Investigating Enzyme Inhibitors Using Catalase

Enzymes are biological catalysts that control reactions in living organisms, but their activity can be slowed or stopped by inhibitors. Using catalase — an enzyme found in many living tissues — students can explore how inhibitors affect enzyme activity and reaction rates.

The Experiment

Catalase breaks down hydrogen peroxide into water and oxygen:

2H_2O_2 \rightarrow 2H_2O + O_2

Students can measure the volume of oxygen produced or the height of foam formed when hydrogen peroxide reacts with catalase from liver or potato extracts.

To investigate inhibition:

Add a competitive inhibitor such as copper sulfate, which competes with the enzyme’s active site.
Add a non-competitive inhibitor like lead nitrate, which alters the enzyme’s shape.
Compare reaction rates with and without inhibitors at controlled temperature and pH.

Method 1: Measuring oxygen production

This method measures the rate of the reaction by quantifying the amount of oxygen produced.

Materials: You will need a catalase source (e.g., yeast, liver, or potato), hydrogen peroxide, a source of inhibitor, test tubes or a conical flask, a gas syringe or ruler to measure foam, and a stopwatch.

Set up the experiment: Place the hydrogen peroxide solution in a test tube or flask. Prepare the enzyme solution by grinding potato with distilled water or using a liquid source like yeast suspension.
Introduce the inhibitor: Add a specific concentration of the inhibitor to the hydrogen peroxide solution. Repeat the experiment with different concentrations of the same inhibitor, and also run a control without any inhibitor.
Start the reaction: Add the enzyme solution to the hydrogen peroxide and inhibitor mixture and immediately start the stopwatch.
Measure the product: Collect the oxygen gas produced in the gas syringe and record the volume at set time intervals, or measure the height of the foam produced.
Analyze the results: A slower reaction rate (less oxygen produced or lower foam height in a given time) indicates a more effective inhibitor.

Method 2: Using a filter paper disk (for qualitative analysis)

This is a simpler method, often used for a qualitative or comparative investigation.

Prepare the enzyme: Make a paste from a source of catalase, such as a potato, and suspend it in a small amount of distilled water.

Prepare the substrate: Place a solution of hydrogen peroxide in a specimen tube.

Prepare the filter paper disks: Dip small filter paper disks into the catalase suspension and tap off any excess liquid.
Add the inhibitor: Apply a drop of the inhibitor to one of the filter paper disks. You can also test disks without any inhibitor (control) and disks with different inhibitors.
Start the reaction: Drop the treated disk into the hydrogen peroxide solution and time how long it takes for the disk to sink to the bottom. The time it takes for the disk to sink is inversely proportional to the enzyme activity.
Analyze the results: If the inhibitor is effective, the disk will sink faster because the catalase is less active.

Prepare reagents: Measure the rate of product formation using a spectrophotometer to measure the absorption of light.

Substrate concentration: Keep the concentration of hydrogen peroxide constant across all experiments.

Method 3: Spectrophotometric analysis

Prepare reagents: Measure the rate of product formation using a spectrophotometer to measure the absorption of light.

Run the experiment: Place the reaction mixture in a cuvette and measure the light absorption of the product at a specific wavelength.
Introduce the inhibitor: Add the inhibitor and record the change in light absorption over time.
Analyze the results: A significant change in the rate of light absorption indicates the inhibitor is effective.
Key variables to control and investigate
This is the easiest to do in a short lesson when results are required quickly and using a @pascoscientific Colorimeter works well.

Substrate concentration: Keep the concentration of hydrogen peroxide constant across all experiments.
Enzyme concentration: Ensure the concentration of the catalase is the same for all trials.
pH and temperature: Maintain a constant pH and temperature, or investigate how inhibitors affect the enzyme activity at different temperatures or pH levels.
Inhibitor concentration: Vary the concentration of the inhibitor to determine its effect on enzyme activity and to calculate the
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value (the concentration of inhibitor required to halve the enzyme activity).

The Science

Enzyme inhibitors reduce the rate of reaction in two main ways:

Competitive inhibitors occupy the enzyme’s active site, blocking the substrate.
Non-competitive inhibitors bind elsewhere, changing the enzyme’s structure so the substrate no longer fits.

By plotting rate against inhibitor concentration, students can see how the rate decreases and learn how enzymes are regulated in cells — and how poisons and drugs work.

Skills Highlight

Designing fair tests with controlled variables
Measuring reaction rates quantitatively using gas volume or sensor data
Analysing graphs to interpret inhibition types
Linking enzyme structure to biochemical function

Why It Works in Teaching

This investigation links practical biochemistry to real-world contexts such as medicine and toxicology. Students can visualise enzyme action, inhibition, and the importance of active site shape — core ideas that underpin much of biology.

Monday, 20 October 2025