Catalase is one of the most efficient enzymes known, breaking down hydrogen peroxide into water and oxygen at rates of up to 40 million reactions per second. In this investigation, you will measure how the rate of catalase activity changes as substrate concentration increases, generating a Michaelis-Menten curve and allowing calculation of the maximum reaction velocity (Vₘₐˣ) and the Michaelis constant (Kₘ). This is a fully quantitative IB Biology HL Internal Assessment investigation.

This practical is suitable for IB Diploma Biology HL and Edexcel IGCSE Biology.

Background Theory

Catalase catalyses the decomposition of hydrogen peroxide:

2H₂O₂ → 2H₂O + O₂

According to Michaelis-Menten kinetics, enzyme reaction rate (v) increases with substrate concentration [S] according to:

v = Vₘₐˣ[S] / (Kₘ + [S])

At low [S], rate is approximately proportional to [S] (first-order kinetics). At high [S], rate approaches Vₘₐˣ asymptotically as all active sites become saturated (zero-order kinetics). Kₘ is the substrate concentration at which rate = Vₘₐˣ/2, and is a measure of enzyme-substrate affinity. A lower Kₘ means higher affinity.

By taking a Lineweaver-Burk double reciprocal plot (1/v vs 1/[S]), the Michaelis-Menten curve can be linearised, allowing precise determination of Vₘₐˣ and Kₘ from the intercepts.

Variables

• Independent variable (IV): Concentration of hydrogen peroxide, H₂O₂ (mol dm⁻³) — e.g. 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00 mol dm⁻³
• Dependent variable (DV): Initial rate of oxygen production (cm³ min⁻¹) measured using a gas syringe or graduated pipette
• Controlled variables (CV): Mass and source of catalase (same potato or liver disc, same mass), temperature (water bath at 25 °C), pH (phosphate buffer at pH 7), total volume of reaction mixture

Equipment

• Fresh potato or liver (source of catalase)
• Hydrogen peroxide solutions at 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00 mol dm⁻³ (prepared by dilution from 1.00 mol dm⁻³ stock)
• Phosphate buffer solution (pH 7.0)
• Cork borer and cutting tile
• Balance (±0.01 g)
• Gas syringe (0–50 cm³) connected to a conical flask via bung and delivery tube, OR a graduated pipette inverted over water
• Stopwatch
• Water bath at 25 °C
• Thermometer

Safety

⚠️ Hydrogen peroxide at concentrations above 0.5 mol dm⁻³ is an irritant and oxidising agent — wear gloves and eye protection. Higher concentrations cause vigorous gas production — ensure the apparatus is not sealed and gas can escape safely. Dispose of all solutions into the appropriate waste disposal bottles provided.

Method

1. Cut uniform potato discs using a cork borer. Weigh each disc to ensure equal mass (e.g. 1.00 ± 0.05 g). Keep potato discs in phosphate buffer until needed to prevent oxidation.
2. Add 20 cm³ of phosphate buffer to a conical flask. Add the potato disc. Connect the gas syringe via a bung.
3. Allow to equilibrate at 25 °C in the water bath for 5 minutes.
4. Add 5 cm³ of H₂O₂ solution at your first concentration through a small hole in the bung (or quickly remove and replace the bung). Start the stopwatch immediately.
5. Record the volume of O₂ produced every 30 seconds for 5 minutes.
6. Calculate the initial rate as the gradient of the steepest part of the volume vs time graph (first 60 seconds).
7. Repeat for each H₂O₂ concentration using a fresh potato disc each time. Take at least three replicates per concentration.

Results Table

[H₂O₂] (mol dm⁻³)	1/[S] (dm³ mol⁻¹)	Rate 1 (cm³ min⁻¹)	Rate 2 (cm³ min⁻¹)	Rate 3 (cm³ min⁻¹)	Mean Rate (cm³ min⁻¹)	1/v (min cm⁻³)
0.05	20.0
0.10	10.0
0.20	5.0
0.40	2.5
0.60	1.67
0.80	1.25
1.00	1.00

Analysis

1. Plot mean rate (y-axis) against [H₂O₂] (x-axis). You should see a hyperbolic Michaelis-Menten curve that plateaus as Vₘₐˣ is approached.

2. Estimate Vₘₐˣ from the plateau of the curve. Read off Kₘ as the [S] at which rate = Vₘₐˣ/2.

3. Construct a Lineweaver-Burk plot: plot 1/v (y-axis) against 1/[S] (x-axis). Draw a best-fit line.

4. From the Lineweaver-Burk plot: y-intercept = 1/Vₘₐˣ, x-intercept = −1/Kₘ, gradient = Kₘ/Vₘₐˣ.

5. Calculate Vₘₐˣ and Kₘ and compare to literature values for potato catalase (Kₘ ≈ 0.06–0.10 mol dm⁻³).

Discussion Points

• Why does the rate increase with [H₂O₂] at low concentrations but level off at high concentrations?
• What does Kₘ tell you about the affinity of catalase for H₂O₂?
• Why is a fresh potato disc used for each concentration rather than reusing the same one?
• Why is it important to measure the initial rate rather than the average rate over the whole experiment?
• How would the Michaelis-Menten curve change if a competitive inhibitor were added?

IA Guidance

The Lineweaver-Burk plot makes this one of the most mathematically sophisticated IB Biology IAs available. To score highly:

• Research Design: Justify your [H₂O₂] range — it should span well below and well above Kₘ to capture both phases of the curve. Explain why a buffer is essential and why temperature must be controlled.
• Data Analysis: Present both the Michaelis-Menten curve and the Lineweaver-Burk plot. Include error bars. Use worst-case lines on the Lineweaver-Burk plot to determine uncertainties in Vₘₐˣ and Kₘ.
• Conclusion: Compare your Kₘ to literature and discuss what it reveals about enzyme-substrate affinity. Note that Lineweaver-Burk plots amplify errors at low [S] — comment on this limitation.
• Evaluation: Discuss whether potato disc mass is a reliable proxy for enzyme concentration, and suggest using a purified catalase solution of known concentration for greater control.

• Practical Biology