Experiment #: 7

Date: 31/01/22

Title: Enzyme Activity II

Aim: To Investigate the Effect of pH on Enzyme Catalyzed Reaction

Apparatus & materials: Test tubes (9), room temperature water bath, measuring cylinder,

test tube rack, ruler, potato, hydrogen peroxide, vinegar, lime

water, distilled water, glass rod

Method: 1) 2g of crushed potato into each of the three test tubes.

2) 3 ml of distilled water was added to the three test tubes with the crushed

potato and mixed with a glass rod.

3) The three test tubes with the potato mixture were placed into a room

temperature water bath and left for 5 minutes.

4) All the tubes were removed from the water bath and then 3 ml of

hydrogen peroxide was added to each tube.

5) Immediately as the hydrogen peroxide was added, the reaction was

timed for 60 seconds.

6) The height of the foam formed after 60 seconds was measured.

7) Steps 2-6 were repeated using vinegar instead of distilled water and

then repeated another time using lime water.

Results:

Title: Table contains results from experiment

pH Medium Theoretical pH Test tube 1 Test tube 2 Test tube 3

Value

Distilled 7.0 28 32 21

Water

Vinegar 2.5 18 24 26
(acetic acid)

Limewater
12.8 20 15 20
(calcium
hydroxide)

Calculation/Result treatment:

1) Determine the average foam formed for each medium

Distilled water

Average foam formed = (28 mm + 32 mm + 21 mm) ÷ 3

= 81 mm ÷ 3

= 27

Vinegar

Average foam formed = (18 mm + 24 mm + 26 mm) ÷ 3

= 68 mm ÷ 3

= 22.67 mm
Lime water

Average foam formed = (20 mm + 15 mm + 20 mm) ÷ 3

= 55 mm ÷ 3

= 18.33 mm

2) Determine the enzyme activity for each medium

Distilled water

Enzyme activity = average height of the foam ÷ time taken for it to develop

= 27 mm ÷ 60 s

= 0.45

Vinegar

Enzyme activity = average height of the foam ÷ time taken for it to develop

= 22.67 mm ÷ 60 s

= 0.38

Lime water

Enzyme activity = average height of the foam ÷ time taken for it to develop

= 18.33 mm ÷ 60 s

= 0.31

3) Plot a graph pH vs enzyme activity

Discussion: Enzymes are protein molecules which can be defined as biological catalysts.

A catalyst is a substance that increases the rate of a chemical reaction while

itself remains unchanged at the end of the reaction. Each enzyme molecule has a

special feature with a precise three-dimensional shaped pocket called the active

site. The enzyme active site is the location on the enzyme surface where another

molecule can bind, and where the chemical reaction catalyzed by the enzyme

occurs. The compounds or molecules the enzyme reacts with are called their

substrates. Enzymes work by lowering the barriers that normally prevent

chemical reactions from occurring by decreasing the required activation energy.

The amount of energy needed before a reaction will proceed on its own is called

activation energy.

Most cells form hydrogen peroxide (H2O2) as a waste product of aerobic

respiration. Although it is produced in small amounts, living things must

detoxify this compound and break it down into non-harmful molecules. Catalase

is a very common enzyme that is present in almost all organisms that are

exposed to oxygen. The purpose of catalase in living cells is to protect them

from oxidative damage, which can occur when cells or other molecules in the

body come into contact with oxidative compounds. Hydrogen peroxide is a toxic

product of many chemical reactions that occur in living things and If allowed to

accumulate it can cause oxidative damage. To prevent such damage, the catalase

enzyme helps get rid of these compounds by breaking up hydrogen peroxide

(H2O2) into harmless water and oxygen.

During the experiment some foaming was observed in all test tubes because

when the enzyme catalase comes into contact with its substrate, hydrogen

peroxide, it starts breaking it down into water and oxygen. Oxygen is a gas and
therefore wants to escape the liquid which causes the foam to form on up the

liquid. The test tubes with the most foam formed after 60 seconds were the tubes

that contained distilled water as the pH medium. This indicates the distilled

water medium produced the greatest enzyme activity; The higher the foam in the

test tube, the greater the enzyme activity.

One factor that affects enzyme activity is pH. The rates of most

enzyme-catalysed reactions are dependent on the pH of the medium in which

they occur. Enzymes have an ideal pH at which enzyme activity is optimal.

Deviations from the optimal pH reduce enzyme activity. Changes in enzyme pH

levels can change the shape of the active site and affect the rate of enzyme

activity. Catalase has a working range of between pH 7-11. Therefore it is

expected that the distilled water would yield the most enzyme activity and the

vinegar and lime water would produce low amounts. However this is not

completely true for the experiment. While the distilled water did have the

greatest enzyme activity and the limewater had a relatively low amount, the

vinegar was a bit different. Comparing the height of the foam in the individual

test tubes it was observed that for test tube 3, the foam formed in the acidic

condition surpasses that of the one in the neutral condition. This could be due to

an error that occurred during the experiment. Generally if the pH level is lower

than 7 or higher than 11, the enzyme becomes denatured and loses its structure;

however each enzyme has a working range of pH values at which it will still

work well but enzyme activity is slowed down.

Conclusion: The structure of the enzyme has a great influence on the activity of the enzyme

therefore changes in the structure of the enzyme affect the rate of chemical

reactions. The pH level that produced the most foam was the distilled water

which had a neutral pH of 7 while the vinegar with an acidic pH of 2.5 and lime

water with a basic pH of 12.8 produced smaller amounts, supporting how an

enzyme’s unique shape can be altered by certain factors causing the enzyme to

be inactivated.

Limitations: 1) Time given to complete the experiment was insufficient

Precaution: 1)

Source of error: 1) Parallax error could occur while taking measurement of the foam in each

test tube.

Reflection: Enzymes can be used outside of cells in many applications such as processing

foods, cleaning clothes, and in diagnostics tests. When using enzymes it is

important to have them work efficiently by creating an environment that allows

high levels of activity, pH value is one variable that can be changed to allow

enzymes to reach their optimal activity. Similar experiments can be conducted to

determine suitable pH conditions for different enzymes. If we would have had

more time to repeat the experiment we could have timed the test tubes more

accurately in order to get the expected results. Over all this lab taught me how

important enzymes truly are in our daily lives.

Experiment #: 6

Skill Being Tested: Analysis & Interpretation

Date: 31/01/22

Title: Enzyme Activity I

Aim: To Investigate the Effect of Temperature on Enzyme Catalyzed Reaction

Apparatus & Materials: test tube (9), Test tube rack, thermometer, water baths, ice bathe,

measuring cylinder, ruler, potato, glass rod, hydrogen peroxide

Method: 1) Three water baths were prepared: 1 room temperature, 2 cold water, 3 hot water

2) 2g of crushed potato into each of the 9 test tubes

3) 3ml of distilled water was added to the potatoes and mixed with a glass rod.

4) Three of the test tubes with the potato mixtures were placed into each of the

respective water baths.

5) The temperature of the water bath was measured then the tubes were left in for 5

minutes.

6) Each of the tubes was removed from the water bath then 2ml of hydrogen

peroxide was added to them.

7) The reaction was timed for 60 seconds as soon as the hydrogen peroxide was

added.

8) The height of the foam foamed after 60 seconds was measured and recorded.
Results:

Title:

Water bath temperature/ Test 1 Test 2 Test 3

℃

Room 28 24 25 15
temperature

Cold water 12 7 8 8

Hot water 70.5 0 0 0

Calculation/Result treatment:

1) Determine the average foam height for each temperature

2) Determine the enzyme activity for each temperature

3) Plot a graph of temperature vs enzyme activity

Discussion: Most chemical reactions are not spontaneous but require an input of energy

to initiate the interaction of the molecules. This energy is called the activation

energy. Enzymes are biological catalysts that function to speed up chemical

reactions in living organisms by decreasing the amount of activation energy

needed to get a chemical reaction started. An enzyme is not permanently altered

or used up during a chemical reaction. They are recycled and can catalyse more

reactions. For two molecules to react they must collide with one another. They

must collide in the right direction and with sufficient energy. Sufficient energy

means that between them they have enough energy to overcome the energy

barrier to reaction (activation energy). Enzymes have an active site. This is part
 of the molecule that has just the right shape and functional groups to bind to one

of the reacting molecules. The reacting molecule that binds to the enzyme is

called the substrate. Small increases in temperature generally increase the rate of

enzyme-catalysed reactions because as temperature increases the kinetic energy

of the enzyme and substrate molecules increases which cause them to have a

greater chance of colliding with each other. Every enzyme has an optimum

temperature; the temperature at which the enzyme activity is greatest However if

temperature increases greatly beyond the enzyme’s optimal temperature this will

disrupt the weak bonds between amino acids and cause the protein to denature

and the substrate will no longer fit into the active site.

This is proven from the experiment as no foam formed in the test tubes that

were placed in the hot water bath which shows that no reaction occurred. The

absence of foaming indicates that the enzyme was denatured as it was unable to

break down the hydrogen peroxide to produce oxygen. The test tubes in the cold

water bath produced a small amount of foam which indicated that the enzyme is

active but reactions are relatively slow as just 7 mm of foam is formed after 60

seconds.This is because at low temperatures the kinetic energy of the enzyme

and substrate are low which means that both the enzyme and substrate molecules

move slower and the frequency of collisions to form enzyme-substrate

complexes are low therefore the rate of enzyme reaction is slow.

Conclusion:

Limitation

Precaution

Source of error: human reaction time

Reflection: The relevance of this experiment is to bring about the correlation between the

enzyme activity and the changes in the enzyme environment. A close focus is placed on the

particular effects of the temperature influences on the enzyme activity. The test tubes in room

temperature water produced the most foam however there was less in one test tube than in the

others, we may obtain more accurate results if we do one test tube at a time instead of

multitasking with all three at once. What also could have been done to take this experiment to

the next level is choose different vegetables with catalase enzymes to see if the results are the

same within each factor tested with the potato.