Catalase Lab
How do enzymes work in living tissues?
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 chemical. 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; 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 (H2O2) 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. 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.
Part 1: Normal Catalase Activity
1.
Place 2 mL of 3% hydrogen peroxide solution into a clean test tube.
Is the hydrogen peroxide bubbling? Yes or No
(circle one)
2. Using forceps and scissors, cut a small piece of liver and add it to the tube. Push it into the
hydrogen peroxide with a stirring rod.
Observe the bubbles. What gas is being released?____________________________
3. Feel the temperature of the tube with your hand.
Has it gotten warmer or colder?
Warmer
/
Colder (circle one)
4. Pour the liquid from this tube into a second tube.
What is this liquid composed of? _______________________________
What do you think if you added more liver to this liquid? Why? _________________________
______________________________________________________________________________
______________________________________________________________________________
5. Add another 2 mL of hydrogen peroxide to the liver remaining in the first tube.
Can you observe any reaction? If so, what happens? ___________________________________
______________________________________________________________________________
What do you think would happen if you poured off this liquid and added more hydrogen
peroxide to the remaining liver?____________________________________________________
______________________________________________________________________________
Are enzyme reusable? Yes
or
No
(circle one)
Part 2: Effect of Temperature on Catalase Activity
Enzymes are unique three-dimensional structures. The active site is the portion of the enzyme that
interacts with the substrate, so that any substance that blocks or changes the shape of the active site
affects the activity of the enzyme. A description of several ways enzyme action may be affected follows:
a. Salt Concentration: If the salt concentration is close to zero, the charged amino acid side chains
of the enzyme molecules will attract each other. The enzyme will denature (change shape). If,
on the other hand, the salt concentration is very high, normal interaction of charged groups will
be blocked, new interactions will occur, and again the enzyme will change shape. An
intermediate salt concentration, such as that of human blood (0.9%) or cytoplasm, is the
optimum for many enzymes.
b. pH: pH is a measure of acidity, or H+ concentration, in a solution. The scale runs from 0 to 14
with 0 being the highest in acidity and 14 the lowest. A pH of 0 to 7 is considered acidic. A pH of
7 to 14 is considered basic. Altering pH can affect the shape of the enzyme. Most enzymes
perform best at a neutral pH and are denatured if the pH is increased or decreased by too much.
Some enzymes, such as pepsin, which actin the human stomach where pH is very low, have a
low pH optimum.
c. Temperature: Chemical reactions tend to speed up as temperature increases. Each enzyme has
an optimum temperature at which it works best. Temperatures that exceed the optimum tend
to denature the enzyme and render it nonfunctional.
Other items can affect the rate at which the enzyme-catalyzed reaction takes place are as follows:
 concentration of enzyme
 concentration of substrate
 source of enzyme (organism)
The rate of a chemical reaction may be studied in a number of ways including the following:
 measure the rate of disappearance of substrate (H2O1 in this experiment)
 measure the rate of appearance of product (O2 which is given off as a gas in this experiment)
 measuring the heat released (or absorbed)during the reaction
Conducting the Temperature Investigation
1. Put equal quantities of liver into 4 clean test tubes. Label the tubes as follows: ice, room temp,
warm and boiling.
2. Put 2 mL of hydrogen peroxide into 4 clean test tubes. Label the tubes as follows: ice, room
temp, warm and boiling.
3. Place the two ice tubes (one liver and one hydrogen peroxide) in ice. Place the two warm tubes
(one liver and one hydrogen peroxide) in the incubator. Place the two boiling tubes (one liver
and one hydrogen peroxide) in the hot water bath – you will have to hold them with a insulated
glove or test tube holders. The two room temp tubes can remain in the test tube rack at your
work area.
4. Tubes should be held at the set temperature for 5 minutes.
5. After 5 minutes, take all tubes back to your work area. Pour each tube of hydrogen peroxide in
the corresponding tube of liver. Observe the reaction (measure the height of the column of
bubbles in the test tube).
6. Estimate how rapidly the solution bubbles with 0 = no reaction, 1 = slow, 2 = moderate, 3 =
above moderate, 4 = fast, and 5 = very fast. Assume that the reaction you saw in part A – step 2
was a rating of 4.
Temperature
Ice (0°C)
Room Temp (24°C)
Incubator (37°C)
Boiling (100°C)
Rate of Enzyme Activity (0 to 5)
Review Questions:
1.
To what group of organic compounds do enzymes belong?
Circle One:
carbohydrate
lipid
nucleic acid
protein
2. What is the name of the enzyme we are studying in this lab?
3. What is the substrate in the reaction we are studying?
4. What are the products in the reaction?
5. What happens if hydrogen peroxide is not broken down in our cells?
6. Explain the results of chart in part 2.
7. What happens when an enzyme is denatured (the 3-demensional shape is changed)?
8. What is the optimum temperature for catalase (according to the fastest/biggest reaction)?