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

• Amylase is an enzyme that digests starch (a polysaccharide of glucose) into maltose (a disaccharide of glucose)
• The effect of different pH levels on the activity of amylase can be investigated

Apparatus

• Spotting tile
• Measuring cylinder
• Test Tube
• Syringe
• Pipette
• Stopwatch
• Buffer solutions
• Iodine
• Starch solution
• Amylase solution

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 2) 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
• Repeat the investigation with buffers at different pH values (ranging from pH 3.0 to pH 7.0) 

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 orange-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

Limitations

• The starch and amylase solutions that need to be used should be placed in a water bath at optimum temperature before being used
• A colorimeter can be used to measure the progress of the reaction more accurately by measuring the absorbance/transmission of light through the coloured solution
  • A control of iodine solution would be used for comparison

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

Applying CORMS to practical work

• When working with practical investigations, remember to consider your CORMS evaluation

CORMS Evaluation

• In this investigation, your evaluation should look something like this:
  • C - We are changing the pH of the environment
  • O - This is not relevant to this investigation as we aren't using an organism
  • R - We will repeat the investigation several times to ensure reliability
  • M1 - We will measure the time taken for
  • M2 - the iodine to stop turning black
  • S - We will control the concentration and volume of the amylase, iodine and starch solution used in the investigation