What effect will changes in substrate concentration
have on enzymatic activity
Standards:
The following PA Educational standards are met or addressed in this
laboratory investigation.
3.2.10.A, 3.2.10.B, 3.2.12.A, 3.2.12.B
Introduction and Background:
Definitions:
A; Enzyme: A substance, usually a protein in biological reactions, which
enable or speed up a chemical reaction in a living cell. Enzymes are usually present in
very small amounts because they are not used up in the reaction they facilitate.
B; Oxidizing agent: A substance which tends to take electrons and
thereby has its own oxidation number reduced. These substances can cause bleaching
and many other undesirable reactions within living cells.
C: Catalase: An enzyme which accelerates the harmless breakdown of
the oxidizing agent Hydrogen Peroxide (H2O2) into water (H2O) and Hydrogen (H2).
D: Substrate: The substance or molecule upon which the catalyst or
enzyme acts.
E: Variables; The data points found on a graph. The Independent
variable, usually plotted on the x axis is the variable whose value determines the value of
the Dependent variable, which is plotted on the y axis. When horizontal lines are drawn
from all of the dependent variable values and vertical lines are drawn from all the
independent variable values, they form points where they cross, which when joined by a
line which can be used to predict other values and to analyze trends and evaluate effects.
Background: Enzymes are biological catalysts. They are usually large
proteins made up of hundreds of amino acids. Enzymes catalyze thousands of chemical
reactions that occur in living cells. Enzymes are highly specific as to which reactions
they participate in so that each enzyme usually speeds up only one type of reaction.
Catalase, also called peroxidase, is found in both plant and animal cells. It is very
abundant in plant storage organs such as potatoes and the fleshy parts of fruits. Catalase
is extremely important in cells because it prevents the buildup of hydrogen peroxide, a
harmful oxidizing agent. Hydrogen peroxide is a strong oxidizer which can upset the
balance of cell chemistry. If too much hydrogen peroxide accumulates, it will kill the
cell.
Several physical or chemical factors affect the actions of enzymes. This
investigation is designed to observe only the effect of changing substrate concentration.
Questions to be answered in this Investigation:
1. How do different concentrations of substrate affect enzymatic activity?
2. How data gathered in this investigation can be graphed to allow interpretation,
analysis, and making predictions?
Materials: Per two person team
Crushed ice
10ml graduated cylinder
glass stirring rod
filter paper discs
50ml beaker
100ml beaker
stop watch
forceps
sheet of paper towel
potato juice
sheet of graph paper
student handouts for each student
hydrogen peroxide solution(concentration to be assigned by instructor)
Safety:
Hydrogen peroxide is a powerful oxidizing agent and poses a serious eye hazard;
therefore, safety glasses are to be worn when it is in use.
Additionally, hydrogen peroxide is a strong bleaching agent. If spilled on
clothing, it will cause serious discoloration. Wearing a lab apron is recommended.
Report any spills or accidents to the instructor, immediately.
2
Procedures:
1. Fill a 10ml graduated cylinder to the 10ml mark with the hydrogen peroxide
concentration from the brown bottle provided by your instructor. Note the
concentration listed on the bottle and write it in the appropriate space in your
data sheet. Refill the graduated cylinder, if necessary, to keep the level at the
10 ml mark.
2. Put about 10-20ml of the potato juice in the 50ml beaker, and place this
beaker in the 100ml beaker over plain crushed ice. Keep the potato extract on
ice throughout the lab.
3. Using forceps, obtain one paper disc and immerse it in the potato juice for 5
seconds.
4. Drain the excess juice from the disc by touching it on the paper towel for 10
seconds.
5. Wet the tip of your glass stirring rod in the potato juice. Stick the disc to the
glass rod, and immerse it, all the way to the bottom of the graduated cylinder,
and remove the rod.
6. As soon as the disc touches the solution in the graduated cylinder, begin
timing and stop the watch when the disc reaches the surface of the solution.
Record the time to the nearest tenth of a second.
7. Repeat steps 3 through 6 up to 9 times, or whenever you get 5 values which
are very close.
8. Calculate the mean of the 5 closest values and record them in Table I of your
handout.
9. Obtain the class data for the substrate concentrations other than your own.
Record these in Table II.
10. Graph the results with the mean times for each substrate concentration as the
dependent variable and the substrate concentrations as the Independent
variable.
DATA TABLES
Observations:
Table I – Group Data
Trial
1
2
3
4
5
Mean
(____%H2O2)
Time (seconds)
3
Table II – Class Data
Conc. H2O2
0%
0.1%
0.5%
1%
3%
Mean Times (seconds)
Calculations:
Use the space below for calculations of mean values.
Questions:
a. What were the observed effects of different concentrations of hydrogen
peroxide substrate?
b. Why was the procedure repeated numerous times?
c. What caused the paper disc to rise?
d. Describe the chemical reaction which takes place and write a balanced
equation?
e. Were there any controls in this investigation?
f. What other conditions, if modified, could alter the rate of reaction?
g. Why is this reaction important to man or any living creature?
4
What Effect Will Changes in Substrate Concentration
Have on Enzyme Activity
Pre Test/Post Test
Name: ________________________
1. Why are enzymes found in cells in tiny quantities ?
2. Enzymes are which type of biological molecule?
3. What do oxidizing agents gain when being reduced?
4. The two breakdown products of Hydrogen Peroxide are?
5. What are the substances which have a similar action to enzymes in
inorganic reactions?
5