Biology AS 3

• Using biochemical tests to identify carbohydrates and protein

  1. Qualitative tests

  2. Determine presence or absence

  3. Do not show quantity
• Carbohydrate

  • Benedict's test - Detects reducing sugars
  • Glucose-specific tests - Detect glucose
  • Iodine test - Detects starch
• Reducing sugars


  Sugars that can donate electrons to (or reduce) Benedict's reagent
• Non-reducing sugars


  Sugars that do not reduce Benedict's reagent initially, but can be hydrolysed into reducing sugars
• Protein


  Biuret test - Detects presence of peptide links
• Identifying amino acids through paper chromatography

  1. Preparing the chromatogram

  2. Running the chromatogram

  3. Developing the chromatogram

  4. Calculating Rf values
• Rf value


  Distance moved by solute / Distance moved by solvent front
• Rf values are approximately the same for the same amino acids separated by chromatography in the same solvent
• Rf value should be less than 1
• Enzyme investigations


  1. Enzyme activity is affected by substrate concentration, enzyme concentration, pH and temperature

  2. Other variables must be controlled when investigating one factor

  3. For example, when measuring effect of pH, enzyme concentration, substrate concentration and temperature must be kept constant
• • In some investigations, increase in product is measured (e.g. oxygen production in hydrogen peroxide-catalase reaction)
  • In others, decrease in substrate is measured (e.g. reduction in starch in starch-amylase reaction)
• Effect of temperature on enzyme activity

  1. Use an appropriate number and range of temperatures (e.g. 20°C, 30°C, 40°C, 50°C, 60°C)

  2. Control and monitor experimental temperatures using thermostatically controlled water baths

  3. Control other variables like pH, enzyme volume/concentration, substrate volume/concentration
• Effect of substrate concentration on enzyme activity

  1. Use a suitable number and range of substrate concentrations

  2. May need to do a pilot investigation to determine appropriate range

  3. Control temperature, enzyme volume/concentration, substrate volume, pH
• • Results may show a linear relationship between substrate concentration and reaction rate
  • But rate may level off as enzyme becomes limiting when substrate is in excess
• Conclusions on effect of substrate concentration

  Rate of formation of enzyme-substrate complexes increases due to more collisions between enzyme and substrate
• Effect of enzyme concentration on enzyme activity

  1. Use a suitable number and range of enzyme concentrations

  2. Control temperature, substrate volume/concentration, enzyme volume, pH
• Conclusions on effect of enzyme concentration

  Rate of formation of enzyme-substrate complexes increases due to more enzyme molecules present
• pH


  • Indication of hydrogen ion concentration
  • Acidic solutions have high H+ concentration, alkaline solutions have low H+ concentration
  • Water has pH 7 (neutral)
  • Many enzymes work best at pH close to 7
• Effect of pH on enzyme activity

  1. Investigate enzyme activity over a suitable range of pH values

  2. Control temperature, enzyme volume/concentration, substrate volume/concentration

  3. Use buffers to maintain desired pH
• • Enzyme activity is highest at optimum pH
  • Activity falls off at pH values above and below the optimum
  • Changes in pH affect charges in enzyme active site, disrupting enzyme-substrate binding
• Enzyme and substrate choice

  • May use purified substrates and enzymes
  • May use unpurified sources like plant/animal tissues which adds variables
  • Factors to consider include type, age, preparation of tissue, measuring reaction rate
• Preparing dilutions


  1. Can use simple arithmetic dilutions like 100%, 80%, 60%, 40%, 20%

  2. Use fresh pipettes/tips for each dilution to prevent carry-over

  3. Thoroughly mix each dilution
• Serial dilutions


  • Each solution is less concentrated than the previous by a set factor (e.g. 1:10 dilution)
  • Allows producing a large range of concentrations
• Serial dilutions are useful for preparing calibration curves when using colorimeters
• Immobilised lactase


  • Lactase enzyme immobilised in alginate beads
  • Can be used to demonstrate breakdown of lactose in milk
• Using a colorimeter


  • Measures change in light intensity passing through a solution
  • Can measure absorbance or transmission
  • Important to calibrate and choose appropriate filter
• When using a colorimeter, it is important to maximise accuracy by ensuring clean cuvettes, correct filling level, consistent orientation, and regular re-calibration
• As an enzyme reaction progresses

  The solution can become more or less turbid (cloudy)
• In a starch-amylase reaction, the percentage of initial starch remaining over time should follow a characteristic curve shape
• Explanation of curve shape

  Rapid rate at start when starch concentration is high, then rate slows as starch is depleted
• Maximising accuracy of readings and hence validity in practical investigations

  • Ensure the cuvettes are clean
  • Rinse and dry the cuvette between samples
  • Ensure the cuvettes are filled to the correct level
  • Ensure the orientation of the cuvette in the colorimeter is correct
  • Re-calibrate every so often by placing the 'blank' back in the colorimeter, and re-calibrate if necessary
• Whether using absorbance or transmission

  The values of the percentage of initial starch remaining against time in a starch-amylase reaction should follow the pattern shown in the diagram
• The course of starch-amylase reaction

  1. Rapid rate at the start when starch concentration is high

  2. Rate decreases over time as starch molecules decrease and product molecules (maltese) 'block' the movement of the enzyme and substrate
• Calibration graph


  Involves measuring transmission or absorbance values for known concentrations of the substance being investigated, to allow colorimeter readings to be expressed in terms of substance concentrations
• Producing a calibration graph

  1. Start with a known 'standard' concentration

  2. Make a range of concentrations

  3. Measure the % transmission (or % absorbance) for each

  4. Plot a graph with % transmission on y-axis and concentration on x-axis
• Graphs


  • Have a title identifying the independent variable, dependent variable and biological material
  • Independent variable on x-axis, dependent variable on y-axis
  • Use suitable scales and label axes
  • Generally use straight lines to join points, but a line of best fit can be used if appropriate
  • Do not extrapolate beyond the range of data points unless there is good reason
• Line graphs


  Used when both IV and DV are continuous, and there is a causal link between them
• Bar charts


  Used when the IV is discontinuous or categoric
• Scattergrams


  Used when both IV and DV are continuous but there is not necessarily a causal link between them
• Calculating rate of change from a linear graph


  Read off values at two time points, divide difference in values by time difference to get average rate