Investigation: Planning and Reporting an Enzyme Experiment

From "what happens" to "how do we know"

You know that temperature affects enzymes. But how would you show it, convincingly enough that another scientist would believe your numbers? That is what this lesson is about: planning, carrying out and reporting a real investigation into a cellular process.

The skills here (writing a hypothesis, controlling variables, handling uncertainty, judging evidence) are exactly the ones first-year university biology labs grade most heavily.

Step 1: Question and hypothesis (A1.1)

Start with a testable question that names one independent and one dependent variable:

How does temperature affect the rate at which catalase breaks down hydrogen peroxide?

Then a hypothesis with a reason:

If the temperature increases from 10 °C to about 40 °C, then the rate will increase, because molecules move faster and collide more often with the active site; above about 40 °C the rate will decrease, because the enzyme begins to denature and its active site loses its shape.

Step 2: Variables and equipment (A1.2)

• Independent variable: temperature (10, 20, 30, 40, 50, 60 °C, using water baths).
• Dependent variable: the time for a filter-paper disc soaked in catalase to float up through hydrogen peroxide. The enzyme produces oxygen bubbles (2 H₂O₂ → 2 H₂O + O₂), which stick to the disc and lift it. Rate = 1 ÷ time.
• Controlled variables: H₂O₂ concentration and volume; disc size and soaking time; enzyme source and concentration (one extract, for example from potato or liver, used for everything); depth of liquid; pH; time each tube spends in the water bath before testing.
• Controls: a disc soaked in water (no enzyme), and a disc soaked in boiled extract, should not float within the test time.
• Equipment: beakers or test tubes, water baths with thermometers, filter-paper hole punch, forceps, stopwatch, graduated cylinders.

Step 3: Procedure and safety (A1.4, A1.5)

1. Equilibrate the H₂O₂ and the enzyme extract at each temperature for 5 minutes.
2. Soak a disc in extract for 5 s, drain it, and drop it into the H₂O₂. Start timing when it touches the bottom; stop when it reaches the surface.
3. Do three trials at each temperature with fresh discs.

Safety: wear goggles. Lab hydrogen peroxide (often 3%) irritates eyes and skin, so rinse spills with water. Use care with hot water baths and glassware. Wash hands after handling raw liver.

Other classic versions of this lab: amylase and starch, sampled every 30 seconds onto a spot plate of iodine until no blue-black colour appears; or a gas-pressure sensor measuring the O₂ produced by catalase.

Step 4: Analysis and conclusion (A1.8, A1.10)

Conclusion: the rate of catalase activity increased from 10 °C to a maximum at about 40 °C and decreased sharply above that, which supports the hypothesis. Use evidence: "The rate at 40 °C (0.100 s⁻¹) was three times the rate at 10 °C (0.033 s⁻¹), and at 60 °C it fell to 0.017 s⁻¹."

Evaluation: ask what could have affected the results.

• Random error: human reaction time with the stopwatch; discs catching on the side of the tube. The wider range at 60 °C suggests extra variability, possibly because the enzyme was denaturing during the trial. More trials would help.
• Systematic error: if the water bath was really 2 °C cooler than the thermometer said, every point would shift. Calibrate thermometers. If discs absorbed different volumes of extract, use a dropper to add a fixed volume.
• Limitations: only six temperatures; the curve's shape between readings is a guess. The disc method measures O₂ production indirectly.
• Improvements: use 5-degree steps near the peak; use a gas-pressure sensor for a direct, continuous measurement.

Never say a result proves a hypothesis; say it supports or does not support it.

Step 5: The lab report (A1.11)

A standard report has: Title; Purpose/Question; Hypothesis; Variables; Materials; Procedure (numbered, past tense or instructions as your teacher asks); Observations (a data table with units and uncertainties, and qualitative notes); Analysis (calculations shown once, then graphs with titles, labelled axes and units); Conclusion (answer the question, refer to data, accept or reject the hypothesis); Evaluation (sources of error, limitations, improvements); and References (in a consistent citation style, such as APA).

Planning your own: a safe home investigation

You can carry out a real enzyme investigation at home with the protease in kiwifruit (called actinidin), which digests gelatin, a protein. Fresh pineapple contains a similar protease (bromelain). In Grade 11 you compared fresh, boiled and canned pineapple. Now you will design a quantitative test of how pre-heating temperature affects the enzyme.

There is a design trap: set gelatin melts if it gets warm (around body temperature), so you cannot test the enzyme at high temperatures on the gelatin itself. Instead, you heat-treat the juice at different temperatures, cool it, and then let every sample act on gelatin in the fridge. The independent variable is therefore the temperature the enzyme was exposed to, which measures denaturation (thermal stability).