Activity 6

CATALASE ENZYME

I. Background Information: Liver and other living tissues contain the enzyme catalase. This enzyme
breaks down hydrogen peroxide, which is a harmful by-product of the process of cellular respiration
if it builds up in concentration in the cells. If we use potato or other tissue containing this enzyme,
we can use this to measure the relative influence of varying several different factors on the activity
of enzymes in living tissue.

In order to obtain energy and building blocks from food, the digestive system must break down proteins, fats
and carbohydrates. In this process, specific enzymes catalyze hydrolysis reactions in which food polymers are
broken up into monomers.

II. Introduction: What would happen to your cells if they made a poisonous chemical? You might think
that they would die. In fact, your cells are always making poisonous chemicals. They do not die
because your cells use enzymes to break down these poisonous chemicals into harmless substances.
Enzymes are proteins that speed up the rate of reactions that would otherwise happen more slowly.
The enzyme is not altered by the reaction. You have hundreds of different enzymes in each of your
cells.

Each of these enzymes is responsible for one particular reaction that occurs in the cell. In this lab, you will study
an enzyme that is found in the cells of many living tissues. The name of the enzyme is catalase (KAT-uh-LAYSS);
it speeds up a reaction which breaks down hydrogen peroxide, a toxic chemical, into 2 harmless substances--
water
and oxygen.

The reaction is as follows: 2H2O2 ----> 2H2O + O2

This reaction is important to cells because hydrogen peroxide (H 2O2) is produced as a byproduct of many normal
cellular reactions. If the cells did not break down the hydrogen peroxide, they would be poisoned and die. In this
lab, you will study the catalase found in liver cells. You will be using chicken or beef liver. It might seem strange
to use dead cells to study the function of enzymes. This is possible because when a cell dies, the enzymes remain
intact and active for several weeks, as long as the tissue is kept refrigerated.

III. Objectives: Demonstrate the activity of an enzyme in living tissues and examine the effects of
environmental changes on enzymatic activity.

IV. Pre-lab: Answer the following questions.

1. What type of biological molecule are enzymes?

- Enzymes are biological proteins. They are catalysts and help speed up chemical reactions
within the body.
 2. Describe how enzyme works and its importance to living organisms.
- Enzymes are proteins that help speed up chemical reactions in our bodies. Enzymes are
essential for digestion, liver function and much more. Too much or too little of a certain
enzyme can cause health problems. Enzymes in our blood can also help healthcare providers
check for injuries and diseases.

3. Define the following terms as they apply to enzymes:

a. Active Site - The part of the enzyme where the substrate binds is called the active site
(since that's where the catalytic “action” happens). A substrate enters the active site of
the enzyme. This forms the enzyme-substrate complex.

b. Substrate - A substrate is a molecule that an enzyme reacts with. The enzyme's active
site, or the location where weak bonds between the two molecules can form, is loaded
with a substrate. An enzyme substrate complex is formed, and the enzyme's pressures on
the substrate drive it to react and become the planned reaction's result.

c. Denature - Enzyme denaturation occurs when an enzyme loses its native conformation, or
three-dimensional structure, rendering it unable to bind to substrate and catalyze product
formation. The two main causes for enzyme denaturation are deviations from optimal
temperature and pH.

V. Materials:

• 6 Test tubes and Test tube holder

• 10-ml Graduated cylinder

• Scissors and Forceps

• Stirring Rod

• Beaker

• Hot Plate

• Hydrogen peroxide

• Ammonia

• Fresh Liver

VI. Procedure

PART A - Observe Normal Catalase Reaction

1. Using forceps and scissors, cut a small piece of liver and put it into the test tube. Push it inside the
test
 tube using a stirring rod.

2. Add 2 ml of hydrogen peroxide into the test tube. Observe the bubbles. Throughout this
investigation, estimate the rate of the reaction (how rapidly the solution bubbles) by using the
following legend below and record it on the data table provide below:

5 – Extremely active bubbling; foamy

3 – Moderately active bubbling
1 – Less active bubbling
0 – non-reactive; no bubbling

3. Feel the test tube. Note that the test tube feels warmer after the reaction. Record this
observation on the data table.

Data Table A. Catalase Reaction

Content of Test Tube Rate of Reaction Remarks/Observation

5 Extremely active bubbling;
Normal Liver foamy

Photo Documentation

PART B – Is Catalase Reusable?

1. Place a small piece of liver into a clean test tube and add 2 ml of hydrogen peroxide solution. Observe
the reaction. Record the rate of reaction and your observation into the data table provided below.

2. Pour off the liquid into a second test tube. Add a piece of liver in this test tube and observe the reaction.
Record the rate of reaction and your observation into the data table provided below.

3. In the first test tube with remaining liver, add another 2 ml of hydrogen peroxide and observe the
reaction. Record the rate of reaction and your observation into the data table provided below.

Data Table B. Catalase Reaction

 Content of Test Tube Rate of Reaction Remarks/Observation
5 Extremely active bubbling;
Normal Liver foamy

Liver Added to Used

0 Non-reactive; no bubbling
Peroxide

Used Catalase
5 Extremely active bubbling;
foamy

Photo Documentation:

PART C - What is the Effect of Temperature on Catalase Activity?

1. Put a piece of liver inside a beaker and soak it in distilled water for several minutes. After
soaking, transfer the piece of liver using forceps into a clean test tube and add 2 ml of hydrogen
peroxide in it. Observe the reaction. Record the rate of reaction and your observation into the
data table provided below.

2. Put a piece of liver inside a test tube, add distilled water and place this test tube in a boiling
water bath for 5 minutes. After which, remove the test tube from the hot water bath, allow it to
air cool, then drain out the water. Add 2 ml of hydrogen peroxide. Observe the reaction. Record
the rate of reaction and your observation into the data table provided below.

Data Table C. Catalase Reaction

Content of Test Tube Rate of Reaction Remarks/Observation

Liver soaked in
3 Moderately active bubbling/ Less
distilled water
 reaction; small bubbles

0 Non-reactive; no bubbling
Boiled liver

Photo Documentation:

PART D - What is the Effect of pH on Catalase Activity?

1. Put a piece of liver inside a beaker and soak it in ammonia for several minutes. After
soaking, transfer the piece of liver using forceps into a clean test tube and add 2 ml of
hydrogen peroxide in it. Observe the reaction. Record the rate of reaction and your
observation into the data table provided below.

Data Table D. Catalase Reaction

Content of Test Tube Rate of Reaction Remarks/Observation

Liver soaked in ammonia

5 Extremely active bubbling;
foamy

Photo Documentation:
VII. Analysis

PART A

1. What gas is being released after the hydrogen peroxide was added to the piece of liver? How can
we confirm it?
- Oxygen Gas. Catalase is one enzyme from liver that breaks down harmful hydrogen peroxide into
oxygen gas and water. When this chemical reaction occurs, you can see the oxygen gas bubbles
escaping and causing the reaction to foam.

2. Is the catalase reaction endothermic or exothermic? Explain.

- Exothermic because it reignites the fire.

PART B

1. What is happening inside the test tube after the hydrogen peroxide was added to the piece of
liver?
- Bubbling, Fizzing, and overflowing over the test tube.

2. Assuming that the reaction is complete in Procedure 2, what do you think is the liquid you
poured into the other test tube composed of? How do you know this?
- Water because after the first reaction occurs the oxygen went into the air and all that was
left was water.

3. After completing procedure 3, do you think catalase is reusable?

- Yes catalase is reusable. When adding the first piece of liver into another 2 ml of H2O2
contain test tube bubbles were formed. Catalase in the liver piece, not going to alter during
the first reaction. Enzyme involves the reaction as a catalyst rather than a reactant.
Therefore, enzymes are not going to alter during the reaction. Active sites of the enzymes
 are responsible for the conversion of the reactant to the products. Shapes of the active sites
are specific to the shape of the reactant molecules, then reactants are combined with the
active sites and reaction is proceed when reaction going to completion reactants are
converted to the products, therefore shapes of the products are not specific to the active
sites hence, products are not bound to the active sites, therefore active sites leave it vacant.
Hence, new reactant molecules can combine with the vacant sites and proceed with the
reaction.

PART C

1. Describe the reaction of the hydrogen peroxide and boiled liver. What is the scientific
explanation for this?
- No bubbles produced. When liver and potato are boiled the catalase enzyme becomes
inactive. The catalase is present in all living cells to protect them from oxidation. Catalase
enzyme breaks the peroxides in water and oxygen. When liver and potato are boiled the
enzyme denatures and they get oxidized because the enzyme does not break peroxide. The
properties of an enzyme depend on its three-dimensional shape. Exposure to heat causes in a
loss of the biological properties of an enzyme.

2. What do you think will happen to the reaction if the liver was kept in a freezer for so long?
- No bubbles produced. Below freezing, crystallization prevents enzymes from functioning.

3. How does temperature affect the activity of catalase?

- The rate of an enzyme-catalyzed reaction increases with an increase in the concentration of
an enzyme. At low temperatures, an increase in temperature increases the rate of an enzyme-
catalyzed reaction. At higher temperatures, the protein is denatured, and the rate of the
reaction dramatically decreases.

4. What is the optimum temperature for catalase?

- Catalase works differently at different temperatures. Catalase activity increases as the
temperature goes from 0'C to around 30'C. Catalase activity decreases as temperature goes
from around 30'C to 100'C.

PART D

1. Describe the reaction of the hydrogen peroxide and liver soaked in ammonia. What is the
scientific explanation for this?
- It bubbles. There is an interaction. When the liver is exposed in ammonia, the amino acids in
the protein bonds break apart and release energy in the form of heat.
 2. What do you think will happen to the reaction if the liver was soaked in a strong acid like
hydrochloric acid?

- No reaction will occur. Strong chemicals can damaged enzymes, thus no longer working.

3. How does pH affect the activity of catalase?

- At extremely high pH levels, the charge of the enzyme will be altered. This changes protein
solubility and overall shape. This change in shape of the active site diminishes its ability to
bind to the substrate, thus annulling the function of the enzyme (catalase in this case).

4. Does there appear to be an optimum pH for catalase to properly function? What is it?
Yes, In humans, catalase works only between pH 7 and pH 11. If the pH level is lower than 7 or
higher than 11, the enzyme becomes denaturated and loses its structure. The liver sustains a
neutral pH of about 7, which creates the best environment for catalase and other enzymes.

VIII. Generalization: What have you learned from this activity?

All living things possess catalysts, or substances within them that speed up chemical reactions and
processes. Enzymes are molecules that enable the chemical reactions that occur in all living things on
earth. In this catalase and hydrogen peroxide experiment, I discovered how enzymes act as catalysts by
causing chemical reactions to occur more quickly within living things. Using a potato and hydrogen
peroxide, we observe how enzymes like catalase work to perform decomposition, or the breaking down,
of other substances. Catalase works to speed up the decomposition of hydrogen peroxide into oxygen
and water. Catalase acts as the catalyzing enzyme in the decomposition of hydrogen peroxide. Nearly all
living things possess catalase, including us! This enzyme, like many others, aids in the decomposition of
one substance into another. Catalase decomposes, or breaks down, hydrogen peroxide into water and
oxygen.