RP4

• Core Practical 4


  Investigate the effect of enzyme and substrate concentrations on the initial rates of reactions
• Factors affecting rate of reaction of an enzyme-controlled reaction

  • Temperature
  • pH
  • Concentration of the substrate
  • Concentration of the enzyme
• Determining the effect of each factor

  1. Changing a single variable

  2. Measuring its effect on the rate of reaction

  3. Keeping all other variables constant
• Initial rate of reaction

  The only point during the reaction when concentration of reactants and products is known
• The rate of an enzyme-controlled reaction is high because enzymes act as biological catalysts, so the concentration of reactants changes rapidly
• Equipment


  • Milk powder solution
  • Trypsin solution (1%)
  • Test tubes
  • Test tube holder
  • Stopclock
  • 5cm3 pipettes
  • Goggles
  • Colorimeter
  • Cuvettes
  • Distilled water
• Method


  1. Dilute stock solution of trypsin with distilled water to produce solutions with concentrations of 0.2%, 0.4%, 0.6% and 0.8%

  2. Make a control by adding 2cm3 of trypsin solution and 2cm3 of distilled water

  3. Add 2cm3 of milk suspension and 2cm3 of the stock trypsin solution, mix, place in the colorimeter and measure absorbance at 15 second intervals for 5 minutes

  4. Rinse the cuvette with distilled water

  5. Repeat step 3 at all trypsin concentrations
• Hazards


  • Broken glass
  • Hot liquids
  • Enzymes
• Casein


  A white protein in milk which, when broken down, causes the milk to turn colourless
• Trypsin


  A protease enzyme which hydrolyses the casein
• As concentration of trypsin increases

  The number of enzyme-substrate complexes forming also increases
• The rate of reaction increases

  Up to the optimum enzyme concentration
• The rate plateaus at the point where all substrates occupy an active site
• Increasing the enzyme concentration won't increase rate as substrate concentration is limiting the rate
• Modification


  Measure the effect of substrate concentration on initial rate of reaction by diluting the milk suspension to produce different concentrations and controlling concentration of trypsin