Biological Molecules » Enzyme pH Practical

What you'll learn this session

Study time: 30 minutes

How enzymes are affected by changes in pH
How to set up and conduct an enzyme pH practical investigation
How to collect, record and analyse data from enzyme experiments
How to interpret results to understand enzyme optimum pH
How to evaluate practical methods and suggest improvements

Introduction to Enzyme pH Practical

Enzymes are biological catalysts that speed up chemical reactions in living organisms. One of the key factors that affects how well enzymes work is pH. In this practical, we'll investigate how different pH conditions affect enzyme activity and discover why this matters in biological systems.

Key Definitions:

Enzyme: A protein that acts as a biological catalyst, speeding up reactions without being used up.
Substrate: The molecule that an enzyme acts upon.
Active site: The region of an enzyme where the substrate binds.
pH: A measure of how acidic or alkaline a solution is (scale of 0-14).
Optimum pH: The pH at which an enzyme works best.
Denaturation: The process where an enzyme loses its shape and function.

🔬 Why pH Matters for Enzymes

pH affects the ionic bonds and hydrogen bonds that maintain an enzyme's 3D structure. Changes in pH can alter the shape of the active site, affecting how well the enzyme can bind to its substrate. If pH changes too much, the enzyme can become denatured and stop working completely.

🎯 Enzyme Specificity

Different enzymes have different optimum pH values. For example, pepsin (a digestive enzyme in the stomach) works best in acidic conditions (pH 2), while amylase (found in saliva) works best at a neutral pH around 7. This specificity is related to where these enzymes function in the body.

The Practical Investigation: Catalase and Hydrogen Peroxide

In this practical, we'll investigate how pH affects the activity of catalase, an enzyme found in most living cells. Catalase breaks down hydrogen peroxide (H₂O₂) into water and oxygen:

2H₂O₂ → 2H₂O + O₂

By measuring the rate of oxygen production at different pH values, we can determine the optimum pH for catalase.

Materials and Equipment

Fresh potato (source of catalase)
Hydrogen peroxide solution (3%)
Buffer solutions of different pH values (pH 3, 5, 7, 9, 11)
Boiling tubes and test tube rack
Measuring cylinder
Cork borer
Scalpel and cutting tile
Gas syringe or inverted measuring cylinder in water bath
Stopwatch
Thermometer

Method

Use a cork borer to cut cylinders of potato of equal size.
Cut each cylinder into 1cm discs and rinse with distilled water.
Label five boiling tubes with the different pH values.
Add 10ml of the appropriate buffer solution to each tube.
Place one potato disc into each tube and leave for 5 minutes to equilibrate.
Set up the gas collection apparatus (gas syringe or inverted measuring cylinder).
Add 5ml of hydrogen peroxide to the first tube, quickly seal with a bung connected to the gas collection apparatus and start the stopwatch.
Record the volume of oxygen produced every 30 seconds for 3 minutes.
Repeat steps 7-8 for each pH buffer solution.
Calculate the rate of oxygen production for each pH (volume of oxygen/time).

Safety First! ⚠

Hydrogen peroxide can irritate skin and eyes. Wear safety goggles and gloves. If contact occurs, rinse immediately with plenty of water. Some buffer solutions may be corrosive, so handle with care and wash hands after the practical.

Results and Analysis

After completing your experiment, you'll need to record and analyse your results to determine the optimum pH for catalase.

📊 Recording Data

Create a table with columns for pH, time, volume of oxygen produced and rate of reaction. For each pH, calculate the rate by dividing the total volume of oxygen by the time taken.

📈 Graphing Results

Plot a graph with pH on the x-axis and rate of reaction on the y-axis. The graph should show a curve with a peak at the optimum pH for catalase (typically around pH 7).

🔍 Interpreting Data

Look for the pH value where the reaction rate is highest - this is the optimum pH. Notice how the rate decreases at pH values above and below the optimum.

Expected Results

Catalase typically has an optimum pH around 7 (neutral). You should observe:

Low activity at pH 3 (acidic conditions)
Increasing activity as pH approaches 7
Maximum activity around pH 7
Decreasing activity as pH increases above 7
Low activity at pH 11 (alkaline conditions)

Explaining the Results

The pattern of enzyme activity across different pH values can be explained by how pH affects the enzyme's structure:

At optimum pH: The enzyme's active site has the perfect shape to bind to the substrate, allowing the reaction to proceed at maximum rate.
At low pH (acidic): H⁺ ions disrupt hydrogen bonds and ionic bonds in the enzyme, changing the shape of the active site. The substrate can no longer fit properly, reducing enzyme activity.
At high pH (alkaline): OH⁻ ions interfere with hydrogen bonds and ionic bonds, also changing the enzyme's shape and reducing its effectiveness.
At extreme pH values: The enzyme becomes completely denatured, losing its 3D structure and function.

Real-World Application: Digestive Enzymes

Your body has different digestive enzymes that work at different pH values:

Amylase in saliva: Works at pH 6.8-7.0 to begin starch digestion in the mouth.
Pepsin in the stomach: Functions at pH 1.5-3.5 to break down proteins in the highly acidic stomach environment.
Trypsin in the small intestine: Works at pH 7.5-8.5 in the alkaline environment of the small intestine.

This pH specificity ensures each enzyme works efficiently in its particular location in the digestive system.

Evaluating the Practical

Like all scientific investigations, this practical has strengths and limitations that should be considered when interpreting your results.

✅ Potential Sources of Error

Variations in potato catalase concentration
Temperature fluctuations during the experiment
Timing errors when measuring oxygen production
Leaks in the gas collection apparatus
Inconsistent potato disc sizes

💡 Improvements

Use a water bath to maintain constant temperature
Use purified catalase instead of potato extract
Test more pH values for a more detailed curve
Use a data logger to record oxygen production continuously
Perform multiple trials at each pH and calculate averages

Conclusion

This practical investigation demonstrates how pH affects enzyme activity, showing that enzymes have an optimum pH where they function most effectively. Understanding this concept is crucial for explaining how enzymes work in different parts of the body and in industrial applications.

For your exam, remember to:

Explain why enzymes have optimum pH values
Describe how pH changes affect enzyme structure and function
Interpret graphs showing enzyme activity at different pH values
Relate enzyme pH optima to their functions in living organisms
Evaluate practical methods for investigating enzyme activity

🧠 Test Your Knowledge!