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First teaching 2014

Last exams 2024

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Skills: Enzyme Experiments (DP IB Biology: SL)

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Practical 3: 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

Catalase experiment, downloadable AS & A Level Biology revision notes

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

Amylase experiment, downloadable AS & A Level Biology revision notes

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 dilutions, downloadable AS & A Level Biology revision notes

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

Examiner Tip

RE-member: RE-peats bring RE-liability to experimental data.

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Phil

Author: Phil

Expertise: Biology

Phil has a BSc in Biochemistry from the University of Birmingham, followed by an MBA from Manchester Business School. He has 15 years of teaching and tutoring experience, teaching Biology in schools before becoming director of a growing tuition agency. He has also examined Biology for one of the leading UK exam boards. Phil has a particular passion for empowering students to overcome their fear of numbers in a scientific context.