required practical 1

Question 1

what is the aim of this practical?

Answer 1

to investigate the effect of a named variable (temperature) on an enzyme-controlled reaction

Question 2

what is the hypothesis for this practical?

Answer 2

that temperature will affect the rate of a trypsin-catalysed reaction

Question 3

what is caesin?

Answer 3

a protein found in milk

Question 4

what is trypsin?

Answer 4

an enzyme which digests caesin - when trypsin is added to a dilute solution of milk powder, the caesin is digested and the solution goes clear

Question 5

what is the risk assessment for the practical?

Answer 5

hazard/risk/prevention
trypsin splashing on skin/allergic reaction/ wear gloves or wash with cold water if contact occurs
trypsin splashing in eyes/ damage to eyes/ wear goggles
milk powder/ inhalation or allergic reaction with skin contact/ wear gloves or wash with cold water if contact occurs, take care not to inhale
buffer solution/ allergic reaction or burns to skin if contact/ wear gloves or wash with cold water if contact occurs
hot water from water bath/ scalds or burns to skin or eyes/ wear eye protection and use heat proof gloves to remove test tubes from water bath

Question 6

what is the method for the practical?

Answer 6

1. use a pen to label three test tubes ‘milk’ and draw an X on the glass halfway down each one
2. measure and add 10cm^3 of milk to each test tube
3. label three test tubes ‘trypsin’ and add 2cm^3 of trypsin solution and 2cm^3 of pH 7 buffer to each tube
4. add all the test tubes to a water bath at 20 degrees and leave for 10 minutes to equilibrate
5. add the trypsin/buffer solution from one tube to the milk in another test tube, start a timer and mix thoroughly (using graduated pipette)
6. put the test tube back in the water bath and time how long it takes for the solution to go clear (by measuring time taken to first see X through solution)
7. repeat this for the other two pre-prepared test tubes and record the times in a table
8. repeat the whole method but for water baths at 30, 40, 50 and 60 degrees
9. calculate a mean time for each temperature and then the rate of reaction, before drawing a graph to display results

Question 7

how do you calculate the rate of reaction?

Answer 7

1/ time (s) x 1000
units = s^-1 x 1000

Question 8

what is the conclusion for the reaction?

Answer 8

initially as the temperature increases, rate of reaction increases because kinetic energy increases so more enzyme-substrate complexes form
the temperature reaches the optimum where rate of reaction peaks, and after this it decreases because bonds in the tertiary structure of the enzyme break so the active site changes shape
eventually the enzyme denatures because the active site changes shape so much that it is no longer complementary to the substrate