How the substrate concentration affects enzyme

activity of catalase
The lab investigates the effects of different enzyme concentrations on
enzyme activity of catalase. I decided to choose this lab because in research
and my attempt to understand the concept of enzymes using online
resources, I came across many examples in relation to catalase. I decided to
further my knowledge and take on this experiment on my own to test how
this reaction will result physically.
Enzymes are proteins with a specific three-dimensional shape. They have an
area designed to match a specific substrate molecule known as the enzyme
substrate and this area is called the active site. The active site only matches
with the specific substrate; an analogy to understand this is to visualize a
lock and key. The lock is the enzyme, whereas the key is the substrate. Only
that substrate can unlock or activate the enzymes ability. The combination
between the enzymes which grabs onto the substrate molecule is called the
enzyme/substrate complex.
Enzymes are biological molecules, only produced by living organisms
that act as catalysts and help complex reactions occur everywhere in life.
They can be used over and over again until they are denatured. There are
several factors that cause denaturation such as the environmental
temperature, pH, enzyme and substrate concentration. When the substrate
is changed from original form, its called catalysis. The substrate is either
broken down or combined with another molecule to make something new,
nevertheless it will break chemical bonds and when done, the result will be
an enzyme product complex. When the enzyme finishes with the product, it
releases it and returns to the original shape in order to work on another
molecule of the substrate.
Most cells in our body contain catalyse. When a tissue is damaged, the
enzyme is released and becomes available to react with the hydrogen
peroxide. If poured on blood, bubbles are produced of oxygen has, however,
if the hydrogen peroxide is poured on the surface of the skin, it wont react
since there is no catalase. Since even without the enzyme, hydrogen
peroxide dissociates at a slow rate, therefore; it has a shelf life. If the
hydrogen peroxide is poured on a cut and no bubbles are formed, there is a
large chance that it is no longer active and is only water
Like all enzymes, catalase also has functions that restrict it from performing
to its best ability. In this experiment, the rate of reaction will be tested in
different concentrations of the substrate (H2O2). Different concentrations of
H2O2 will be diluted along with the use of filter paper to float up. This will help
calculate the time of reaction and how different concentration affect this

rate, but it is known that the rate of enzyme activity increases as the
substrate concentration is increased. However only to a certain degree
where the enzyme becomes saturated and the rate cant increase. The trend
is illustrated in figure 1.
Figure 1 expected graph of rate of reaction against concentration of
hydrogen peroxide solution
Research question
To investigate how the substrate concentrations affect the rate if
enzyme activity in the breakdown/decomposition of hydrogen peroxide.
Hypothesis
It is hypothesized that the rate if reaction will increase (less time will
be taken to breakdown the substance) as the concentration of the substance
is increased, the catalase reduces the activation energy for the reaction to
occur so its speeds up. As long as the other factors that affect enzyme
activity are kept constant (pH, temperate, enzyme concentration, air
pressure). However at very high substrate concentration, the rate if enzyme
activity will not increase since the enzyme being used will become saturated.
(occupied with other substrate molecules) the rate of decomposition of
hydrogen peroxide will stop increasing.
Variables
Variable
measured
Independ Concentration of
ent
hydrogen
peroxide solution

Method to control
Hydrogen peroxide solutions are prepared
by using 6% hydrogen peroxide. Certain
values of water are added to dilute the
H2O2 to a specific concentration. Diluting
it to certain concentrations
-

Dependa
nt

Rate of reaction

Controlle
d

substrate (H2O2)
concentration and
quantity

Concentrations: 6%, 4%, 3%, 2%,

0%
- 0% is only water, it serves as the
control concentration
The time for the filter paper to reach the
surface. This will be timed and recorded
for each concentration. This will be
directly related to the amount of
concentration.
All trials will use exactly 10ml of H2O2 and
water solution, in order to keep the
volume constant throughout the
experiment.

Amount of
enzyme (catalase)

Size/volume of
test tubes

Filter paper discs

temperature

One potato/water solution is created will

have the same diameter, length, and size
to maintain a constant volume. They will
be cleaned and dried before a new trial is
begun in order to reduce error.
All test tubes used in this experiment will
have the same diameter, length and size
to maintain a constant volume. They will
be cleaned and dried before a new trial is
begun in order to reduce error.
Same branded filter paper is used and the
discs are created by the use of the same
hole puncher, so they all have the same
dimensions. Essentially, they are identical.
Lab performed in the same room, in
the same time frame so the room
temperature remains constant
throughout the lab.

Material
-

test tubes (5)

tape
potato (1)
knife
filter paper (2)
beaker (400ml)
hole puncher
safety goggles
micro pipet

test tube rack

stop watch
distilled water (at least
100ml)
6% h202 (at least 100 ml)
mortar and pestle
petri dish
graduated cylinder 10 ml
(2)

procedure
prepare 5 different solutions of different concentrations of the
substrate (hydrogen peroxide)
3)water needed to make each concentration. The final
concentration of each should be 10 ml in order to have a
constant volume throughout.
Eg. For 4 %
C1V1 = C2V2
0.06 x V1 = 0.04 x 10 cm3
V1 = 6.67 cm3
Water: 10 cm3 - 6.67 cm3 = 3.33 cm3
test tube
number

1
2
3
4
5

Concentra
tion of
hydrogen
peroxide
(%) (
0.02)
0
2
3
4
6

Volume of
6%
hydrogen
peroxide
(cm3) (
0.01)
0
3.33
5
6.67
10

Volume of
water
added
(cm3) (
0.01)

10
6.67
5
3.33
0

2. Peel the potato and cut ten small pieces (preferably 1cm by 1cm for
easy crushing)
3. Crush the potato pieces in the mortar and pestle
4. Add 5ml of distilled water and homogenize until an even liquid
solution is observed
5. Place a filter paper over a beaker and dump the potato water
solution over in order to remove access water and the solution is
placed into a petri-dish.
6. Using filter paper, hole-punch several pieces
7. Submerge two disc pieces and place them into the first solution
(control)
8. Start a timer using the stopwatch immediately and record the time
that the filter paper took to reach the top.
9. Repeat step 7) and 8) with the rest of the H2O2 concentrations (2%,
3%, 4%, 6%)
10. Conduct four more trials for each hydrogen peroxide concentration
and take the average of all five
11. Determine the rate of reaction by dividing using the formula

average time ( seconds ) taken for filter paper discs

1
Rate of reaction= reach the surface
12. Ensure all trials are performed in the same room for similar
environment for each trial
Amendment
create a potato and water solution using blender instead of manually
by use of mortar and pestle
o add 20 ml of distilled water and half of a potato.
o Blend for 20 seconds
Safety
When conducting an experiment hat involves chemical and glass, there
must always be precautions to be taken care to avoid any potential
injuries that could be prevented. Instructors and students should
always adhere to general laboratory safety procedures and wear
proper footwear, safety googled or glasses, laboratory coats and
gloves.
In the case that your hands come into contact with the chemical, wash
hands immediately with soap. Always keep safety goggles on during an
experiment to avoid coming into contact with your eyes
Dispose of any broken glass in the proper container
Hydrogen peroxide
Clear, colourless liquid, slight odour
Warning! Strong oxidizing agent and body tissue irritant
Always handle hydrogen peroxide with care since it is a chemical that
constrains acid contents
Use ventilation to keep airborne concentrations below exposure limits,
have approved eyewash facility, safety shower, and fire extinguisher
readily available. Wear chemical splash goggles and chemical resistant
clothing such as gloves and aprons. Wash hands thoroughly after
handling material and before eating or drinking
First aid measures:
Always seek professional medical attention after first aid measures
provided.
Eyes: immediately flush eyes with excess water for 15 minutes, lifting
lower and upper eyelids occasionally.
Skin: immediately flush eyes with excess water for 15 minutes while
removing contaminated clothing
Ingestion: call poison control immediately. Do not induce vomiting.
Rinse mouth with cold water. Give victim 1-2 cups of water or milk to
drink
Inhalation: remove to fresh air. If not breathing, give artificial
respiration

Handling and storage:

Handling: use with adequate ventilation and do not breathe dust of
vapor. Avoid contact with skin, eyes, or clothing. Wash hands
thoroughly after handling.
Storage: store in general storage area with other items with no specific
storage hazards. Store in cool, dry, well-ventilated, locked store room
away from incompatible materials.
Since the concentrations of the reactive materials in this laboratory
drain. The concentrations used in the investigations are deemed to be
safe by all chemical standards, but recall tat any compound has the
potential to harm the environment
Data collection qualitative data
- Time taken for the filter paper disc
- Concentration of
to reach the
hydrogen
- surface / s
- peroxide solution
- /%
m
- ( 0.02)
- 1
2
3
4
5 ean
-

0.00

Rate of
reaction
- / s-1

2.1

1.

1.5

0.0472

3.6

3.

6.0

3.8

2.0

3.

9.6

10.

0.0867

0.0806

5.9

6.

12.

11.

2.00

3.00

4.00

6.00

0
-

0
-

0
-

0.0976

0.1426