no negative control: boiled extract run at 37 °C, or hydrogen peroxide with no extract at all, would show what zero activity looks like. Without those, the partner is inferring absence of enzyme from absence of signal, which is the same error as calling a failed stain a negative result.