4: Enzymes and Metabolism - Practical

Question 1

4.1: Demonstration of the action of catalase on hydrogen peroxide

Cubes of tissue are added to 5cm³ hydrogen peroxide solution in different test tubes.
What is the dependent variable, and how is it measured and manipulated? (3)

Answer 1

The dependent variable is the breakdown of the hydrogen peroxide, which is measured by the presence of oxygen. This is manipulated by observing whether the glowing splint relights when it is inserted into the test tube containing hydrogen peroxide and tissue. If the glowing splint relights, then oxygen is present.

Question 2

4.1: Demonstration of the action of catalase on hydrogen peroxide

Cubes of tissue are added to 5cm³ hydrogen peroxide solution in different test tubes.
List 4 important controlled variables. (4)

Answer 2

Volume and concentration of the hydrogen peroxide solution,
Dimensions / mass of the tissue (surface area is indirect, because it is derived from dimensions),
Temperature of the hydrogen peroxide solution,
Time of immersion of the tissue inside the hydrogen peroxide solution.

Question 3

4.1: Demonstration of the action of catalase on hydrogen peroxide

Cubes of tissue are added to 5cm³ hydrogen peroxide solution in different test tubes.
What is the purpose of setting up a test tube with no tissue added?

Answer 3

It is a control to show that no oxygen is given off from hydrogen peroxide solution without tissue.

Question 4

4.1: Demonstration of the action of catalase on hydrogen peroxide

Cubes of tissue are added to 5cm³ hydrogen peroxide solution in different test tubes. The presence of oxygen is tested.
List 3 sources or error and possible improvements of the investigation.

Answer 4

1. The tissue may not be fresh enough; the catalyse inside the tissue might have lost their catalytic ability -> Replace all tissue with fresh tissue
2. There was not enough oxygen to relight the glowing splint -> Increase the time of immersion, which increases the chance that the reaction is complete; or use quantitative tests to detect the presence of oxygen.
3. Repeat and do more trials to increase the reliability of the results

Question 5

4.1: Demonstration of the action of catalase on hydrogen peroxide

How can the experiment be modified to show that the breakdown of hydrogen peroxide is due to the catalytic action of an enzyme?

Answer 5

Boiled tissues, instead of fresh tissues, can be used in a further investigation. If the boiled tissues have no catalytic action, it is more likely that the reaction is catalysed by an enzyme.

Question 6

4.2: Temperature’s effects on enzyme activity

The test tubes of amylase and starch are left in water baths at different temperatures for 10 minutes before mixing.
What is the significance of this step?

Answer 6

This is to allow the solutions to reach the set temperatures before mixing.

Question 7

4.2: Temperature’s effects on enzyme activity

Why should a clean dropper be used to transfer each mixture?

Answer 7

To prevent contamination by any residue inside the used dropper.

Question 8

4.2: Temperature’s effects on enzyme activity

Explain the change of amylase activity with temperature with reference to the graph. (4)

Note: blue-black colour does not disappear at 100ºC

Answer 8

Amylase is inactive at low temperatures. Its activity increases with temperature and it the highest at 40°C. Afterwards the activity decreases and stops at 100°C.
When temperature increases, the kinetic energy of amylase and starch molecules increases. The speed of the movement of amylase and starch molecules increases,
the molecules collide more frequently and the chance of successful collision between them increases to form enzyme-substrate complexes. Therefore, the rate of the enzymatic reaction increases.
As the temperature increases further, more enzymes are denatured and the reaction rate decreased; at 100°C, all amylase is denatured and no reaction takes place.

Question 9

4.2: Temperature’s effects on enzyme activity

Predict and explain the time taken for disapearance of the blue-black colour if the starch-amylase mixture at 0ºC was warmed to 37ºC. (4)

Answer 9

The time taken for the disappearance of the blue-black colour would decrease as temperature of the starch-amylase mixtures increases from 0ºC to 37ºC.
The inactive amylase becomes active and increases its activity when the temperature rises. As temperature increases, there is more kinetic energy for the starch and amylase, so they vibrate faster.
As a result, there is a higher chance of successful collision between starch and amylase, forming more starch-amylase complexes. The rate of the enzymatic reaction increases.
Therefore, starch is catalysed by amylase to break down into maltose at a higher rate; the colour of the iodine solution will turn form blue black to brown faster.

Question 10

4.2: Temperature’s effects on enzyme activity

Predict and explain the time taken for disapearance of the blue-black colour if the starch-amylase mixture at 100ºC was cooled to 37ºC. (3)

Answer 10

The blue-black colour of the iodine solution will not disappear. Amylase is denatured at 100°C and the conformation of the active site of the amylase is permanently changed.
Even when the mixture is cooled, the activity of the amylase would not be restored. Starch can no longer fit into the active site of the amylase, so no starch-amylase complex is formed.
Therefore, starch cannot be catalysed by amylase to break down into maltose, and the blue-black colour of the iodine solution would remain.

Question 11

4.3: Design an investigation of the effect of pH on enzyme activity

What is the biological principle behind the design of the investigation? (3)

Answer 11

To study the effect of pH on amylase activity, starch-agar plates can be used.
When incubated with filter paper discs soaked with buffer solutions at different pH values and amylase, the starch in the starch-agar will be broken down and clear zones will be observed.
The larger the diameter of the clear zone, the higher the amylase activity.

Question 12

4.3: Design an investigation of the effect of pH on enzyme activity

What is the independent variable of the investigation, and how is it manipulated? (2)

Answer 12

The pH of the solutions used to soak the filter paper discs, manipulated by adding buffer solutions of different pH into the solutions.

Question 13

4.3: Design an investigation of the effect of pH on enzyme activity

What is the dependent variable of the investigation, and how is it measured? (2)

Answer 13

The activity of the amylase,
which is measured by the diameter of the clear zone on the starch agar plate, using a ruler.

Question 14

4.3: Design an investigation of the effect of pH on enzyme activity

List 4 important controlled variables of this investigation. (4)

Answer 14

Volume / concentration of the buffer and amylase solutions added into the wells of the spot plate,
Time for incubating the starch agar plate with filter paper discs,
Temperature for incubating the starch agar plate with filter paper discs,
Dimensions of the filter paper discs

Question 15

4.3: Design an investigation of the effect of pH on enzyme activity

Explain whether it is necessary to set up a control.

Answer 15

No, because the investigation aims to study the quantitative relationship between pH and activity of amylase.

Question 16

4.3: Design an investigation of the effect of pH on enzyme activity

Explain one important assumption of this investigation.

Answer 16

The diameter of the clear zone on the starch-agar plate has a positive correlation with the activity of the amylase.

Question 17

4.3: Design an investigation of the effect of pH on enzyme activity

List 4 important precautions to take in the investigation. (4)

Answer 17

Shake the bottle of amylase before it is used because enzymes tend to settle at the bottom.
Ensure the filter discs are placed at equal distances apart from each other on the starch-agar plate.
Rinse the pair of forceps before transferring each soaked filter disc into the starch-agar plate to avoid contamination.
Slightly press the filter discs onto the starch-agar plate to ensure there is a good contact between the soaked filter discs and the agar plate.

Question 18

4.3: Design an investigation of the effect of pH on enzyme activity

Explain the formation of clear zones in the starch-agar. (3)

Answer 18

Amylase in the paper discs diffuses to the starch-agar and catalyses the breakdown of starch.
When iodine solution is added, the part of the starch-agar plate containing starch turns blue-black while the part without starch becomes brown.
Therefore, clear zones are formed in the starch-agar. (clear zone ≠ colourless because iodine solution is brown without starch.)

Question 19

4.3: Design an investigation of the effect of pH on enzyme activity

Predict and explain the results if the paper disc was soaked in a buffer solution at pH 1. (2)

Answer 19

No clear zone would be formed,
this is because extremely low pH would denature the amylase and stop its activity.

Question 20

4.3: Design an investigation of the effect of pH on enzyme activity

Predict and explain the results if the paper disc was soaked in a buffer solution at pH 11. (2)

Answer 20

No clear zone would be formed,
this is because extremely high pH would denature the amylase and stop its activity.

Question 21

4.4: Effect of inhibitors on enzyme activity

What is the colour change of bromothymol blue indicator in different pH?

Answer 21

It is yellow in an acidic medium and blue in an alkaline medium.

Question 22

4.4: Effect of inhibitors on enzyme activity

Tube A consists of acidic urea solution, bromothymol blue indicator, urease solution, and mercuric chloride (inhibitor) solution. Tube B consists of the same volume of these substances except the mercuris chloride is replaced by an equal amount of distilled water.
Explain the results of the investigation. (3)

Answer 22

Urea is still detected in tube A at the end of the investigation, showing that there is little to no breakdown of urea by urease. The inhibitor in tube A inhibits the activity of the urease, causing little to no urea broken down,
so the pH is not high enough to show the colour change (reaction slows down / stops with the inhibitor).
In tube B, the urease is active and it catalyses the breakdown of urea into ammonia in the absence of mercuric chloride.

Question 23

4.6: Design an investigation of protease activity in fruit juices

What is the aim of this investigation?

Answer 23

To compare the action of protease in different juices.

Question 24

4.6: Design an investigation of protease activity in fruit juices

What is the problem of this investigation?

Answer 24

Which fruit juice has the highest protease activity?

Question 25

4.6: Design an investigation of protease activity in fruit juices

Milk-agar is used in this investigation.
Explain the biological principle behind the design of the investigation. (3)

Answer 25

The milk agar contains a source of proteins.
A well is made by the cork borer to put in the juice. They are put in a constant temperature in an incubator.
The level of protease activity is measured by the diameter of the clear zone after the incubation.

Question 26

4.6: Design an investigation of protease activity in fruit juices

What is the independent variable of the investigation, and how is it manipulated?

Answer 26

The type of juice used, which is manipulated by adding different types of juices into the different wells of the agar plate.
(mention important procedures concerning independent variable)

Question 27

4.6: Design an investigation of protease activity in fruit juices

What is the dependent variable of the investigation, and how is it measured?

Answer 27

The protease activity, which is indicated by the diameter of the clear zone, measured with a ruler.

Question 28

4.6: Design an investigation of protease activity in fruit juices

List 3 important controlled variables of this investigation. (3)

Answer 28

Temperature for incubation,
volume of juice,
time allowed for incubation

Question 29

4.6: Design an investigation of protease activity in fruit juices

Explain whether it is necessary to set up a control. (2)

Answer 29

Yes, a control set-up is obtained by replacing the fruit juice with distilled water.
This is to confirm that the formation of clear zones are due to proteases present in the juice.

Question 30

4.6: Design an investigation of protease activity in fruit juices

Explain two important assumptions of this investigation. (2)

Answer 30

Other chemicals present in the fruit juice will not affect the formation of the clear zone,
the diameter of the clear zone has a positive correlation with the enzyme activity.