RPQ 1

• measuring the rate of an enzyme controlled reaction
• can measure how fast the product of the reaction appears and use this to compare rate of reaction under different conditions
• catalase catalyses the breakdown of hydrogen peroxide into water and oxygen - easy to measure the volume of oxygen produced and to work out how fast it is given off
• oxygen released displaces water from the measuring cylinder
• 1. set up boiling tubes containing the same volume and concentration of hydrogen peroxide - to keep pH constant - add equal volumes of a suitable buffer solution to each boiling tube
• 2. set up apparatus
• 3. put each boiling tube in a water bath set to a different temperature along with another boiling tube containing catalase - wait 5 minutes before moving on to the next step so the enzyme gets up to temperature
• 4. use a pipette to add the same volume and concentration of catalase to each boiling tube then quickly attach the bung and delivery tube
• 5. record how much oxygen is made in the first minute of the reaction - use a stopwatch
• 6. repeat the experiment at each temperature 3 times and use the results to find the mean volume of oxygen produced
• 7. calculate the mean rate of reaction at each temperature by dividing the volume of oxygen produced by the time taken

  units in cm3 s-1
• you can measure how fast the substrate is broken down and use this to compare the rate of reaction under different conditions
• enzyme amylase catalyses the breakdown of starch to maltose - can test for the presence of starch with iodine in potassium iodine solution
• Experiment to observe amylase activity

  1. Put a drop of iodine in potassium iodine solution into each well on a spotting tile

  2. Label the wells to help read results

  3. Mix together a known concentration and volume of amylase and starch in a test tube

  4. Use a dropping pipette to put a drop of this solution/mixture into one of the wells containing the iodine solution at regular intervals

  5. Observe the resulting colour - iodine solution goes dark blue-black when starch is present but remains its normal browny-orange colour where there is not starch

  6. Record how long it takes for the iodine solution to no longer turn blue-black when the starch/amylase is added

  7. Repeat experiment using different concentrations of amylase

  8. Repeat the experiment three times at each amylase concentration and use your results to find the mean time taken
• Iodine solution


  Goes dark blue-black when starch is present, remains browny-orange where there is no starch
• Amylase activity


  Can be observed by recording how long it takes for the iodine solution to no longer turn blue-black when the starch/amylase is added
• variables: can alter these experiments to investigate the effect of a different variable, such as pH (by adding a buffer solution with a different pH to each test tube), substrate concentration (use serial dilutions to make substrate solutions with different concentrations)

  key to experiments like this is to remember to only change one variable at a time
• You can use a tangent to estimate the initial rate of reaction from a graph. The initial rate of reaction is the rate of reaction at the start of the reaction, close to time = 0 on the graph
• to work out the initial rate of reaction:

  1. draw a tangent to the curve at t=0 - position the ruler so it is an equal distance from the curve at both sides of where it is touching it - have to estimate where the curve would continue if it carried on below zero
  2. calculate the gradient of the tangent - this is the initial rate of reaction - gradient = change in y / change in x
  3. need to work out units of the rate - vary depending on what was measured in experiment - divide units of y axis by units of x axis
• why is it important to add the buffer solution to the enzyme before adding the starch

  • because the reaction will begin immediately
  • you would be measuring the effect after the reaction had started - results not valid
• Testing amylase activity


  1. Place a single drop of iodine solution in rows on the tile

  2. Label the test tube with the pH to be tested

  3. Use the syringe to place 2cm3 of amylase into the test tube

  4. Add 5cm3 buffer solution to the test tube

  5. Use another syringe to add 2cm3 starch to the amylase/ buffer solution - start the stopclock and leave throughout the test - mix using a plastic pipette

  6. After 10 seconds use the plastic pipette to place one drop of the mixture on the first drop of iodine - should turn blue-black

  7. Wait another 10 seconds then remove second drop and add to next drop of iodine

  8. Repeat until iodine and amylase/buffer /starch mixture remain orange

  9. Could prepare control drop

  10. Count up how many iodine drops used - each one equals 10 seconds

  11. Repeat whole procedure with another of the pH buffers
• investigation could be improved through use of colorimter, glass pipette (graduated) - one drop not always same size
• when hydrogen peroxide is broken down, oxygen, and water are released
• when trypsin breaks down, gelatine, amino acids are formed
• the difference between a time taken and rate of reaction graph is that time taken will form a u shape and rate of reaction will form a bell-shaped - bell shape easier to describe and analyse
• % uncertainty = uncertainty/ value X 100
• % uncertainty = uncertainty/mean value X 100 (in repeated measures)