RP 5 - Enzymes

• (5) Enzymes practical aim:

  Use continuous sampling with iodine solution to determine the time taken for starch (the substrate) to be completely digested, hence calculate the rate of enzyme activity at different pH values.
• (5) Enzymes practical equipment:

  ● test tubes

  ● a test tube rack

  ● water baths (electrical or Bunsen burners and beakers)

  ● spotting tiles

  ● a 5 cm3 measuring cylinder

  ● syringes

  ● a glass rod

  ● a stopwatch

  ● starch solution

  ● amylase solution

  ● buffered solutions

  ● iodine solution

  ● thermometer
• (5) Enzymes practical equipment method 1-6:

  1. On a tile, 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
• (5) Enzymes practical equipment method 7-11:

  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.
• (5) Enzymes practical sources of error:

  Intervals between testing may be too long to accurately find the time taken for starch to be completely broken down.
• (5) Enzymes practical safety precautions:

  Be careful using hot water. If using a Bunsen burner tie long hair back and wear goggles.