Practical: Investigating pH & Enzyme Activity (Edexcel IGCSE Biology): Revision Note

Practical: Enzymes & pH

• Amylase is an enzyme that digests starch into maltose

• The effect of different pH levels on the activity of amylase can be investigated using buffer solutions at different pHs

Apparatus

• Spotting tile

• Measuring cylinder

• Test Tube

• Syringe

• Pipette

• Stopwatch

• Buffer solutions at different pH levels

• Iodine

• Starch solution

• Amylase solution

Safety

• Both iodine solution and amylase solution can cause irritation to eyes - wear safety goggles

• Amylase solution can cause irritation to the skin - wear gloves

Method

• Add a drop of iodine to each of the wells of a spotting tile

• Use a syringe to place 2 cm³ of amylase into a test tube

• Add 1cm³ of buffer solution (at pH 3) to the test tube using a syringe

• Use another test tube to add 2 cm³ of starch solution to the amylase and buffer solution, start the stopwatch whilst mixing using a pipette

• Every 10 seconds, transfer a droplet of the solution to a new well of iodine solution (which should turn blue-black)

• Repeat this transfer process every 10 seconds until the iodine solution stops turning blue-black (this means the amylase has broken down all the starch)

• Record the time taken for the reaction to be completed by counting the number of tiles up to when the iodine stops changing colour and remains yellow-brown

• Repeat the investigation with buffers at different pH values (eg. pH 5.0, 7.0, 9.0 and 11.0) 

Investigating the effect of pH on enzyme activity

Results and Analysis

• Amylase is an enzyme which breaks down starch

• When the iodine solution remains orange-brown, all the starch has been digested

• This investigation shows:

  • At the optimum pH, the iodine stopped turning blue-black and remained yellow-brown within the shortest amount of time

    • This is because the enzyme is working at its fastest rate and has digested all the starch

• At higher or lower pH's (above or below the optimum) the iodine took a longer time to stop turning blue-black or continued to turn blue-black for the entire investigation

  • This is because on either side of the optimum pH, the enzymes are starting to become denatured and as a result are unable to bind with the starch or break it down

A graph showing the optimum pH for an enzyme from a region of the small intestine

Applying CORMS to practical work

• CORMS can be used to plan a valid investigation on the effect of pH on enzyme activity

CORMS Evaluation

• The CORMS for an investigation into the effect of pH on enzyme activity might look like this:

  • C - change the pH of the environment

  • O - same concentration of enzyme

  • R - repeat the investigation several times to ensure reliability

  • M1 - measure the time taken by counting the number of wells for

  • M2 - the iodine to stop turning blue-black (1 well = 10 seconds)

  • S -control the temperature and volume of the amylase, iodine and starch solution used in the investigation