Catalase Kinetics Under Time and Volume Constraints
A Year 13 student has 25 minutes of laboratory time to investigate how hydrogen peroxide concentration affects catalase activity in potato tissue. Her equipment is 30 mL of 3% hydrogen peroxide, one potato, a cork borer, a 10 mL measuring cylinder, a stopwatch and a water bath set at 25 °C. No gas syringe is available, so she plans to count oxygen bubbles released from a 5 mm potato disc over 30 seconds and use the count as a measure of reaction rate.
In a pilot run at 3% peroxide she recorded bubble counts of 62, 41 and 55 across three repeats. Room temperature during the pilot drifted between 21 and 24 °C. She noticed bubbles clinging to the disc and to the tube wall, and that the disc began to disintegrate after about 90 seconds in the strongest solution.
She needs data for at least five concentrations, each with enough repeats to support a defensible conclusion.
1. Identify the two most serious threats to the validity or reliability of this investigation and describe how she could modify her procedure to overcome them within her time and equipment constraints.
Read the worked answer
Threat 1: the measurement itself. Bubble counting is a discontinuous, observer-dependent proxy. Bubbles vary in size, coalesce, and cling to glass and tissue, and some oxygen dissolves before a bubble ever forms. The pilot spread (41–62 at a single concentration) is larger than most of the differences she hopes to detect, so the method would produce noise, not a trend. The best fix within her kit is the filter-paper disc method: saturate a 6 mm disc with crushed potato, drop it into the peroxide, and time how long it takes to float as oxygen collects on it. Time to float uses the stopwatch, gives a continuous number, and removes the counting judgement. If she keeps counting, she must tap the tube to dislodge bubbles and use one fixed observer.
Threat 2: temperature. A 3 °C drift changes enzyme rate by roughly 20%, which is comparable to the effect she is trying to measure. The 25 °C bath was available and unused; she must equilibrate both the peroxide and the tube in it for five minutes before each trial.
Hidden constraint. Five concentrations × three repeats × 10 mL requires 150 mL, but she only has 30 mL. She must scale down to about 2 mL per trial in a small tube and check that arithmetic before starting.
2. Explain the biological reason why the rate of reaction should change with substrate concentration, and predict what her data should look like at the highest concentrations. Link your answer to what she actually observed.
Read the worked answer
Catalase catalyses 2H₂O₂ → 2H₂O + O₂. Rate depends on how often hydrogen peroxide molecules collide with active sites. At low concentration, most active sites are empty most of the time, so adding substrate increases the frequency of successful collisions and the rate rises steeply. As concentration climbs, active sites become saturated, and the rate levels off at Vₘₐₓ, set by how much enzyme is present, not by how much substrate is present. So a graph of rate against concentration should be a hyperbolic curve flattening at higher concentrations.
Her data are unlikely to show a clean plateau, for two measurement reasons. First, at the highest concentration the reaction is fast enough that bubbles merge and counting becomes impossible, so the recorded count underestimates the true rate and the curve appears to flatten or even fall. Second, she saw the disc disintegrate after about 90 seconds at 3%; fragmentation increases exposed surface area, so the later part of a 30-second count is not measuring a constant amount of enzyme. There is also a genuine biological effect: catalase shows substrate inhibition at very high peroxide concentrations, because excess substrate binds and blocks the active site. A defensible conclusion must therefore state that the apparent plateau above about 2% is partly a measurement artefact, and that only the rising portion of the curve can be interpreted as enzyme kinetics.
3. She ran a tube containing 3% hydrogen peroxide with no potato and saw no bubbles; a classmate says this is sufficient control. Evaluate that claim and describe one further control she should include.
Read the worked answer
The no-potato tube is a valuable negative control: it establishes that the peroxide does not break down appreciably on its own, at that temperature, over that timescale. Without it she could not claim that any bubbles seen are the product of a biological catalyst. It also confirms the water bath is not hot enough to drive thermal decomposition.
It does not establish that the bubbles come from catalase specifically. Potatoes contain peroxidases and other enzymes, and dissolved gas or air trapped in the tissue could contribute. A better control is a disc of boiled potato (or potato treated with a protein denaturant) run under identical conditions. Denaturing the enzyme removes catalytic activity while leaving the physical structure, dissolved gases and any non-protein chemistry unchanged. If the boiled disc produces the same bubble count or the same float time as live tissue, the measured oxygen is not enzyme-derived and the whole method is invalid. If it produces none, she has isolated enzymatic activity as the cause.
She should also state the controlled variables explicitly - disc diameter and thickness, volume and concentration of peroxide, blotting of the disc, equilibration time, and the same potato tuber - because these are what make the comparison fair across concentrations.