Name
Grade and Section:
Understanding Enzymes
CONTENT:
Catalase is an enzyme found in nearly all
living organisms exposed to oxygen, where it
plays a crucial role in protecting cells from
oxidative damage. It catalyzes the
breakdown of hydrogen peroxide, a harmful
byproduct of metabolic processes, into water
and oxygen. This reaction occurs rapidly,
allowing cells to neutralize hydrogen
peroxide before it can cause harm. Catalase
is highly efficient, capable of converting
millions of hydrogen peroxide molecules per
second. Its activity is vital for maintaining
cellular health, particularly in tissues that
generate high levels of reactive oxygen
species, such as the liver and red blood cells.
Activity 1: Testing Catalase
Materials:
Fresh liver sample (raw)
3% hydrogen peroxide solution (H₂O₂)
Scalpel or knife
Test tubes or small beakers
Tweezers
Pipette or dropper
Safety goggles and gloves
Stopwatch or timer
Steps:
1. Prepare the Liver Sample:
o Cut a small piece of raw liver (about the size of a pea) using a
scalpel or knife. Place it on a clean surface or in a dish.
2. Transfer the Liver to a Test Tube:
o Using tweezers, carefully place the liver piece into a test tube
and place it in the test tube rack
3. Add Hydrogen Peroxide:
o Using a pipette or dropper, add about 5-10 mL of the 3%
hydrogen peroxide solution directly onto the liver in the test
tube.
4. Observe the Reaction:
o Immediately after adding the hydrogen peroxide, observe the
liver sample. Bubbling or foaming should occur as oxygen gas is
released due to the catalase breaking down the hydrogen
peroxide into water and oxygen.
o Touch the test tube, and determine whether the test tube is
warm or not.
5. Measure Reaction Speed:
o Start the stopwatch as soon as the hydrogen peroxide is added
and note how quickly bubbles form. The rate of bubbling is an
indication of catalase activity.
6. Record Observations:
o Record the intensity and duration of the reaction, noting the
amount of bubbling and whether the reaction slows down over
time.
OBSERVATIONS
1. Write the chemical equation of this set-up.
2. What is the effect of adding hydrogen peroxide to the liver sample?
3. Is the reaction of catalase, energy giving or energy absorbing? Is is
endothermic or exothermic? Explain your answer.
Procedure:
7. Add 5-10ml hydrogen peroxide up to three times in the test tube
containing the liver, what can you say about the amount of bubbles
produced everytime? Is there a difference?
OBSERVATIONS
4. What can you say about catalase as an enzyme based on procedure
number 7?
PART TWO: Effect of Heat.
Steps:
1. Preheat the water bath to 50°C.
2. Place a small piece of liver in a test tube.
3. Using tongs, immerse the test tube in the water bath for 5 minutes.
4. After 5 minutes, carefully remove the liver sample and let it cool
slightly.
5. Add 5-10 mL of hydrogen peroxide to the liver sample and observe the
reaction.
6. Record the intensity of bubbling and reaction time.
7. Repeat steps 1-6 for liver samples at 60°C and 70°C, using new liver
pieces for each temperature.
8. Compare the reaction rates at different temperatures with the control.
Observations
5. What are the differences in the amount of bubbles produced in the
different samples? Present your answers in a tabulated form in your lab
report.
6. One can say that the enzyme catalase is the second experiment is
denatured. What is meant by this? Explain your answer.
Experiment 3: Catalase Activity in Acidic Conditions (pH ~2-3)
Steps:
1) Dilute muriatic acid (HCl) with distilled water to create a ~1% solution.
Test the pH with a pH strip or meter to ensure it’s around pH 2-3.
2) Prepare a liver sample and place it in a test tube.
3) Add 5-10 mL of the dilute muriatic acid to the liver in the test tube and
allow it to sit for 2 minutes.
4) After 2 minutes, add 5-10 mL of hydrogen peroxide to the test tube.
5) Observe and record the reaction.
6) Compare the results to the neutral pH control.
Experiment 3: Catalase Activity in Basic Conditions (pH ~10-11)
1) Dilute bleach with distilled water to create a ~1% solution. Test the pH
with a pH strip or meter to ensure it’s around pH 10-11.
2) Prepare a liver sample and place it in a test tube.
3) Add 5-10 mL of the dilute bleach solution to the liver in the test tube
and allow it to sit for 2 minutes.
4) After 2 minutes, add 5-10 mL of hydrogen peroxide to the test tube.
5) Observe and record the reaction.
6) Compare the results to the control and the acidic pH experiment.
OBSERVATIONS
1. What observations did you make in the reaction at neutral pH?
Consider the intensity and speed of the reaction.
2. How did the catalase reaction in acidic conditions (using
muriatic acid) differ from the reaction at neutral pH?
Did the bubbling slow down, stop, or become more intense?
3. What changes did you observe in the reaction when bleach
(basic pH) was used?
How did the basic condition affect the catalase activity compared to
the control and acidic conditions?
4. Why do you think the enzyme catalase behaves differently at
extreme pH levels?
Relate this to the enzyme's structure and how pH affects protein
function.
5. What do you think would happen if you used a more extreme
pH (even stronger acid or base)?
Predict how further changes in pH might influence the enzyme’s
activity.
6. Based on your observations, at what pH level does catalase
seem to work best?
Identify the optimal pH for catalase activity and explain why this might
be important for biological systems.
7. What could be the implications of pH changes in living
organisms on enzymes like catalase?
Discuss how organisms might regulate pH to maintain proper enzyme
function.
Investigating the Effect of Substrate Concentration on Liver Catalase Activity
Catalase Activity at Low Substrate Concentration (1% Hydrogen
Peroxide)
Steps:
1. Prepare a small piece of liver (about the size of a pea) and place it in a
test tube.
2. add 5-10 mL of 1% hydrogen peroxide solution to the liver sample
using a pipette or graduated cylinder.
3. Observe the reaction (bubbling intensity and duration) as the catalase
breaks down the hydrogen peroxide.
4. Record the speed and amount of bubbling as a measure of catalase
activity at this low substrate concentration.
Experiment 2: Catalase Activity at Medium Substrate Concentration
(3% Hydrogen Peroxide)
Steps:
1. Prepare a new liver sample and place it in a clean test tube.
2. Add 5-10 mL of 3% hydrogen peroxide solution to the liver.
3. Observe and record the reaction, noting the intensity and duration of
bubbling.
4. Compare the results with the 1% hydrogen peroxide solution.
5. Repeat the steps with new liver samples for 6%, 9%, and 12%
hydrogen peroxide solutions.
6. For each concentration, add 5-10 mL of the respective hydrogen
peroxide solution to a test tube containing a liver sample.
7. Observe the reaction, noting the intensity, speed, and duration of
bubbling for each concentration.
8. Compare all results, identifying trends in catalase activity as the
substrate concentration increases.
Analysis:
1. Graphing Results (Optional):
o After gathering data from all experiments, plot the substrate
concentration (x-axis) against the rate of reaction (y-axis). The
rate can be estimated from how quickly the bubbles form or the
total volume of gas produced if you have measuring tools.
2. Processing Questions:
3. What observations did you make at the lowest substrate concentration
(1% hydrogen peroxide)?
4. How did the intensity and duration of bubbling compare to the other
concentrations?
5. How did the catalase activity change as the hydrogen peroxide
concentration increased from 1% to 3%, 6%, and 9%?
6. Describe any noticeable differences in the speed and amount of
bubbles.
7. What did you observe when using the highest concentration of
hydrogen peroxide (12%)?
8. Did the rate of reaction continue to increase, or did it level off? Why
might this happen?
9. At what substrate concentration do you think the catalase enzyme
reaches its maximum activity (saturation point)?
10. Explain how enzyme saturation might affect the results.
11. How do you think increasing the substrate concentration further
(above 12%) would impact catalase activity?
12. Predict whether catalase activity would continue to increase or if
it would slow down and why.
GENERALIZATIONS:
Reflection:
