Required Practical 1 - Enzyme-controlled reactions

Required Practical 1

• To measure the rate of an enzyme-controlled reaction we can:
• Measure how fast the product of a reaction appears and use this to compare the rate of reaction under different conditions
• Measure how fast the substrate is broken down and use this to compare the rate of reaction under different conditions

Method 1- how fast product appears

• Catalase catalyses the breakdown of hydrogen peroxide into water and oxygen, we can measure the volume of oxygen produced and measure how fast its given off.
• Set up boiling tubes containing the same volume and concentration of hydrogen peroxide and a suitable buffer solution.
• Set up apparatus as shown in diagram.
• Put each boiling tube in a water bath set to a different temperature.
• Use a pipette to add equal volume and concentration of catalase to each boiling tube.
• Record how much oxygen is produced after 60s.
• Repeat experimen 3 times at each temp. and use results to calculate mean vol. of oxygen produced
• Calculate the mean rate of reaction
• 

Method 2 - how fast substrate is broken down

• Amylase catalyses the breakdown of starch to maltose. By using iodine in potasium iodide solution you can test for the presence of starch - this can be used to work out the rate of reaction for different concentrations of enzyme.
• Put a drop of iodine in potassium iodide solution into each well on a spotting tile. Label the wells.
• Mix together a known concentration and volume of starch and amylase in a test tube.
• Use a dropping pipette to put a drop of the mixture into one of the wells at regular intervals e.g 10 seconds.
• Observe the resulting colour. If the iodine solution goes a blue-black colour then starch is present, if it stays browny-orange no starch is present.
• Record how long it takes for the iodine solution to no longer turn blue-black.

Variables

• The previous 2 experiments can be altered to investigate the effect of a different variable e.g. pH or substrate concentration.
• However, only one variable can be altered at a time.