3.3.3 Enzyme Rate Practical

Aim:

To investigate how a factor (e.g., temperature, pH, or substrate concentration) affects the rate of enzyme activity.

Equipment:

• Enzyme solution (e.g., amylase for starch digestion).
• Substrate solution (e.g., starch).
• Buffer solutions (to control pH).
• Water bath (to control temperature).
• Iodine solution (to test for the presence of starch).
• Timer.
• Pipette or syringe.
• Test tubes and racks.

Method: Example for Amylase Activity

1. Prepare Solutions:

• Add a known volume of starch solution and buffer solution to a test tube.

2. Set Conditions:

• Place the test tube in a water bath to maintain a constant temperature (e.g., 37°C for optimal enzyme activity).

3. Add Enzyme:

• Add a fixed volume of amylase solution to the starch and buffer mixture.
• Start the timer immediately.

4. Test for Starch Breakdown:

• Every 30 seconds, take a small sample of the mixture and add it to a spotting tile containing iodine solution.
• Iodine turns blue-black in the presence of starch. When the iodine no longer changes colour, the starch has been fully broken down.

5. Record Results:

• Record the time taken for the starch to be fully hydrolysed.

6. Repeat:

• Repeat the experiment at different temperatures, pH levels, or substrate concentrations to observe how they affect the rate.

Variables:

1. Independent Variable:

• The factor being investigated (e.g., temperature, pH, or substrate concentration).

2. Dependent Variable:

• Time taken for the enzyme to break down the substrate.

3. Control Variables:

• Volume of enzyme solution.
• Volume and concentration of substrate solution.
• Consistent method of measuring time and using iodine.

Expected Results:

1. Effect of Temperature:

• Optimum Temperature: Enzyme activity is fastest (e.g., ~37°C for human enzymes).
• Low Temperatures: Enzyme activity is slower due to reduced kinetic energy.
• High Temperatures: Enzyme activity decreases as the enzyme denatures.

2. Effect of pH:

• Optimum pH: Enzyme activity is highest (e.g., ~pH 7 for amylase).
• Extreme pH Levels: Enzymes denature, and activity decreases.

3. Effect of Substrate Concentration:

• Low Concentration: Fewer collisions between enzyme and substrate.
• Higher Concentration: Rate increases until enzymes become saturated.

Calculations:

1. Rate of Reaction:

• Use the formula:

$$\text{Rate of Reaction} = \frac{1}{\text{Time Taken (s)}}$$

2. Plot graphs to visualise relationships:

• Temperature vs Rate: Bell-shaped curve.
• pH vs Rate: Bell-shaped curve.
• Substrate Concentration vs Rate: Plateaus when enzymes are saturated.

Tips for Accuracy:

• Use a water bath for precise temperature control.
• Use a buffer solution to maintain a constant pH.
• Repeat the experiment multiple times and calculate a mean for reliable results.

Potential Sources of Error:

1. Human Error:

• Delays in starting/stopping the timer.

2. Temperature Control:

• Fluctuations in water bath temperature.

3. Inconsistent Mixing:

• Uneven enzyme-substrate mixing.