Factors affecting enzyme reaction

• 4 factors that affect enzyme activity

  • enzyme concentration
  • substrate concentration
  • temperature
  • pH
• control set up when measuring enzyme activity

  • replace the enzyme solution with distilled water or boiled enzyme solution
• how a colorimeter determines the rate of reaction of trypsin and milk

  • as trypsin digests the proteins milk, milk becomes more translucent and more light passes through
  • decrease in absorbance can be measured by colorimeter
• Procedure to test enzyme concentration on enzyme activity

  1. dilute a solution with distilled water to produce 4 concentrations
  2. zero the colorimeter using 2 cm^3 of milk and 2cm^3 of distilled water
  3. to another cuvette, add 2 cm^3 of milk and 2 cm^3 of enzyme solution
  4. mix and place in the colorimeter and measure absorbance at 15 second intervals for 5 minutes
  5. rins cuvette
  6. repeat for all enzyme concentrations
• rate of reaction = 1 / time
• effect of enzyme concentration on enzyme activity

  • as enzyme concentration increases, successful collisions to form enzyme substrate complexes increase
  • beyond optimum temperature, rate plateaus as substrate concentration becomes limiting
• effect of substrate concentration on enzyme activity investigation

  1. Prepare a simple dilution of milk/substrate concentrations
  2. add each solution to 1cm^3 of trypsin of a fixed concentration
  3. record the absorbance immediately and every 15 seconds for 5 minutes
• effect of substrate concentration on enzyme activity

  • enzyme activity increases initially as substrate concentration increases
  • higher concentration results in more successful collisions to form enzyme substrate complexes
  • beyond a certain substrate concentration, enzyme acticity plateaus, as all the enzyme active sites are saturated and enzyme concentration is limiting
• pH on enzyme activity investigation

  1. add fixed volumes of buffer solutions with a range of pH values to 1 cm^3 of trypsin and 2 cm^3 of milk
  2. record the absorbance immediately and every 15 seconds for 5 minutes
• effect of pH on enzyme activity

  • enzyme activity is at highest at the optimum pH
  • above or below the optimum pH, enzyme activity decreases
  • the unsuitable pH disrupts it tertiary structure and changes the shape of its active site, causing partial denaturation
  • complete denaturation may occur at extreme pH values
• effect of temperature on enzyme activity investigation

  1. prepare water baths with a range of temperatures
  2. place 2cm^3 of trypsin solution and 2cm^3 of milk in each water bath
  3. leave for 5 minutes to allow the solutions to reach the temperature of the water bath
  4. mix together and record the absorbance immediately and every 15 seconds for 5 minutes
• effect of temperature on enzyme activity

  • increasing temperature increases enzyme activity to an optimum
  • enzyme and substrate gain kinetic energy and move faster so there are more successful collisions to form enzyme substrate complexes
  • beyond optimum temperature, enzyme activity decreases as the high temperature disrupts the tertiary structure of enzymes and denatures them
• hazards and safety precautions for enzyme activity

  • may have allergic reactions to enzymes so avoid contact with skin and eyes and wear eye protection
  • glassware is sharp
  • water baths can cause scalding
  • milk can be in lab for a while so dont drink it