Requires Practical 1: Enzymes

Question 1

What are 5 possible factors that you could investigate in this experiment (of seeing how rate of enzyme action changes with a specific factor)?

Answer 1

• Enzyme concentration
• Substrate concentration
• Temperature
• pH
• Inhibitors

Question 2

What is the most common enzyme that is investigated in this required practical?

Answer 2

• Tryspin.

Question 3

If we were to investigate effect of temp/ pH on tryspin, what would be the aim of this experiment?

Answer 3

• Aim would be to find optimum pH/ temperature for enzyme activity and the range of pH/ temperature values trypsin works within.

Question 4

What is the protein found in milk called? What happens when tryspin enzyme breaks down this protein in this investigation?

Answer 4

• PROTEIN: caesin.
• When tryspin is added to the dilute solution of milk powder, the caesin is digested and the solution goes clear.

Question 5

Whatare the 8 steps for investigating the effect of temp on the breakdown of caesin protein in milk by enzyme tryspin?

Does not have to be word for word!!

Answer 5

1.) Draw X on the halfway down three test tubes.
2.) Add same volume of solution of milk powder to the three test-tubes (ensure that the solution is above the X mark.)
3.) Add same volume of tryspin/ buffer solution in three other test-tubes.
4.) Leave all test-tubes in water bath at 20⁰C for 10 mins.
5.) Add solution of trypsin/ buffer solution to each test-tube with solution of milk powder. Return 3 test-tubes to water bath.
6.) Start a stopwatch/ record time (for each test-tube) to see X through solution. Make sure to mix the solution throughout this time.
7.) Calculate the mean time taken to see X through the solution.
8.) Repeat experiment with different temperature water baths.

Question 6

How do you calculate rate of reaction given mean time taken for you to see X through the solution in test-tube after tryspin was added?

Answer 6

Do: 1/ mean time taken.

Question 7

What do we do after we have obtained results for time taken to see X through milk powder solution for each temperature investigated?

Answer 7

• Record data in table.
• Calculate the rates of reaction.
• Plot graph of rate of reaction (y axis) against whatever factor was investigated (ie. temp/ pH) –> x axis.

Question 8

Why do we do a dot- to- dot on the graph of rate of reaction against whatever factor you investigated in the practical?

Answer 8

• Because we don’t have enough data points/ intermediate values to actually predict the pattern.

Question 9

If you have a graph with rate of reaction against pH and the graph peaks at 9, does this necessarily mean that the optimum pH for tryspin activity is 9? Explain why/ why not?

Answer 9

• No.
• Because not enough intermediate values were tested between pH 8.1 and 9.9.

(refer to Miss Estreuch vid if needs be.)

Question 10

What are limitations to this RP method? ASUME that the rate of reaction didn’t reach 0 at any point

Answer 10

1.) End- point for when X was seen was subjective –> leads to innacurrate “time taken” measurements.
2.) Water bath not thermostatically controlled/ temp would have decreased during practical.
3.) Not enough intermediate values are tested to identify the ACTUAL optimum pH/ temperature (OR WHATEVER FACTOR!!)
4.) Range of tested values was not wide enough as point as which trypsin denatures was not identified (test beyond your maximum temp/ pH - or whatever factor.)

Question 11

What is the best equipment to use to add the volumes of milk powder solution/ pH 7 buffer solution and tryspin solution to the test-tubes? Why?

Answer 11

• Graduated pipette.
• Because it has the highest resolution - reduces percentage uncertainty.

Question 12

Why do we leave 6 test-tubes in water bath before adding tryspin/ pH buffer solution to the test-tubes containing milk powder solution?

Answer 12

• To ensure that all solutions reach the same temperature/ equilibrate.

Question 13

How could we ensure that temp of water bath remains as constant as possible when timing how long it takes to see X through the milk powder solution?

Answer 13

• Place thermometer in water bath and add hot/ cold water when the temp rises/ falls.

Question 14

What is the risk of using tryspin? Give safety precuation?

Answer 14

• May cause allergies.
• Avoid contact with skin/ eyes. WEAR GOGGLES.

Question 15

What is the risk of hot liquids in this RP? Give safety precuation?

Answer 15

• May cause burning.
• Use tongs to remove test-tubes from hot water baths/ wear goggles.

Question 16

Why may we draw the X a third from bottom of test-tube rather than halfway down?

Answer 16

• Perhaps you are using smaller volumes, so in order to see X through the solution, the X must be lower down on the test-tube.