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LAB TWO: ENZYME

CATALYSIS
ADRIENNE
HARREVELD
PERIOD 4
AP BIO
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Introduction A: Enzymes are catalytic proteins that speed up chemical reactions, but
do not produce them. These enzymes are not permanently altered or used up in the
process. In an enzyme-catalyzed reaction, the substrate or the substance acted on,
binds reversibly to the active site of the enzyme. The enzymes are able to achieve
this catalysis by lowering the activation energy (the energy required for activation).
This makes a reaction occur more easily. Each enzyme is specific for a particular
reaction because of unique amino acid sequences. Enzymes have a variety of ways for
causing catalysis. One is in regards to salt concentration, when the concentration is
close to zero, the charged amino acid side chains of enzyme molecules will attract
each other. The enzymes will denature and form an inactive precipitate. If the salt
concentration is high, normal interaction of charged groups will be blocked, new
interactions will occur and the enzyme will precipitate (Biology Lab Manual).
Ph is scale that measures acidity, it is based on logarithms. The scale runs from 0 to
14, 0 being the most acidic. The scale from 0-7 a substance is said to be acidic, at
about 7 the solution is neutral, past 7-14 the substance is said to be basic. An enzyme
will range between very acidic and very basic. The amino side chains readily gain or
lose H+ ions.
Most enzymes perform well at their neutral state.
Temperature: Enzyme reactions will increase with increasing temperature. However,
once this enzyme exceeds its temperature optimum the conformation of enzyme
molecules is disrupted. Many proteins are denatured at about 40-50 degrees Celsius.
Activations and Inhibitors. If a molecule that is not an enzyme speeds up the rate of
reaction it is called an activator. If it decreases the reaction rate it is an inhibitor.
These molecules can regulate how fast an enzyme acts.

Introduction B: The base line is an index of the initial concentration of something in a

solution. In this case, the initial concentration of H202

Introduction C: In exercise C you determine the rate of a reaction without a

catalysis. Meaning a reaction without any activations or inhibitors, changes in salt
concentration, pH, or temperature.

Introduction D: When catalase is added to a solution the rate of decomposition

changes. You can find out how much of something has been consumed by titrating the
solution in time intervals.

Question A: Will the addition of catalase cause a physical change in a substrate

showing decomposition?
Will a high amount of heat denature an enzyme?
Is catalase present in living tissue?
Question B: will you be able to determine an index of initial concentration of H2O2
in the solution?
Question C: Will uncatalayzed H2O2 decompose into H2O and O2 within 48 hrs?
Question D: How will the rate of H2O2 decomposition change at different time
intervals when catalyzed by the purified catalase extract?
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Hypothesis A: I believe that the addition of catalase to H2O2 will cause bubbles to
form showing that a gas (o2) evolved from the peroxide.
I also believe that when heated with boiling water the enzyme will become
denatured.
When peroxide begins to decompose bubbles form, I believe that the catalase in the
potato with hydrogen peroxide will cause bubbling.
Hypothesis B: If you assay for the amount of H2O2 properly then you will be able to
establish a base line.
Hypothesis C: I believe that 100% of the H2O2 will decompose within 48hrs and open
to the .
Hypothesis D: I believe the reaction rate of the amount of H2O2 decomposed by time
will gradually decrease with increasing time intervals.

Procedure: A:
Transfer 10 ml of 1.5% mL (.44M) H202 into a 50-mL beaker and
add 1 mL of the freshly made catalase solution. The bubbles coming from the reaction
mixture are O2, which results from the breakdown of H2O2 by catalase.
Keep the Freshly made catalase solution on ice at all times.
What is the enzyme in this reaction: Blended chicken liver
What is the substrate in this reaction? Hydrogen Peroxide
What is the product in this reaction? Water and Oxygen
How could you tell that the gas evolved is O2? The bubbles forming
Transfer 5 mL of purified catalase extract into a tube and place it in boiling water
bath for five minutes.
Transfer 10 mL of 1.5% H202.
What do you observe? What do you think would happen if the potato was boiled
before being added to the H2O2?
The high temperature had denatured the enzyme making it useless because it
exceeded its temperature optimum.
Cut 1 cm3 of potato
Macerate it
Transfer it into a 50 mL glass beaker containing 10 mL of 1.5% H2O2.
What do you observe? What do you think would happen if the potato was boiled
before being added to the H2O2?
The H2O2 reacts with the potato fizzing up. If the liver were boiled there would be
minimal reactions occurring.

B: Put 10 mL of 1.5% H202 into a clean glass beaker.

2. Add I mL of H20 (instead of enzyme solution)
3. Add 10 mL of H2SO4 (1.0 M). Use extreme care in handling reagents.
4. Mix well
5. Remove a 5 mL sample.
6. Place this sample into another beaker and assay for the amount of H202 as follows.
Place the beaker containing the sample over a piece of white paper. Use a pipette
and add KMnO4 a drop at a time until a persistent pink or brown color is obtained.
* Remember to swirl the solution after each drop.
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** Make sure you understand the calibrations on the syringe

7. Record your readings

C: 1. Put about 15 mL of H2O2 into a beaker.

2. Store the 15 mL of H2O2 uncovered at room temperature for 24 hours.
3. Repeat steps 5-7 in exercise 2B
4. Record your readings

D: 1. 10 seconds
a. Put 10 mL of H2O2 into a 50 mL glass beaker
b. Add 1 mL of catalase extract
c. Swirl gently until time is up
d. At the end of the time interval ( in this case 10)
2. 30,60,90,120,180,360
a. Each time, repeat steps a-d, except allow the reactions to proceed for 30, 60,
90, 180, 360 seconds, respectively, while swirling gently.

Data : 2B
Base Line Calculation:
Final reading of the burette: 1mL
Initial reading of the burette: 5 mL
Base line: 4 mL
2C:
Uncatalyzed H202 decomposition
Final reading of the burette: 1 mL
Initial reading of the burette: 1 mL
Amount of KMnO4: I drop
Amount of H2O2spontaneously decomposed: 4 mL
What percent of the H202 spontaneously decomposes in 24 hours? 100%

2D: Base Line Calculation:

Final reading of the burette: 1mL
Initial reading of the burette: 5 mL
Base line: 4 mL

KMnO4 (ml) Time (seconds)

10 30 60 90 120 180 360

Base Line 4 4 4 4 4 4 4
Final Reading 3 2 1.9 1.8 1.65 4.2 4.2
Initial Reading 5 3 2 1.9 1.8 5 5
Amount of KMnO4 2 1 .1 .1 .15 .8 .8
consumed
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Amount of KMnO4 2 3 3.9 3.9 3.85 3.2 3.2

Used

H2O2 Decomposed by Time

4.5

3.5

2.5 Amount of H2O2 used

1.5

0.5

0
10 30 60 90 120 180 360

Results A: In the first section of the procedure you could observe physical changes
when adding our catalase which was blended chicken liver. When adding the catalase
the hydrogen peroxide (our substrate) began to bubble. It was also observed that
after the enzyme reached its temperature optimum it becomes denatured and does
not have its intended effect.

Results C: After titrating the solution we found out when the Hydrogen Peroxide was
laid out, uncovered for forty eight hours the H2O2 decomposed into water (H20) and
oxygen (O2).

Results D: After titrating the solution at different time intervals, as the amount of
time elapsed increased, the amount of KMnO4 consumed should have decreased. This
happened for the most part until after 180 and 360 seconds the amount of KMnO4
consumed slightly increased. For the most part, as time increased the reaction rate
was slower.

Analysis:
The independent variable was time.
The dependent variable was the amount of H2O2 decomposed.
The control was in section A showing that catalase will cause Hydrogen to start to
decompose.
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Time Intervals (seconds)

Initia 10- 30- 60-90 90-120 120-180 180-360
l 0-10 30 60
Rates .2 .05 .03 0.0 -.000166 -.01083 0
(H202/sec) 7

2. When is the rate the highest? Explain why.

The reaction ate is the highest within the first time interval. When the hydrogen
peroxide and catalase are first mixed because the substrate concentration is high and
the molecules meet the active sites often and accurately.

3. When is the rate the lowest? For what reason is the rate low?
The reaction rate is the slowest near the end because within those high time intervals
the Hydrogen Peroxide is beginning to decompose into water and Oxygen. This means
there is less of the substrate to bind with the active site.

4. Explain the inhibiting effect of sulfuric acid on the function of catalase. Relate this
to enzyme structure and chemistry.
Adding sulfuric acid denatures the enzyme inhibiting its catalytic capability. Enzymes
are specially shaped to fit into the substrate it reacts with. When the acid is added it
changes the shape of the enzymatic protein so it can’t catalyze the H2O2.

5. Predict the effect of lowering the temperature would have on the rate of enzyme
activity. Explain your prediction.
The decomposition would slow down if the temperature was reduced. As the
temperature decreases the amount of kinetic energy also decreases and the enzyme is
less likely to bind with the active site. If the temperature gets low enough it is
possible that the substrate freezes and the enzyme becomes denatured.

6. Design a controlled experiment to test the effect of varying pH, temperature, or

enzyme concentration.

In order to test the effect of pH on rate of reaction, one needs to set up a series of
beakers with varying levels of pH. For this hypothetical experiment, use pH 7.0, pH
3.0, pH 5, pH 9, pH 11, pH13; for purposes of this experiment, we will call pH7 the
control because it is average pH of water. The first step is to establish a baseline by
mixing 10.0ml of H2O2 with 10.0ml of H2SO4 and 1.0ml H2O, swirl for five seconds,
extract 5.0ml and assay with KMnO4. Add the catalase and start the timer; swirl for
five seconds and then allow to react for 55 additional seconds (total reaction time 60
seconds), after one minute, add the proper syringe of sulfuric acid and swirl for 5
seconds to completely denature the catalase. Repeat for the remaining six pHs. Now,
for each ending solution transfer 5.0ml to a separate container and use a burette to
find out how much KMnO4 it takes to react with the H2O2 and subtract this from your
baseline. This is how much H2O2 was used up. Record and analyze your data.
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Determine the base line of the H202 present in moles.

Determine the amount of H202 remaining after 180 seconds in moles.

Determine the amount of H2O2 decomposed after 180 seconds in moles.

Conclusion: In this lab we were able to establish a base line, show that enzymes can
be denatured, show that enzymes are present in living tissues and show how the
addition of enzymes speed up decomposition. In section A we saw that heating the
catalase caused to become denatured making my hypothesis correct. When The
heated catalase was added to the hydrogen peroxide, the peroxide didn’t bubble and
the catalase kind of congeals. When adding a cooled catalase the hydrogen peroxide
bubbles, showing that decomposition is occurring. The fact that the potato bubbled
shows that there is catalase present in living tissues.
We were able to establish 4 mL as our baseline. After finding that our baseline was
four mL we found that after forty eight hours hydrogen peroxide decomposes on its
own into water and oxygen. Within the given time intervals the reaction rates
decreased for the most part and then increased after 180 seconds, so my hypothesis
was partially correct. The one area with the most error was the section D titrations.
Because the drops were so small and the syringe only read mL, we couldn’t really tell
how much KMnO4 was in each drop making our results and reaction rates a little off
center. If we were able to have syringes that were more precise we would have been
able to retrieve more accurate data. A change in experimental design could be the
time intervals. If the time intervals were on a scale i.e. increasing by 10 seconds each
time, we would have been able to determine a better understating of the reaction
rate and maybe we would have had less error and our graph would show a more
distinct trend.