Potato Catalase and pH: Rescuing a Flawed Investigation
A student investigates how pH affects catalase activity in potato tissue. She cuts 30 potato disks with a cork borer, each 1 cm in diameter and 2 mm thick. She prepares buffer solutions at pH 3, 5, 7, 9, and 11. For each pH, she places 5 disks in a 50 mL tube with 20 mL of buffer and 5 mL of 3% hydrogen peroxide. She measures foam height after 2 minutes. She runs each pH once, then repeats only the pH 7 trial three more times. Foam heights are 4, 11, 18, 9, and 2 mm from pH 3 through pH 11. The three extra pH 7 trials give 17, 18, and 26 mm. She concludes that pH 7 is optimal and that pH 11 denatures catalase. Class data show one group used room-temperature peroxide and another used refrigerated peroxide. Her teacher asks her to justify both conclusions and to revise the investigation before submitting it to the science fair.
1. Identify the independent variable, the dependent variable, and two variables the student should control. For each controlled variable, explain why it matters when comparing foam heights across pH levels.
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Independent variable: pH of the buffer (3, 5, 7, 9, 11). Dependent variable: foam height after 2 minutes, used as a proxy for catalase activity.
Two variables to control:
- Potato disk size and number. She uses 5 disks of 1 cm diameter and 2 mm thickness per tube, so surface area and enzyme amount are comparable. If disks differ in size or number, foam height could change for reasons unrelated to pH.
- Peroxide volume and concentration. She uses 5 mL of 3% H2O2. If concentration or volume varies, the amount of substrate varies and the reaction rate changes.
Temperature and time also matter. The class data show some groups used room-temperature peroxide and others refrigerated peroxide, so temperature must be standardized because enzyme activity depends on temperature. Foam height also depends on how long she waits, so a fixed 2-minute interval is needed. Controlling these variables makes pH the only major difference among trials.
2. Evaluate the conclusion that pH 7 is optimal. Use the five pH values and the repeated pH 7 trials, including the 26 mm result. State whether the evidence is reliable enough for the science fair and why.
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The single-run data put the highest foam height at pH 7 (18 mm), with lower values at pH 3 (4 mm), pH 5 (11 mm), pH 9 (9 mm), and pH 11 (2 mm). That pattern is consistent with pH 7 being near the optimum, but the evidence is weak because each non-7 pH was tested only once.
The repeated pH 7 trials were 17, 18, 18, and 26 mm. The 26 mm result is an outlier relative to the other three (17, 18, 18 mm), which cluster tightly. The outlier could come from inconsistent timing, a temperature difference, a larger effective disk surface, or a bubble measurement error. The mean of the four pH 7 trials is 19.75 mm, but the median is 18 mm; the median better represents the typical result. Without repeats at pH 5 and pH 9, she cannot tell whether pH 7 is significantly better or whether the difference is within trial variation.
For a science fair, she should repeat every pH at least three times, use the same protocol, and report means and ranges. As it stands, the conclusion is plausible but not reliable enough to be definitive.
3. Evaluate the claim that pH 11 denatures catalase. What additional evidence would make this claim stronger, and what redesign would produce that evidence?
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The 2 mm foam height at pH 11 shows very low activity, but low foam alone does not prove denaturation. Low activity could result from the enzyme being denatured, but it could also result from the enzyme working slowly at that pH, from the buffer interfering with foam formation, or from the potato disks losing enzyme during preparation.
To support denaturation, she needs evidence that the enzyme's structure or function is irreversibly lost. One approach is to expose catalase to pH 11 for a set time, then return it to pH 7 and measure activity. If activity remains low after returning to pH 7, that supports irreversible denaturation; if activity recovers, the pH 11 effect was reversible inhibition or suboptimal conditions. Another approach is to compare the pH 11 result with a boiled-enzyme control, which should show near-zero activity, and with a no-peroxide control to check for foam from the buffer alone. A redesign should include at least three trials per pH, a recovery test, and controls for temperature, disk size, and peroxide concentration.