Required Practical: Measuring Enzyme Activity (AQA AS Biology): Revision Note

Required practical: measuring enzyme activity

• 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 over a set time

• The rate of reaction can then be calculated

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 (the substrate) into maltose and glucose

  • Amylase functions best at pH 7 and 37 ^°C (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 (e.g. by altering pH, temperature, enzyme concentration or starch concentration), and the reaction rates can then be compared

Investigating the effect of starch concentration on amylase activity using colorimetry

• A colorimeter can measure light absorbance (how much light is absorbed) or light transmission (how much light passes through) a substance

• Colorimetry can be used in any enzyme-catalysed reaction that involves a 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 colorimeter 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:

  • Colorimeter calibration is an important step in a colorimetric investigation, and in this case, a weak iodine solution can be used to calibrate the colorimeter as the endpoint (or 100 % transmission)

  • Preparation of a starch solution of known concentration (stock solution), from which a range of concentrations is made using serial dilutions

  • 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 colorimeter, 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)

Controlling variables

• Enzyme rate experiments are carried out to determine the effect of changing a particular variable on the rate of a reaction

• Variables that can affect the rate of an enzyme-catalysed reaction include:

  • temperature

  • pH

  • enzyme concentration

  • substrate concentration

• During enzyme rate experiments, only one of these variables must be changed

  • This is known as the independent variable

• All other variables must be controlled (they must stay the same)

  • These are known as the control variables

• For example, if investigating the effect of temperature on the rate of reaction, the pH, enzyme concentration and substrate concentration must be kept constant each time the experiment is carried out

• If these control variables are not kept constant, they could affect the results of the experiment

  • This would make the results invalid

Calculating uncertainty

• Uncertainty is the amount of error your measurements might contain

• Results from experiments (including enzyme rate experiments) always contain some error (they are never perfect)

• There will always be a small degree of uncertainty in your readings or measurements

  • This is often because the sensitivity of the apparatus being used is limited

  • E.g. If using a gas syringe, measuring results to the nearest 1 cm³, there may be a degree of uncertainty if the real volume produced is 0.5 cm³ smaller or larger. The syringe, therefore, has an uncertainty of ± 0.5 cm³

    • A ‘±’ sign tells you the range in which the true value lies; this range is called the margin of error

• For enzyme rate experiments, you may need to calculate the percentage error of your measurements

  • As long as you know the uncertainty value of your measurements, the percentage error can be calculated using the following formula

percentage error = (uncertainty ÷ measured value) x 100