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 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 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