Tan Ri Shen

P20012314
Experiment 4 - Enzymes

Title – Enzymes are proteins that speed up the rate of biological reaction. The reaction that
will be investigated in this experiment is the decomposition of hydrogen peroxide.

Objectives – To identify the effect of temperature, pH and enzyme concentration on enzyme

activity. Lastly, to identify catalase activity on different tissues.

Materials [1m]
Procedures

Results
A. Control
Enzyme Activity (1-5) Description

Positive Control 5

Negative Control 1

1. Did the reaction give off heat?

B. Effect Of Temperature On Enzyme Activity

Temperature (C) Enzyme Activity (1-5)

Tube 1 (ice bath) 2

Tube 2 (room temperature) 3

Tube 3 (37 C) 4

Tube 4 (100 C) 0

1. Is there a temperature at which the catalase activity was the most effective? Was it the
temperature you were expecting? If it was not, how can you explain the results?

2. Why put the tubes of H2O2 (and not just the liver tubes) into the water baths?

3. Graph

C. Effect Of pH On Enzyme Activity

Enzyme Activity (1-5)

pH 3 3

pH 7 4

pH 11 5

1. At what pH does catalase exhibit the greatest activity?

2. Plot a graph (enzyme activity vs pH) that corresponds with the results of your test.
D. Effect Of Enzyme Concentration On Enzyme Activity
[Enzyme] Enzyme Activity (1-5) Description

100% 5

10% 4

1% 2

1. Were the results of this experiment as you expected? If they were not, can you explain
why they were not?

2. How effective is catalase? In this experimental setup, do you think you would detect a
difference in reaction rates between a 100% and a 50% liver solution?

E. Catalase Activity In Different Tissues

Tissue Enzyme Activity (1-5) Description
Liver 4
Chicken 2
Apple 0
Potato 2
Carrot 2
1. What do your results tell you about the functions of the different types of tissues?

2. Why was it important that the puree of each substance is obtained by blending the same
amount of substance in a similar volume of water?

Discussion
Enzymes         [2m]
Factors affecting enzyme activities    [2m]

QUESTIONS

1. List the conditions you tested to determine which catalase exhibited the greatest activity.
How do these conditions compare to those of a cell in the body?

2. H2O2 is commonly used as a disinfectant for scrapes and cuts. What exactly are you
trying to do when you apply H2O2 to your scraped knee? What on your knee causes the
H2O2 to bubble up when you apply it?

3. The browning that occurs when fresh potatoes and apples are cut is a result of this
reaction:
catecholas
Catechol + O2
e
Benzoquinone
(colorless)
Why do mashed potatoes stay(reddish
white?brown)
4. Some people put fresh lemon juice on fruit salad to keep it from browning  what might
the chemical explanation be for this practice?

5. (a) The U.S. Food & Drug Administration recommends that cooked beef be refrigerated
for no more than 3-5 days before it's eaten; for cooked fish they recommend only 1-
2 days. Why do you think fish might not keep as long in the refrigerator?
(b) It is recommended that uncooked ground beef is refrigerated for only 1-2 day's as
well. Why might this be?

6. Fever is a common symptom of a viral or bacterial infection. What are two different
functions of a rise in body temperature in this case? What would the danger be if the
temperature got too high (above about 42 C, in humans)?

7. Lizards and snakes may often be found sitting in sunny spots (on exposed rocks, in the
middle of a road) in the morning. They do not use the heat generated by their bodies to
heat themselves: they obtain heat from the environment. After a cold night, they are
sluggish and must heat up before they can be active.
(a)What do they need heat for?
(b) From where does the body heat from mammals and birds come? (The Second Law
of Thermodynamics might help you answer this one.)

Conclusion:

----------------------------------------------------------------------------------------------------------------

EXPERIMENT 4

ENZYMES

INTRODUCTION

Even when you are sitting still, the 10 trillion living cells of your body are busy. Muscle cells
in your heart are contracting; red blood cells are picking up oxygen from your lungs and
distributing it throughout your body. Many cells are growing and dividing. And all cells are
performing the functions necessary to keep themselves alive: breaking down sugars,
disposing of wastes, building membranes, producing enzymes, etc. All the chemical reactions
that accomplish these activities require energy.

According to the First Law of Thermodynamics, energy cannot be created or destroyed; it

can only be changed from one form to another. So cells must use the energy from exergonic
(energy releasing) reactions to power the endergonic (energy consuming) reactions. To
synchronize these two types of reactions cells use large molecules, usually proteins, called
enzymes. Enzymes have several characteristics:
 Enzymes speed reactions between other molecules  without them, most reactions would
occur too slowly to sustain life.
 Enzymes speed reactions by lowering the activation energy (minimum energy required
for a reaction to occur) of reactions. They accomplish this by participating directly in the
reaction, by orienting the substrate molecules to increase the chances of their interaction,
or by straining the bonds of the substrate to pave the way for new bonds.
 Enzymes are not changed by the reactions they speed.
 Enzymes are specific: each enzyme lowers the activation energy for one type of
reaction.

As with all proteins, the three-dimensional shape of an enzyme molecule is critical to its
performance. Part of its structure includes an active site, a place where the enzyme binds to
the reacting molecules, called substrates. It is this binding that lowers the activation energy.
If the shape of the enzyme is altered, the active site will not fit and bind to its substrate (see
Figures 1 and 2).

Figure 1. Induced fit model of enzyme-substrate interaction.

Both the enzyme and the substrate change shape, straining bonds during the reaction. The
strained form of a substrate the transition state exists for as little as a billionth of a second.

Figure 2. How does an enzyme catalyze a reaction between two other molecules?
The enzyme brings both substrates close together in the proper orientation and also strains
the covalent bonds of the substrates, lowering the activation energy of the reaction.
The liver makes many different enzymes. Some are metabolic, working to convert fructose
and other sugars into glucose or making glucose and proteins into glycogen for storage. Some
enzymes convert toxic compounds like alcohol and nitrates into less toxic compounds or into
compounds that can be easily cleared from the body. Hydrogen peroxide (H 2O2) is a product
of many of these types of reactions. It is highly reactive and can easily damage or kill cells if
left intact. Cells use the enzyme catalase to break H2O2 into harmless products. In a cell,
catalase is contained in a special type of organelle called a peroxisome.

The reaction you will be testing is the conversion of H2O2 into water and oxygen gas by the
enzyme catalase:
catala
2H2O2 se 2H2O
+ O2
Because H2O2 is a by-product of many biochemical reactions, many cells have catalase
activity. The liver, with its detoxifying functions, has higher levels of catalase than most
cells. When H2O2 is added to ground beef liver, there will be vigorous bubbling.

In this experiment, you will test the activity of the enzyme catalase under different
temperatures, concentrations, and pH levels and in different types of tissues

For all these experiments, use a number scale from 0 to 5 to describe the level
of activity, with 0 = no reaction and 5 = vigorous bubbling.

PROCEDURE

A. Control
1. Take two test tubes: label one "+" (positive control) and the other "" (for negative
control).

2. Measure 1 mL of liver puree with the pipette. Insert the pipette into one test tube, and
lower the tip as close to the bottom of the test tube as possible before squeezing the liver
puree out. If liver is smeared on the sides of the tube, the amount available for reaction
will not be consistent among your tests.

3. Put 1 mL of liver puree in the other tube, using the same method.

4. Into the first test tube put 3 mL of deionised water. Record your observations.

5. Into the other test tube put 3 mL of 3% (v/v) H2O2. Watch the reaction for several
seconds, noting the height the bubbles travel as well as the speed with which the reaction
occurs. Assign this level of bubbling "4". These will be the standards by which you
compare the other reactions today.

6. According to the Second Law of Thermodynamics, the amount of energy available to

do work decreases in the course of any process. Often the energy that is unavailable to do
work is in the form of heat. Feel the test tube with your hand  did this reaction give off
heat?

7. Record your observations.

B. Effect Of Temperature On Enzyme Activity

Increased temperature generally increases the effectiveness of enzymes because as the
enzyme and substrate molecules move around more rapidly, the chances of their colliding and
interacting increase. However, as with all proteins, the three-dimensional structure is in
danger when the temperature gets high enough to break weak bonds. When bonds are broken
and the enzyme loses its three-dimensional structure, the enzyme is denatured: it loses its
activity.
Every enzyme has a range of temperatures at which it is the most effective. Most enzymes in
the human body work best at body temperature, about 37 °C.
1. Using the thermometer placed on the instructor’s bench find the room temperature of the
laboratory in C, and record it.

2. Check the thermometer in the ice bath and record its temperature.

3. Put 1 mL of liver puree into four clean test tubes using the method described in step 2 of
the control experiment, above. Label each tube "1" to "4" respectively and use them at
the temperatures listed below:
Tube 1: temperature of ice bath
Tube 2: room temperature of laboratory
Tube 3: 37 °C
Tube 4: 100 °C

4. Into four more clean test tubes, put 3 mL of 3% (v/v) H 2O2. Label each tube "1" to "4"
respectively and use them at the same temperatures as the liver tubes listed above.

5. Leave the room temperature tubes of liver and H2O2, in the test tube rack on your bench.
Put each of the other six tubes into the appropriate water baths. After 4 minutes, remove
the tubes from the boiling water and 37 C water baths, and bring them back to your
bench.

6. Add each tube of H2O2 to the corresponding tube of liver. Rate the reactions and record
your results. Make a graph of the activity rate versus temperature.

C. Effect Of pH On Enzyme Activity

The pH of a solution is a measure of how acidic or basic it is. Pure water with a neutral pH
(in which the H+ and OH concentrations are equal) has a pH value of 7. An acidic solution
has a higher concentration of H+ ions and a lower pH value (<7); a basic solution has a lower
concentration of H+ ions and a higher pH (>7).
Enzymes usually have a narrow range of pH values at which they work best, just like they
have a certain optimum temperature range. The pH may alter the shape of the enzyme or add
or remove hydrogen ions from the enzyme, changing the way it binds to the substrate.
1. Label three test tubes "pH 3", "pH 7", and "pH 11". Carefully put 1 mL of liver puree
into each tube.

2. Add 2 mL of 0.1 M sodium citrate buffer solution at the appropriate pH into the
respective test tubes. Wait for about 20 minutes.

3. Add 3 mL of 3% (v/v) H2O2 to each tube. Rate the reactions, and record your results.

D. Effect Of Enzyme Concentration On Enzyme Activity

In this experiment, you will rate catalase activity at full strength (100%), 10%, and 1%.
1. Get five clean test tubes. Label the tubes "100%", "10%", "l%", "stock 10%", and
"stock l%".

2. Into the "100%" tube, put 1 mL of liver puree, being careful to get all liver puree into the
bottom of the tube.
3. Into the "stock 10%" tube, put 1 mL of liver puree. Add 9 mL of deionised water and
mix by squeezing the pipette several times. You have now diluted the liver to 10% of the
original concentration. Put 1 mL of this mixture into the "10%" tube.

4. Into the "stock l%" tube, put 1 ml of the "stock 10%" solution and 9 mL of deionised
water. Mix with the pipette and put 1 ml of this mixture into the "l%" tube.

5. Add 3 mL of 3% (v/v) H2O2 to each tube: "100%", "10%", and "l%". Rate the reactions
and record your results.

E. Catalase Activity In Different Tissues

1. Label five test tubes "liver", "chicken", "apple", "potato", and "carrot". Transfer the
puree of each substance into the respective tubes to a depth of 1 cm. (Note: The puree of
each of the substances is obtained by blending the same mass in a similar volume of
deionised water.)

2. Add 3 mL of 3% (v/v) of H2O2 to each tube. Watch for several seconds, rate the
reactions, and record your results.