Required practical - Investigating enzymes (AQA GCSE Biology Combined Science): Revision Notes
Required practical - Investigating enzymes
What is this practical about?
This practical helps you understand how pH affects enzyme activity. You'll investigate how different pH levels change how quickly the enzyme amylase breaks down starch.
Key fact: Amylase is an enzyme that breaks down starch into simple sugars.
Aim
To investigate the effect of pH on amylase activity using continuous sampling.
Method
Here's what you need to do step by step:
- Set up your spotting tile - Add one drop of iodine solution to each well
- Prepare your test tube - Add:
- 2 cm³ of pH buffer solution
- 2 cm³ of amylase solution
- 2 cm³ of starch solution
- Mix and start timing - Use a glass rod to mix, then start your stopwatch
- Test every 30 seconds - Use the glass rod to transfer one drop to a well on the spotting tile, then rinse the rod
- Keep testing - Repeat step 4 until the iodine solution no longer changes colour, then stop the clock and record the time
- Test different pH values - Repeat steps 1-5 using different pH buffer solutions
Critical Steps for Accurate Results:
- Always rinse the glass rod between tests to prevent contamination
- Start timing immediately after mixing all solutions
- Test at exactly 30-second intervals for consistency
Important points about the method
Temperature Control: All solutions should stay at the same temperature (25°C works well). Use a water bath for this to ensure accurate and reliable results.
Buffer Solutions: These are mixtures that resist changes to their pH. They help keep the pH steady during your experiment, ensuring that the pH remains constant throughout the reaction.
Understanding the Colour Change: When iodine no longer changes colour, all the starch has been digested into simple sugars. This is your endpoint indicator.
Results and analysis
Recording your results
Create a table with these columns:
- pH of buffer
- Time taken to digest starch (in seconds)
- Rate of reaction (in s⁻¹)
Calculating reaction rates
The rate of reaction shows how fast the enzyme works, with higher rates meaning faster reactions.
Worked Example: Calculating Reaction Rate
Formula: Rate =
Given: Reaction takes 200 seconds to complete
Calculation: Rate = = 0.005 s⁻¹
Interpretation: This means the enzyme processes the substrate at a rate of 0.005 reactions per second.
Finding the optimum pH
- Calculate the rate of reaction for each pH value using the formula above
- Plot a graph with:
- Rate of reaction on the vertical axis
- pH on the horizontal axis
- The optimum pH is where the rate is highest - this is where amylase works best
Key Insight: The optimum pH gives the greatest rate of reaction. This represents the pH at which the enzyme's active site has the ideal shape for maximum substrate binding.
What your results will show
Your experimental data will reveal a characteristic pattern:
- At very low or very high pH values, amylase works slowly (low rate)
- At the optimum pH, amylase works fastest (high rate)
- This creates a curve on your graph with a peak at the optimum pH
This bell-shaped curve is typical for enzyme activity versus pH graphs. The enzyme becomes denatured at extreme pH values, explaining the reduced activity away from the optimum.
Why control temperature?
Temperature affects enzyme activity just like pH does. By keeping temperature constant, you can be sure that any changes in reaction rate are only due to pH changes. This makes temperature a crucial control variable in your experiment.
Key Points to Remember:
- Amylase breaks down starch into simple sugars
- Buffer solutions keep pH steady during the experiment
- Rate of reaction =
- The optimum pH gives the fastest reaction rate
- Control variables like temperature to get reliable results
- The colour change indicates when all starch has been digested