Chapter 5 LAB 5.

PROPERTIES OF ENZYMES:
BIOLOGICAL CATALYSTS

Objectives:
Students must be able to:
1. Explain how an enzyme functions;
2. Describe the effect of concentration and
temperature on enzyme activity;
3. Define: catalyst, enzyme, activation energy,
substrate, product and active site.

Enzymes are biologically active molecules, usually

proteins, which mediate chemical reaction rates in
the cell. They function by lowering the amount of
energy (activation energy) necessary for a chemical
reaction to occur (cf. figure below). By lowering the
activation energy, an enzyme affects the rate at
which a reaction occurs. Enzyme-mediated
reactions may proceed from 105 to 107 times faster
than without the enzyme!

Enzymes and Activation Energy

In an enzyme-catalyzed reaction, the reactant – the substance being acted upon -- is called the
substrate. Substrate molecules temporarily combine with enzyme molecules to form an enzyme-
substrate complex. As a result of the reaction, a product(s) is (are) formed, and the enzyme molecule
is released without itself having undergone permanent chemical change. A simplified schematic of the
relationship between enzyme and substrate is presented in the following figure.

Enzyme - Substrate Relationship

S = substrate; E = enzyme; A = active site of enzyme; ES = enzyme-substrate complex; P = product

As an introduction to some of the properties of

enzymes, you will experiment with catalase, an
enzyme whose substrate is hydrogen peroxide, a toxic compound which, if not degraded, could
accumulate during cellular respiration:

COMPARING THE CATALASE CONTENT OF PLANT AND ANIMAL CELLS

Most living cells contain the enzyme, catalase. One easy way to determine the amount of catalase
present within cells is to compare the amount of oxygen bubbles released when these cells are placed in
hydrogen peroxide. In this investigation, we will compare the amount of catalase in plant and animal
cells. Also, we will determine if boiling the cells impacts the activity of catalase.

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Read the Protocol Below and Answer the Following Questions:

1. Which will show more catalase activity: plant tissue or animal tissue? Explain your answer.

2. Will raw tissues or cooked tissues show more catalase activity? Explain your answer.

3. Based upon what you know about enzyme activity, and your answers to the questions above, state
a succinct hypothesis for the experiment described below:

Materials (for each lab group):

5 test tubes
Test tube rack
3% hydrogen peroxide
10mL graduated cylinder
Chunks of raw and cooked carrots, potatoes, liver, ground beef (make sure the chunks are all of
equivalent size)

Procedure:
Part A:
1. Number the test tubes 1-5 and place them in the rack.
2. Add the following to each tube:
a. Small chunk of raw carrot to tube 1
b. Small chunk of raw potato to tube 2
c. Small chunk of raw liver to tube 3
d. Small chunk of raw ground beef to tube 4
e. Empty tube
3. Using a small graduated cylinder, add 5 mL of H2O2 to each tube.
4. Observe the amount of bubbling or foam (O2 gas) in each tube.
5. Using a scale of 0-6, record the amount in your data table.
Part B: Repeat steps 1-5 above using the same foods that were cooked prior to lab

Scale:
0= No visible O2 released
1= Few bubbles
2= Bubbles 1/4 way up the tube
3= Bubbles 1/2 way up the tube
4= Bubbles 3/4 way up the tube
5= Bubbles to top of tube
6= Bubbles over the top of the tube

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 TYPE OF CELL Catalase activity RAW Catalase activity COOKED
Carrot

Potato

Liver
Ground Beef

Control

Enzyme Activity
Enzyme activity is affected by environmental conditions, including time course of the reaction,
concentration, temperature and pH. Due to time constraints, you will be able to investigate only the
affect of concentration and temperature on catalase activity.

For the following experiments with catalase, it is critical that you keep all glassware clean -- washing
and rinsing with tap water followed by a final rinse with diHOH both before and after each use!

You will be provided with (1) a stock solution of catalase at a concentration of mg/ml; (2) a
stock solution of hydrogen peroxide; (3) a quantity of diHOH; and (4) a stock solution of 3N sulfuric
acid. BE CAREFUL WITH THE SULFURIC ACID! NOTE: The stock solution of catalase must be kept
in an ice bath!

At the conclusion of each experiment, sulfuric acid is added to the reaction system to deactivate the
enzyme; subsequently, additional hydrogen peroxide -- the substrate -- is not degraded to water and
oxygen. The amount of hydrogen peroxide remaining can be determined by titration with 0.25%
potassium permanganate. When all the remaining hydrogen peroxide has reacted, a pink color -- the
end point -- will appear. The amount of titrant required is directly proportional to the amount of
hydrogen peroxide remaining in the reaction system at the conclusion of the experiment. Thus, one
has an indirect measure of the amount of hydrogen peroxide degraded during the experiment.

Titration Reaction

Your instructor will demonstrate the proper use of a calibrated buret you will be using for titrations.

For the following exercises, work in groups of four as designated by your instructor.

CATALASE ACTIVITY/TEMPERATURE

1. Number two clean, dry test tubes.

2. Using a 10.0 ml graduated cylinder, place 3.0 ml of hydrogen peroxide into each tube.

3. Place Tube #1 into an ice bath (4C) and Tube #2 into a test tube rack on your desk (24C).
(Your instructor my propose you set up a third tube designated at a temperature above ambient)

4. Allow each tube to “acclimate” at its respective temperature regime for 20 min.!!

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5. Using a 1.0 ml Pipetman, transfer exactly 0.50 ml of cold catalase stock solution into each test
tube. Record the time! Mix thoroughly by gently shaking the tubes once. DO NOT CONTINUE
SHAKING THE TUBES!
6. Immediately return each tube to its respective temperature regime.

7. Exactly 6 min after adding the catalase, stop the reaction in each tube by adding enough sulfuric
acid to double the volume of liquid in each tube. Mix thoroughly by gently shaking the tubes.

8. Allow the four tubes to “sit” at room temperature for 10 min.

9. Transfer the contents of a tube into a small beaker and determine the amount of substrate
remaining by using a buret to titrate the contents with 0.25% potassium permanganate. Add the
permanganate slowly, rotating the beaker gently, until a pink color, the endpoint, just appears.
[NOTE: If, upon addition of permanganate to the beaker, a brownish-purple color appears, discard the
contents and start over!]. Record the amount of titrant required for each tube in the table below and
on the blackboard.

10. Titrate the contents of the remaining tubes, rinsing the beaker with diHOH between titrations.

Enzyme Activity and Temperature: Titrant Required (ml)

Group Tube #1 (4°C) Tube #2 (24°C) Tube #3 °C
I
II
III
IV
V

Based upon your Group’s results and those of other Groups conducting this experiment, what
conclusion(s) do you reach regarding the effect of temperature upon the activity of catalase?

Using the standard graph paper section (last page), plot your Group’s results with temperature (°C)
on the horizontal axis and titrant required (ml) on the vertical axis.

CATALASE ACTIVITY/ENZYME CONCENTRATION

1. Number three clean, dry test tubes.

2. Using a 10.0 ml graduated cylinder, place 3.0 ml of hydrogen peroxide into each tube.

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3. Treat each tube, individually, as follows:
Tube #1:
a. Using a 1.0 ml Pipetman, add exactly 0.50 ml of cold catalase solution to the tube. Record
the time! Mix thoroughly by gently shaking the tube. Note the evolution of gas. What gas is being
evolved? Place the tube into your test-tube rack, leaving it undisturbed for 6 min.
b. After exactly 6 min, stop enzyme activity by adding enough sulfuric acid to double the
volume of liquid in the tube. Mix thoroughly by shaking the tube.
c. Allow the tube to “sit” at room temperature for 10 min.
d. Determine the amount of hydrogen peroxide remaining in the tube by titrating with 0.25%
potassium permanganate. Add the permanganate slowly, rotating the beaker gently, until a pink color,
the endpoint just appears.
e. Record the amount of titrant (ml) required and the concentration (cf. formula) of catalase in
the reaction mixture* in the following table.

* ml stock catalase + ml diHOH (if any) + ml hydrogen peroxide *

Tube #2: As for Tube #1, except, using a 1.0 ml Pipetman, add exactly 0.20 ml of cold diHOH and
0.30 ml of catalase stock solution to the tube. Record titrant value and concentration of catalase in
the above Table. (First pipet the catalase solution in each tube, then use a new pipet tip for the
diHOH)
Tube #3: As for Tube #1, except, using a 1.0 ml Pipetman, add exactly 0.40 ml of cold diHOH and
0.10 ml of catalase stock solution to the tube. Record titrant value and concentration of catalase in
the above Table.

Enzyme Activity and Concentration

Concentration of Enzyme in Reaction Mixture (mg/ml) *stock enzyme concentration is 20µg/ml

Enzyme Activity and Concentration

Concentration of Enzyme in Titrant Required
Reaction Mixture (mg/ml)
Tube #1
Tube #2
Tube #3
Tube #4 0.0 Not performed

State a general hypothesis to cover the experiment on the relationship between catalase
concentration and activity.

Did your experimental results support the hypothesis? YES NO.

Explain!

Using the standard graph paper section below, plot your Group’s results of
the experiment on catalase concentration with enzyme concentration (mg/ml) on the horizontal axis
and titrant required (ml) on the vertical axis. Other variables being constant, is enzyme concentration
linearly related to enzyme activity?

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YES NO. Explain your choice

Graph

Record, in the following Table, the results of all Groups completing the two experiments on catalase
activity.

Class Results: Effect of Temperature and Concentration Upon Catalase Activity

Experiment/Group Tube #1 Tube #2 Tube #3 Tube #4
Temperature (4°) (24° ) (34° ) (60° )
I
II
III
IV
V
Catalase (ml) (0.50) (0.30) (0.10) (0.0)
I
II
III
IV
V

You will most likely observe considerable variation in results obtained by different Groups. Identify the
most likely sources that would account for the variable results obtained in the two experiments.

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Graph

“The word enzyme comes from Greek: "in leaven". As early as the late 1700s and
early 1800s, the digestion of meat by stomach secretions and the conversion of starch
to sugars by plant extracts and saliva were known. However, the mechanism by which
this occurred was unknown. In the 19th century, when studying the fermentation of
sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation
was catalyzed by a vital force contained within the yeast cells called "ferments", which
were thought to function only within a living organisms. He wrote that "Alcoholic
fermentation is an act correlated with the life and organisation of the yeast cells, not
with the death or putrefaction of the cells."

In 1878, German physiologist Wilhelm Kühne (1837-1900) coined the term "enzyme,"
meaning "in leaven," to describe this process. The word enzyme was used later to refer to non-living
substances such as pepsin, and the word ferment was used to refer to chemical activity produced by living
organisms. In 1897, Eduard Buchner began to study the ability of yeast extracts to ferment sugar, despite the
absence of living yeast cells. In a series of experiments at the University of Berlin he found that the sugar was
fermented, even when there were no living yeast cells in the mixture. He named the enzyme that brought about
the fermentation of sucrose "zymase". It was not until 1926, however, that pepsin became the first enzyme to
be obtained in pure form by Northrop, Sumner and Stanley. Their successful purification and crystallization of
pepsin proved that enzymes were proteins and they were awarded the 1946 Nobel prize for Chemistry.”

From http://en.wikipedia.org/wiki/Enzyme and http://en.wikipedia.org/wiki/Pasteur

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