RP4 - PH's EFFECT ON ENZYME REACTIONS

• Investigating Enzymatic Reactions

  You'll soon know how to investigate the effect of pH on the rate of enzyme activity
• Investigating the Effect of pH on Enzyme Activity

  1. Put a drop of iodine solution into every well of a spotting tile

  2. Place a Bunsen burner on a heat-proof mat, and a tripod and gauze over the Bunsen burner. Put a beaker of water on top of the tripod and heat the water until it is 35 °C

  3. Use a syringe to add 1 cm³ of amylase solution and 1 cm³ of a buffer solution with a pH of 5 to a boiling tube. Put the tube into the beaker of water and wait for five minutes

  4. Use a different syringe to add 5 cm³ of a starch solution to the boiling tube

  5. Immediately mix the contents of the boiling tube and start a stop clock

  6. Use continuous sampling to record how long it takes for the amylase to break down all of the starch

  7. Repeat the whole experiment with buffer solutions of different pH values to see how pH affects the time taken for the starch to be broken down

  8. Control any variables each time (e.g. concentration and volume of amylase solution) to make it a fair test
• Amylase

  Enzyme that catalyses the breakdown of starch to maltose
• Iodine solution


  Changes from browny-orange to blue-black in the presence of starch
• You could use an electric water bath, instead of a Bunsen and a beaker of water, to control the temperature
• You could use a pH meter to accurately measure the pH of your solutions
• Method


  1. Label each well with the time (from 0 onwards) and add a drop of iodine solution to each well

  2. Add 2 cm3 of each buffer solution using a syringe (ranging from pH 3.0 to 7.0) into each labelled test tube

  3. Immerse the starch solution, amylase solution, and the test tubes of buffer solution in a water bath at 25°C

  4. Allow a few minutes for the temperature to equilibrate

  5. Use a syringe to add 2 cm3 of amylase into a test tube of buffer solution

  6. Use a syringe to add 2 cm3 of starch into the same test tube and start timing immediately

  7. Use the glass rod to transfer a drop of the mixture to the well labelled '0' on the tile

  8. Repeat step 6 every 30 seconds, rinsing the glass rod in between every test, until the iodine solution remains brown and does not turn blue-black

  9. Calculate the rate of enzyme reaction by using 1/ time taken for iodine solution to remain brown

  10. Repeat steps 2-8 for buffer solutions with different pH values

  11. Plot a graph of the rate of enzyme reaction against pH