RP5: Effect of PH on Amylase

Question 1

What is the aim of the experiment?

Answer 1

• Use continuous sampling with iodine solution to determine the time taken for starch (substrate) to be completely digested, hence calculate the rate of enzyme activity at different pH values

Question 2

Describe how you would investigate the effect of pH on the rate of reaction of the amylase enzyme (method)

Answer 2

1) place one drop of iodine solution in each well of spotting tile
2) 3 test tubes - one with 2cm³ starch solution, one with 2cm³ amylase solution + one with 2cm³ pH 5 buffer solution
3) place all three in a 30º water bath and leave for 10 minutes to allow solutions to reach correct temperature
4) combine all solutions into one test tube and mix with stirring rod. return to water bath and start stopwatch
5) after 30 seconds use stirring rod to add one drop of solution to one well in spotting tile which contains iodine
6) the iodine should turn blue-black showing presence of starch
7) take sample every thirty seconds and continue until iodine remains orange; this tells us starch is no longer present (reaction has completed)
8) record time and repeat whole experiment several times using different pH buffers

Question 3

What are some problems with the experiment?

Answer 3

• only taking samples every thirty seconds; we only have an approximate time for the reaction to complete
• SOLUTION: take samples every ten seconds
• we are looking for the time when iodine does not go blue-black; this is not always obvious. some wells might have a small amount of blue-black mixed with orange so it can be difficult to see when the reaction has finished
• SOLUTION: ask several people to look at the spotting tile and decide when the reaction has completed

Question 4

Which enzyme catalyses the breakdown of starch in the human digestive system?

Answer 4

• Amylase to maltose

Question 5

Why must samples be left in the water bath before solutions are mixed?

Answer 5

• To allow temperature to equilibrate

Question 6

What is a buffer solution?

Answer 6

• A solution which can resist changes in pH

Question 7

What are the potential risks?

Answer 7

• iodine solution is an irritant
• hot water

Question 8

Name some sources of error

Answer 8

• Intervals between testing may be too long to accurately find the time taken for starch to be
  completely broken down.