Required Practical 1

• Examples of variables that could affect the rate if an enzyme controlled reaction are:

  • Enzyme concentration or volume
  • Substrate concentration or volume
  • Temperature of solution
  • pH of solution
  • Inhibitior concentration

  These are also the independent variables
• Temperature can be controlled using a thermostatically controlled water bath. It is also monitored using a thermometer at regular intervals and add hot and cold water if the temperature fluctuates
• pH can be controlled using a buffer solution and monitiored using a pH meter at regular intervals
• Enzymes and substrate solutions left in the water bath for 10 mins before mixing so the solutions equilibriate and reach the temperature of the water bath
• A control experiment would be to use denaturede enzymes by boiling. Everything else should be kept the same as the experiment, like the same concentration or volume of substrate at the start and also the enzyme, same type of buffer solution and same temperature.
• The rate of an enzyme controlled reaction can be measured by:

  • Measuring the time taken for the reaction to reach a set point, e.g, concentration/ volume/ mass/ colour of substrate or product
  • Measure the concentration/ volume/ mass/ colour of substrate or product at regular intervals throughout the reaction
• Rate of reaction = 1/ time units= s-1
• A safety risk would be handling enzymes may cause an allergic reaction and to reduce this risk you would have to avoid contact with skin by wearing gloves and eye protection
• Using a colorimeter to measure colour changes is better than comparison to colour standards because it is not subjective and it is more accurate
• A procedure that could be used to stop each reaction would be to:

  1. Boil and add either a strong alkali or acid in order to denature the enzyme
  2. Then put it in ice to lower the kinetic energy so no enzyme substrate complexes form
  3. Add high concentration of inhibitor so no enzyme substrate complexes form
• The rate of reaction decreases over time throughout each experiment because the initial rate is highest as the substrate concentration is not limiting so many enzyme substrate complexes form. The reaction then slows as the substrate is used up and often stops as there is no substrate left.