Practical: Investigating Enzymatic Reactions

Section 1 of 7The reaction being studied

Amylase and starch

• Amylase and starch Amylase is an enzyme that digests starch into the sugar maltose.

Iodine test

• Iodine test Iodine solution is orange-brown, but turns blue-black when starch is present.
• The reaction is finished when a sample no longer turns blue-black.

Measuring activity

• Measuring activity The time taken for the starch to disappear is recorded;
  • a shorter time means a faster rate.

Section 2 of 7Effect of pH: method

1

Place a drop of iodine in each well of a spotting tile.

2

Put amylase solution into a test tube and add a buffer solution of a chosen pH.

3

Add starch solution, start the stopwatch and mix.

4

Every 10 seconds transfer a drop of the mixture into a fresh well of iodine.

5

Stop when the iodine stays orange-brown and record the time.

6

Repeat with buffers at other pH values (for example pH 3 to pH 7).

Section 3 of 7Effect of temperature: changes to the method

Feature	Detail
Independent variable	Temperature, set using a water bath
Dependent variable	Time for the starch to be digested
Control variables	Volumes and concentrations of enzyme and starch, pH (use a buffer)
Sampling	A drop is tested in iodine every minute
Improvement	A colorimeter can measure the colour change more objectively

Section 4 of 7Interpreting results

Enzyme activity

Optimum

• Optimum At the optimum pH or temperature, the starch disappears in the shortest time.

Too acidic or too alkaline

• Too acidic or too alkaline The enzyme starts to denature;
  • the active site changes shape, so starch cannot bind.

Low temperature

• Low temperature Particles have less energy, so there are fewer collisions;
  • the enzyme is slow but not denatured.

High temperature

• High temperature Above the optimum the enzyme denatures and activity stops.

Section 5 of 7Calculating rate

1

Rate = change ÷ time, where the change is the amount of substrate used up or product formed.

2

Example: 15 g of starch digested in 2 hours gives 15 ÷ 2 = 7.5 g per hour.

3

Example: 45 cm³ of oxygen released in 5 minutes gives 45 ÷ 5 = 9 cm³ per minute.

4

If only the time is known, use rate = 1 ÷ time.

Section 6 of 7Key terms

Term	Meaning
Enzyme	A biological catalyst, usually a protein, that speeds up a reaction.
Denatured	Permanently changed in shape so the active site no longer fits the substrate.
Optimum	The condition at which the enzyme works fastest.
Buffer solution	A solution that keeps the pH constant.
Substrate	The substance that an enzyme acts on.

Section 7 of 7Exam technique

1

Saying low temperatures denature enzymes - they only slow them down.

2

Forgetting to keep pH constant when changing temperature, or temperature constant when changing pH.

3

Saying that blue-black means the starch has gone - it means starch is still present.

4

Mixing the iodine dropsuse a fresh well for each sample.