Catalase Kinetics and the Limits of a Student's Claim
A preservice teacher reviews a lab notebook from a general biology section. Three groups measured catalase activity using 10 g of potato tissue blended in 20 mL buffer, 5 mL of 3% H2O2, and filter disks; they counted bubbles rising in 30 s. Group A varied temperature (10, 25, 40, 60 °C) at pH 7. Group B varied pH (3, 5, 7, 9) at 25 °C. Each condition was run once, except 25 °C/pH 7, which was run three times (counts 42, 61, 38). The 60 °C trial produced 3 bubbles; the pH 3 trial produced 2. Group A concluded the optimum is 40 °C (count 55). Group B concluded the optimum is pH 7. The notebook notes that potato batches differed between groups and that timing began when the disk was dropped, not when it floated. The teacher must decide whether to let the groups present these conclusions as valid and what revision to require.
1. Do the data support the two optimum claims? Identify at least two specific confounds and explain how each could shift the apparent optimum.
Read the worked answer
No - the claims are suggestive, not established. The replicated condition alone shows a count range of 38–61 at 25 °C/pH 7, so a single count of 55 at 40 °C could easily be noise rather than a peak. Two confounds are especially serious:
- Different potato batches: catalase specific activity varies with tuber age and storage. Because temperature and pH groups used different tissue, batch differences are confounded with the variable being tested and could move the apparent optimum up or down.
- Timing from disk drop rather than float: the lag before the disk contacts the substrate varies with temperature and viscosity. A 30 s count therefore measures different effective reaction intervals across conditions.
A third problem is that bubble counting is not a linear rate: bubbles coalesce, and gas solubility falls as temperature rises, which can inflate apparent activity at higher temperatures. The very low counts at 60 °C and pH 3 are consistent with denaturation, but one trial cannot distinguish denaturation from substrate depletion, pH drift, or procedural delay.
2. Design a revised protocol that would allow the class to estimate temperature and pH optima with confidence. Include controls, replication, and the measurement you would use.
Read the worked answer
Use an initial-rate assay with a single homogenate and proper controls. Grind one large potato batch in ice-cold buffer, filter it, and keep it on ice; use the same homogenate for all conditions. Measure catalase activity as the initial linear rate of H2O2 disappearance (spectrophotometer at 240 nm) or as initial gas pressure change, not endpoint bubble counts. Pre-equilibrate enzyme and substrate separately for 5 min at each temperature; mix and record the first 20–30 s. For temperature, hold pH constant with a verified buffer, test 10–60 °C in 5–10 °C steps, and include a boiled-enzyme negative control and a no-substrate blank. For pH, use overlapping buffers to cover pH 3–9 at a fixed 25 °C and verify final pH in each tube. Run 3–5 independent replicates per condition in randomized order. Report mean initial rate ± SD and fit a curve; the optimum is the peak of the fitted rate, not the highest single count.
3. The 25 °C/pH 7 replicates vary widely. Propose one biological and one procedural explanation and explain how you would distinguish them.
Read the worked answer
Biological explanation: catalase concentration or microbial load differed among tissue aliquots, so each disk carried different enzyme activity. Procedural explanation: disks varied in size or soak time, or the 30 s interval was started inconsistently relative to mixing. To distinguish them, use a single homogenate and divide it into identical aliquots; measure total protein or catalase concentration in each aliquot. If variability persists within the same homogenate and protein concentration, the cause is procedural. Also randomize run order and record time to disk float; if variability tracks run order or float time, procedural drift is likely.