• The?progress?of?enzyme-catalysed reactions?can be investigated by:
  • Measuring the?rate of formation of a product?using catalase
  • Measuring the?rate of disappearance of a substrate?using amylase

   

Investigating catalase activity

• In this investigation, the rate of?product formation?is used to measure the rate of an enzyme-controlled reaction:
  • Hydrogen peroxide?is a common but?toxic?by-product of metabolism
  • This means it must be?broken down?quickly
  • Catalase?is an enzyme found in the cells of most organisms that?breaks down hydrogen peroxide into water and oxygen
  • Hydrogen peroxide and catalase are combined and the?volume of oxygen generated?is measured in a set time
  • The?rate of reaction?can then be calculated

   

Experimental set-up for investigating the rate of formation of a product using catalase

Investigating amylase activity using iodine

• In this investigation, the rate of?substrate disappearance?is used to compare rates of reaction under different conditions
• Amylase?is a digestive enzyme that?hydrolyses starch into maltose and glucose
• Amylase functions best at pH 7 and 37oC (all enzymes operate best under specific conditions)
• Amylase and starch are combined?and this reaction mixture is then?tested for starch?at regular time intervals
• This can be done by taking samples from the reaction mixture at each time interval and adding each sample to some?iodine in potassium iodide solution
  • Starch forms a?blue-black?colour with this solution
  • If no starch is present, the iodine solution remains yellow-brown

   

• In this way, the time taken for starch to be broken down can be measured
• The investigation can be repeated under a variety of conditions?(eg. by altering pH, temperature, enzyme concentration or starch concentration) and the?reaction rates can then be compared

Experimental set-up for investigating the rate of disappearance of a substrate using amylase

Investigating the effect of starch concentration on amylase activity using colourimetry

• A?colourimeter?is able to measure?light absorbance?(how much light is absorbed) or?light transmission?(how much light passes through) a substance
• Colourimetry can be used in any enzyme-catalysed reaction that involves colour change
• As the colour breaks down the?transmission increases?or?light absorption decreases?and this can be used to?measure the rate of the reaction
• For example, a colourimeter can be used to follow the progress of a?starch-amylase catalysed reaction?as the amylase breaks the starch down into maltose
• This can be carried out as follows:
  • Colourimeter calibration: this is an important step in a colourimetric investigation and in this case a weak iodine solution can be used to calibrate the colourimeter as the end point (or 100% transmission)
  • Preparation of a starch solution of?known concentration?(stock solution), from which a range of concentrations are made using?serial dilutions?(method outlined in diagram below)
  • Following calibration and switching on the red filter (to maximise the percentage transmission or absorbance), the colourimeter is used to?measure the percentage absorbance or percentage transmission values
  • Sometimes a reagent or indicator is used to produce the colours detected by the colourimeter and sometimes the solutions themselves absorb light waves
  • A?calibration graph?is then plotted of starch concentration (X-axis) vs percentage absorbance or percentage transmission (Y-axis)

   

Serial dilution of starch to make a range of concentrations