Catalase activity: pH optimum, a suspect replicate, and copper inhibition
A student grinds 20 g of potato in buffer to make an extract and measures catalase activity. In each trial 5.0 cm³ of extract is added to 20 cm³ of 2.0 % H₂O₂ at 20 °C, and the volume of O₂ collected in the first 60 s is recorded. Results, in cm³: pH 4, 3.2; pH 5, 9.8; pH 6, 18.2; pH 7, 24.6; pH 8, 12.4. At pH 7 the three repeats gave 24.6, 25.1 and 9.9. Adding CuSO₄ to give 1.0 mmol/L in the pH 7 mixture reduced the volume to 6.1 cm³. The student concludes that pH 7 is the optimum, that the 9.9 cm³ value should be deleted as "an outlier", and that Cu²⁺ is a competitive inhibitor. A second experiment in which H₂O₂ concentration was varied in the presence of Cu²⁺ was planned but never carried out.
1. Assess how precisely the optimum pH is located by these data, and state what the conclusion at pH 7 can and cannot claim.
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The data locate the optimum only within a gap between tested points. Activity rises from 3.2 at pH 4 to a peak of 24.6 at pH 7, then falls to 12.4 at pH 8. With no measurement at pH 6.5 or 7.5, the true optimum could lie anywhere in roughly 6.5–7.5; the curve is also clearly asymmetric (only 12.4 at pH 8 against 18.2 at pH 6), so the peak may sit slightly to the acid side of 7.
The valid claim is therefore: "the optimum lies between pH 6 and pH 8, most probably near pH 7". The invalid claim is "the optimum is pH 7".
A further limitation is that each pH point came from a single trial apart from pH 7, so no uncertainty is attached to the four other values. Also, the buffer used to set each pH is itself changed - different buffer ions and ionic strengths can affect enzyme shape independently of pH, so part of the trend could be a buffer artefact. The fix is to test pH 6.4, 6.7, 7.0, 7.3, 7.6 in the same buffer system, in triplicate, and plot activity against pH with error bars.
2. Judge whether the 9.9 cm³ reading should be discarded, showing how the decision affects the mean.
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The reading must not be deleted on the grounds that it "looks wrong" - that is fitting the data to the hypothesis. It is more than 50 % below the other two (24.6 and 25.1, which agree to within 2 %), so the question is whether the procedure failed, not whether the result is inconvenient.
Plausible procedural causes: the bung was not sealed in time so gas escaped before collection began; the extract had stood and warmed, or had been diluted; the H₂O₂ was added before the extract was in place; the syringe/measuring cylinder was read at the wrong moment.
The defensible route is: check the record for that trial. If a procedural fault is documented, exclude it and state the reason. If not, repeat at pH 7 several times. Only if the repeat values cluster with 24.6 and 25.1 should the 9.9 be treated as an outlier (and even then a formal test such as Grubbs' should be cited).
Effect on the mean: including it gives (24.6 + 25.1 + 9.9)/3 ≈ 19.9 cm³; excluding it gives ≈ 24.85 cm³. Either way pH 7 remains the highest point, so the qualitative conclusion survives, but the reported rate changes by about 20 % - enough to matter if it feeds a calculation of initial rate or of Vmax.
3. Evaluate the claim that Cu²⁺ is a competitive inhibitor, and describe the experiment that would test it.
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The claim is not supported by the data. A single substrate concentration cannot distinguish inhibition types: competitive inhibition raises the apparent Km while leaving Vmax unchanged, and non-competitive inhibition lowers Vmax while leaving Km unchanged. At one [H₂O₂] you measure only a single lowered rate (6.1 versus ~24.6 cm³), which is compatible with either, with mixed inhibition, or with Cu²⁺ denaturing the enzyme or reacting with H₂O₂ or with the assay.
Experiment needed: at pH 7 and 20 °C, measure initial rate at a series of H₂O₂ concentrations (say 0.25, 0.5, 1.0, 2.0, 4.0 %) both with and without 1.0 mmol/L Cu²⁺, keeping extract volume and total volume constant.
Interpretation: plot rate against [S] and then a reciprocal plot (1/v against 1/[S]). If the inhibited line meets the control line on the 1/v axis (same Vmax, higher Km), inhibition is competitive - consistent with Cu²⁺ binding the active site. If it meets the 1/[S] axis (same Km, lower Vmax), it is non-competitive. Parallel-shifted lines with both parameters altered indicate mixed inhibition.
A control with Cu²⁺ but no enzyme, and a control with boiled extract, would check that Cu²⁺ is not simply interfering with the assay chemistry.