IB DP Biology: SL复习笔记2.4.3 Skills: Enzyme Experiments

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

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

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

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

Exam Tip

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

 

 

 

 

 

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