Enzyme Catalysis Lab

Hypothesis
The enzyme catalase, under optimum conditions, effectively speeds the decomposition of
hydrogen peroxide.

Method
Exercise 2A: Test of Catalase Activity

In Part 1, 10 mL of 1.5% H2O2 were transferred into a 50-mL beaker. Then, 1 mL of fresh
catalase solution was added and the reaction was observed and recorded. In Part 2, 5 mL of
catalase was placed in a test tube and put in a boiling water bath for five minutes. 10 mL of 1.5%
H2O2 were transferred to a 50-mL beaker and 1 mL of the boiled catalase was added. The
reaction was observed and recorded. In Part 3, 10mL of 1.5% H2O2 were transferred to a 50 mL
beaker. 1 cm3 of liver was added to the beaker and the reaction was observed and recorded.

Exercise 2B: The Baseline Assay

10 mL of 1.5% H2O2 were transferred to a 50-mL beaker. 1 mL of H2O was added instead of
catalase, and then, 10 mL of H2SO4 were added. After mixing well, a 5 mL sample was removed
and placed over a white sheet of paper. A 5-mL syringe was used to add KMnO4, 1 drop at a
time until a persistent brown or pink color was obtained. The solution was swirled after every
drop, and the results were observed and recorded. The baseline assay was calculated.

Exercise 2C: The Uncatalyzed Rate of H2O2 Decomposition

A small quantity of H2O2 was placed in a beaker and stored uncovered for approximately 24
hours. To determine the amount of H2O2 remaining, 10 mL of 1.5% H2O2 were transferred to a
50-mL beaker. 1 mL of H2O was added instead of catalase, and then, 10 mL of H2SO4 were
added. After mixing well, a 5 mL sample was removed and placed over a white sheet of paper. A
5-mL syringe was used to add KMnO4, 1 drop at a time until a persistent brown or pink color
was obtained. The solution was swirled after every drop, and the results were observed and
recorded. The percent of the spontaneously decomposed H2O2 was calculated.

Exercise 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition

The baseline assay was reestablished following the directions of Exercise 2B. Before starting the
actual experiment a lot of preparation was required. Six labeled cups were set out according to
their times and 10 mL of H2O2 were added to each cup. 6 mL of catalase were placed in a 10-mL
syringe, and 60 mL of H2SO4 were placed in a 60-mL syringe. To start the actual lab, 1 mL of
catalase was added to each of the cups, while simultaneously, the timer was started. Each of the
cups were swirled. At 10 seconds, 10 mL of H2SO4 were added to stop the reaction. The same
steps were repeated for the 30, 60, 120, 180, and 360 second cups, respectively.

Afterwards, a five 5 mL sample of each of the larger cups were moved to the corresponding
labeled smaller cups. Each sample was assayed separately by placing each over a white sheet of
paper. A 5-mL syringe was used to add KMnO4, 1 drop at a time until a persistent brown or pink
color was obtained. The solution was swirled after every drop, and the results were observed and
recorded.

Results

Table 1
Enzyme Activity

Activity Observations

Enzyme activity The solution only bubbled slightly and

slowly.

Effect of Extreme temperature The catalase had no reaction with the H2O2;
there were no bubbles

Presence of catalase The solution foamed up immediately

Table 2
Establishing a Baseline

  Volume

Initial reading 5.0 mL

Final reading 1.0 mL

Baseline ( final volume – initial volume) 4.0 mL

Table 3
Rate of Hydrogen Peroxide Spontaneous Decomposition

  Volume

Initial KMnO4 5.0 mL

Final KMnO4 1.2 mL

Amount of KMnO4 used after 24 hours 3.8 mL

Amount of H2O2 spontaneously 0.4 mL

decomposed
( ml baseline – ml after 24 hours)
 Percent of H2O2 spontaneously 9.5%
decomposed
( ml baseline – ml after 24 hours/
baseline)

Table 4
Rate of Hydrogen Peroxide Decomposition by Catalase

Time ( Seconds)
  10 30 60 120 180 360
Baseline 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL
KMnO4
Initial 5.0 mL 5.0 mL 5.0 mL 5.0 mL 5.0 mL 5.0 mL
volume
KMnO4
Final 2.2 mL 1.4 mL 2.0 mL 1.7 mL 2.4 mL 2.3 mL
volume
KMnO4
Amount 2.8 mL 3.6 mL 3.0 mL 3.3 mL 2.6 mL 2.7 mL
KMnO4
used
(baseline –
final)
Amount 1.2 mL 0.4 mL 1.0 mL 0.7 mL 1.4 mL 1.3 mL
H2O2 used
(KMnO4 –
initial)

Amount of Hydrogen Peroxide Decomposed by Catalase

Exercise 2A: Test of Catalase Activity

1. Observing the reaction of catalase on hydrogen peroxide:

   a. What is the enzyme in this reaction?  catalase

    b. What is the substrate in this reaction? Hydrogen peroxide

    c. What is the product in this reaction? Oxygen & water

    d. How could you show that the gas evolved is O2? The gas could be shown to be O2 if the
gas were collected in a tube, and a glowing splint was held in the tube. If the splint glowed, it
would prove the gas was oxygen.

2. Demonstrating the effect of boiling on enzyme action:

   a. How does the reaction compare to the one using the unboiled catalase? Explain the
reason for this difference. While the unboiled catalase caused bubbles to form in the solution,
the boiled catalase did not react at all because boiling an enzyme causes the protein to unfold and
therefore denatures it.

3. Demonstrating the presence of catalase in living tissue:

   a. What do you think would happen if the potato or liver was boiled before being added
to the H2O2? The catalase in the liver would have been denatured by the boiling and would not
have reacted with the H2O2.

Analysis of Results

1. Determine the initial rate of the reaction and the rates between each of the time points.

Time Intervals (Seconds)

  Initial 0 to 10 10 to 30 30 to 60 60 to 120 120 to 180 180 to 360

Rates 0.12 mL/sec -0.04 0.02 -0.005 0.01167 -0.00083

mL/sec mL/sec mL/sec mL/sec
mL/sec

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

The rate is the highest in the initial ten seconds because the concentration of catalase is at its
highest. As more of the product is formed, it blocks the reaction between the catalase and the
hydrogen peroxide.
3. When is the rate the lowest? For what reasons is the rate low?

The rate is lowest during the 180-360 seconds time period because of the law of mass action.
This law says that when there is a high concentration of product as in this period, the enzymes
will be blocked by the product (water) from reaching and reacting with the substrate (H2O2).

4. Explain the inhibiting effect of sulfuric acid on the function of catalase. Relate this to
enzyme structure and chemistry

Sulfuric acid has an inhibiting effect on catalase function because it causes the pH level in the
solution to lower considerably. Acidic solutions cause the protein structure of the enzyme to gain
H+ ions causing it to denature.

5. Predict the effect lowering the temperature would have on the rate of enzyme activity.
Explain your prediction.

Lowering the temperature of the catalase would slow the rate of reaction until it finally caused
the enzyme to denature, and it would no longer react with the substrate. Most enzymes are only
affective in a temperature range between 40° - 50° C.

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

Part 1: Enzyme Activity at Room Temperature

Add 10 mL of 1.5% H2O2 to a 50-mL beaker, and add 1 mL of room temperature catalase. Mix
well and add 10 mL of H2SO4. Watch the reaction and record the results.

Part 2: The Effect of Excessive Heat on Enzyme Activity

Put 5 mL of catalase into a test tube and heat thoroughly over a Bunsen burner. Add 1 mL of the
heated catalase to 10 mL of 1.5% H2O2 in a 50-mL beaker. Add 10 mL of H2SO4. Watch the
reaction and record the results.

Error Analysis
Any number of factors in this lab could have affected the results of this experiment. To get the
desired results all of the measurements had to be precisely accurate and fully planned before
hand, and although I thought mine were they may not have been. In Exercise D especially, the
factor of planning became increasingly essential. The first attempt at 2D was unsuccessful due to
several reasons. First of all, the measurements, which were taken, could have possibly been
inaccurate and the 60-mL syringe containing H2SO4 also dripped into one of the cups early which
did not allow the reaction to fully take place. There was also some confusion on the operation of
the timer and precise planning in its use. The second attempt at 2D contained errors as well. The
measurements were still not as accurate as they should have been, and the solution did not appear
entirely uniform. In one cup, for example, the first drop of KMnO4 left a persistent pink color,
and then after over a minute, it returned back to being clear. It then took several milliliters more
to get it back to a pink color.

Discussion and Conclusion

This lab showed how catalase increased the rate of decomposition of hydrogen peroxide. In 2A,
it was shown that catalase causes a visual reaction with H2O2, that when boiled catalase is no
longer reactive, and that catalase is present in living tissue. Lab 2C shows that the natural
decomposition of H2O2 is much slower than the enzymatic reaction. Lab 2D showed the
decomposition of H2O2 over just a period of six minutes, and it had already decomposed more
than the uncatalyzed H2O2 had done in 24 hours.