2.4.3 Skills: Enzyme Experiments

Design of experiments to test the effect of temperature, pH and substrate concentration on the activity of enzymes

• Three different independent variables can be tested
  • Temperature
  • pH
  • Substrate concentration

• You should plan how the dependent variable is going to be measured
  • With appropriate units

• Also, what intervals of the independent variable are going to be chosen
• These factors dictate the choice of apparatus and other equipment required for the experiment
• The control variables need to be identified and monitored eg. temperature when measuring the effect of pH

Investigating the effects of temperature or pH on catalase activity

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

• 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

• If measuring the effect of temperature on enzyme activity, the conical flask containing potato pieces can be held in a water bath at the required temperature
  • The water level in the water bath must be higher than the level of H₂O₂ in the conical flask, to ensure even heating
  • The conical flask can also be swirled gently to mix the contents and maintain an even temperature

Investigating the effect of substrate concentration on 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 37^oC (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 different starch concentrations and the reaction rates can then be compared
  • This experiment also can be adapted to measure the effects of altering pH, temperature or enzyme concentration

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

Investigating the effect of starch concentration on amylase activity using colorimetry

• A colorimeter is able to 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: this 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 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 colorimeter 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)

Serial dilution of starch to make a range of concentrations

NOS: Experimental design; accurate, quantitative measurements in enzyme experiments require replicates to ensure reliability

• Accurate measurements mean data that are close to the true value
• Quantitative measurements must be made
  • A qualitative measurement might state that, "the enzyme worked at a faster rate at the higher temperature", whereas
  • A quantitative measurement for the same experiment might state that, "the enzyme worked at a rate of 2.3 mmol product minute^-1 at 40°C, versus 1.6 mmol product minute^-1 at 25°C"
  • Quantities, using numbers and appropriate units, are quoted in the experimental results

• Reliable data are generated from repeated experiments
  • Anomalies can be identified and eliminated
  • A reliable mean can be calculated from the data that remain