Biological Molecules » Enzyme Temperature Practical

What you'll learn this session

Study time: 30 minutes

How enzymes are affected by temperature
How to set up and conduct an enzyme temperature practical
Methods for measuring enzyme activity
How to collect and analyse data from enzyme experiments
How to explain results using biological knowledge
Common errors and how to improve experimental accuracy

Introduction to Enzyme Temperature Practical

Enzymes are biological catalysts that speed up chemical reactions in living organisms. Unlike most catalysts, enzymes are highly sensitive to their environment, particularly temperature. In this practical, we'll investigate how temperature affects the rate of enzyme activity.

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.
Denaturation: The process where an enzyme loses its 3D shape and function.
Optimum temperature: The temperature at which an enzyme works most efficiently.

🔥 Temperature Effects on Enzymes

As temperature increases from 0°C:

Enzyme and substrate molecules gain kinetic energy
More frequent collisions between enzyme and substrate
Reaction rate increases
At very high temperatures, the enzyme denatures
The 3D shape of the active site changes
Substrate can no longer fit and the reaction stops

📊 Typical Enzyme Activity Graph

A typical graph of enzyme activity vs temperature looks like this:

Low activity at low temperatures
Steady increase as temperature rises
Peak activity at optimum temperature (often 37-40°C for human enzymes)
Rapid decline after optimum as denaturation occurs
Little to no activity at high temperatures

Planning Your Enzyme Temperature Practical

A common enzyme practical involves using catalase (found in potato, liver, or yeast) to break down hydrogen peroxide. The reaction produces oxygen gas, which can be measured to determine the rate of reaction.

Materials You'll Need

Fresh potato (source of catalase enzyme)
Hydrogen peroxide solution (substrate)
Boiling tubes or test tubes
Delivery tubes and bungs
Measuring cylinders
Water baths at different temperatures (e.g., ice bath, room temperature, 30°C, 40°C, 50°C, 60°C, 70°C)
Stopwatch
Thermometer
Cork borer or knife
White tile
Gas syringe or inverted measuring cylinder filled with water

Experimental Method

Use a cork borer to cut cylinders of potato of equal size (e.g., 2cm long).
Place each potato piece in a different water bath for 5 minutes to reach the test temperature.
Set up the apparatus to collect oxygen gas (using a gas syringe or inverted measuring cylinder).
Add 10ml of hydrogen peroxide solution to a test tube.
Place the test tube in the same water bath as the potato piece for 2 minutes.
Add the potato piece to the hydrogen peroxide, quickly seal with a bung connected to the gas collection apparatus.
Start the stopwatch and record the volume of oxygen produced every 30 seconds for 3 minutes.
Repeat for each temperature being tested.
Calculate the rate of reaction (volume of oxygen produced per minute).

⚙ Variables

Independent: Temperature of water bath

Dependent: Volume of oxygen produced per minute

Control: Size of potato, concentration of H₂O₂, time of reaction

💡 Safety Tips

Hydrogen peroxide can irritate skin and eyes - wear goggles and gloves

Take care with hot water baths - use tongs to handle hot test tubes

Ensure gas collection apparatus is secure to prevent leaks

📈 Data Collection

Record in a table with columns for:

- Temperature (°C)

- Volume of O₂ at 30s intervals

- Total volume after 3 minutes

- Rate (ml/minute)

Alternative Methods for Measuring Enzyme Activity

There are several ways to measure enzyme activity in the lab:

🟢 Amylase and Starch

Amylase breaks down starch into maltose. You can track this reaction using iodine solution, which turns blue-black in the presence of starch.

Method:

Set up test tubes with starch solution at different temperatures
Add amylase to each tube and start timing
Every 30 seconds, remove a drop and test with iodine on a spotting tile
Record the time taken for the iodine to stop turning blue-black (indicating starch is gone)

🟥 Lipase and Milk

Lipase breaks down fats in milk, producing fatty acids that lower the pH. This can be tracked using a pH indicator like phenolphthalein.

Method:

Add milk and phenolphthalein (pink) to test tubes at different temperatures
Add lipase and start timing
Record the time taken for the solution to turn colourless (as pH drops)
Faster colour change indicates faster enzyme activity

Analysing Your Results

After collecting your data, you'll need to analyse it to draw conclusions about how temperature affects enzyme activity.

Plotting Your Graph

Plot temperature (°C) on the x-axis
Plot rate of reaction (ml oxygen/minute or time taken) on the y-axis
Draw a line of best fit (usually a curve)
Identify the optimum temperature (peak of the curve)

Interpreting Your Results

Your graph should show:

Low activity at low temperatures (0-10°C)
Increasing activity as temperature rises to around 37-45°C (for most enzymes)
Maximum activity at the optimum temperature
Decreasing activity above the optimum as denaturation occurs
Very little or no activity at high temperatures (above 60-70°C)

Case Study: Why Different Enzymes Have Different Optimum Temperatures

Enzymes from different organisms often have different optimum temperatures that reflect their natural environment:

Human enzymes: Typically have an optimum around 37°C (normal body temperature)
Bacteria in hot springs: Have enzymes with optima of 70°C or higher
Arctic fish enzymes: Can function efficiently at temperatures close to 0°C

This is an example of adaptation - organisms evolve enzymes suited to their environment. Thermophilic bacteria from hot springs are used in biological washing powders because their enzymes remain active at high washing temperatures.

Common Errors and Improvements

Potential Sources of Error

Temperature fluctuations in water baths
Inconsistent size of potato pieces
Gas leaks in collection apparatus
Timing errors
Variation in enzyme concentration between potato pieces

Improving Your Experiment

Use a thermostatically controlled water bath
Use a cork borer to ensure uniform potato pieces
Check for gas leaks by testing with soapy water
Use a digital timer for accuracy
Consider using purified enzyme solution instead of potato
Repeat each temperature test three times and calculate an average
Include more temperature points for a more detailed curve

Exam-Style Questions to Consider

Why does enzyme activity increase with temperature up to the optimum?
Explain why enzyme activity decreases rapidly above the optimum temperature.
How would you modify this experiment to investigate the effect of pH on enzyme activity?
Why might the optimum temperature for human digestive enzymes differ from those found in bacteria?
How could you determine if the enzyme has been completely denatured or just temporarily inhibited?

Remember, a well-conducted practical not only helps you understand enzyme function but also develops your scientific skills in planning, data collection, analysis and evaluation - all essential for success in your IGCSE Biology exams!

🧠 Test Your Knowledge!