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1B. ENZYME ACTIVITY (OXYGEN)

Background
Cells have to carry out thousands of chemical reactions very quickly to sustain life. Enzymes are vital to this
operation. Enzymes are protein catalysts—they increase the rate of the reaction by lowering the activation energy of
the reaction. An enzyme's shape is critical to its ability to catalyze reactions.
Enzymes are hyper-specific, that is, usually an enzyme interacts with only one substrate. Like catalysts in other
chemical reactions, enzymes are not consumed during the reaction but help turn the substrate into the final product.
Notice in the following reaction that the enzyme is present before and after the reaction.
enzym e(a q)  2H 2 O 2 (a q) 
enzym e complex
 2H 2 O(l)  O 2 (g)  enzyme(a q)

Hydrogen peroxide (H2O2) is a byproduct of aerobic respiration in cells and is used in cell signaling and apoptosis.
Hydrogen peroxide is highly reactive and can produce free radicals that damage nucleic acids, so cells must
carefully regulate its concentration. To remove excess hydrogen peroxide, cells produce an enzyme (such as
catalase or peroxidase) which breaks down H2O2 into oxygen (O2) and water (H2O), as shown above. This reaction
proceeds spontaneously without the enzymes at a very slow rate. This uncatalyzed reaction will serve as the
baseline, or control, in the initial investigation.

Driving Question
How does the catalyzed decomposition rate of hydrogen peroxide compare with the spontaneous (uncatalyzed)
decomposition rate?

Materials and Equipment

Use the following materials to complete the initial investigation. For conducting an experiment of your own design,
check with your teacher to see what materials and equipment are available.

 Data collection system  Magnetic stirrer and stirring bar

 Oxygen gas sensor  Base and support rod
 Sampling bottle, 250-mL  3-Finger clamp
 Graduated cylinder, 25-mL  1.5% Hydrogen peroxide (H2O2), 20.0 mL
 Pipet, 1-mL  Catalase suspension, 2.0 mL

Safety
Follow these important safety precautions in addition to your regular classroom procedures:
 Wear safety goggles at all times.
 If using a hot plate, use caution to prevent burns.

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 1B. ENZYME ACTIVITY (OXYGEN) / STUDENT HANDOUT

Initial Investigation
Complete the following investigation before designing and conducting your own experiment. Record all
observations, data, explanations, and answers in your lab notebook.

1. Put on your safety goggles.

2. Open the 1B ABI Enzyme-oxygen lab file. Connect the oxygen sensor to your device.
NOTE: If the lab file is not available, create a graph of Oxygen gas (ppm) versus Time. Set the sample rate to
15 seconds and set an auto-stop condition for three minutes.

3. Calibrate the oxygen gas sensor.

4. Put the magnetic stirring bar in the sampling bottle.

5. Set up the equipment as shown in Figure 1.

6. Use a graduated cylinder to transfer 20.0 mL of 1.5% H2O2 into the clean 250-
mL sample bottle or flask. If the bottle or flask is on a stir plate, set the stir
speed to a medium setting.

7. Use a pipet to add 2.0 mL of catalase and quickly seal the bottle or flask with
the sensor stopper. Begin data collection.
NOTE: Loosely plug the sample bottle or flask with the sensor. Stir
continuously on a medium setting or gently swirl the bottle or flask, making
sure the solution does not come in contact with the sensor.

8. Why does the addition of the yeast suspension cause a change in the oxygen Figure 1: Setup with stirrer
concentration inside the sampling bottle?

9. Copy Table 1 into your lab notebook to record the results.

10. When data collection has stopped, calculate the rate of the reaction in ppm/min and record it in your copy of
Table 1.

11. The spontaneous decomposition of hydrogen peroxide is very slow, less than 0.5%/day. When the
decomposition was measured in a controlled experiment over several days with the pressure and oxygen
sensors, the following data was obtained.
Table 1: Comparison of hydrogen peroxide decomposition rate with and without a catalyst
Spontaneous Rate of Catalyzed Rate of Increase in the Catalyzed
Sensor
Decomposition Decomposition Rate
Pressure sensor 4.26 × 10–5 kPa/min 1.00 kPa/minRECORD ANSWERS
Oxygen sensor 0.33 ppm/min & DATA IN YOUR NOTEBOOK.

a. Complete Table 1. How much faster is the catalyzed reaction you observed compared to the spontaneous
rate of decomposition?

b. Explain why the reaction is so much faster when an enzyme is present.

12. Is the rate of the reaction constant for the 180 seconds of data collection? Support your answer with evidence.

13. If the reaction continued to run, do you predict the reaction rate to be constant? Explain your thinking.

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Design and Conduct an Experiment

Many factors that affect the structure and function of enzymes and the reaction rate of enzyme-catalyzed reactions
can be easily manipulated in the lab. Identify one of these factors and design an experiment to determine how that
factor affects the rate of an enzyme-catalyzed reaction.

Design and carry out your experiment using either the Design and Conduct an Experiment
Worksheet or the Experiment Design Plan. Then complete the Data Analysis and Synthesis
Questions.

Design and Conduct an Experiment: Data Analysis

1. From your observations and data,

a. Describe how the independent variable you manipulated affected the rate of decomposition of hydrogen
peroxide. Does the data support your hypothesis? Justify your claim with evidence from your experiment.

b. Based on the evidence you collected, explain why the results occurred.

2. Is there any evidence in your data or from your observations that experimental error or other uncontrolled
variables affected your results? If yes, is the data reliable enough to determine if your hypothesis was
supported?

3. Identify any new questions that have arisen as a result of your research.

Synthesis Questions
1. If you were to double the amount of catalase in the initial investigation, how would the reaction rate change?
Explain your reasoning.

2. Many organisms, such as fungi, animals, and plants, have catalase.

a) What does this indicate about the enzyme?

b) Catalase is just one of thousands of different enzymes found in yeast cells and other organisms. Why do
organisms need so many different types of enzymes?

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3. The graphs below show the relative activity of α-amylase from two different species. Amylase is an enzyme
that breaks down complex carbohydrates, like starch, into simple sugars that are used in cell respiration. Figure
1 shows data obtained using α-amylase samples from the bacterium Bacillus subtilis, found in the gut of
termites across the southern United States.1

Figure 2 shows data for an α-amylase sample taken from the copepod Heliodiaptomus viduus. This organism is
found mainly in the Indian Ocean around hot vents. In each case, the enzyme was incubated at a given
temperature and then tested for activity at regular intervals.2

Figure 1. Amlyase activity in Bacillus subtilis Figure 2. Amylase Activity in Heliodiaptomus viduus

a. Discuss how and why temperature affects enzyme activity.

b. Explain why the optimal temperature for α-amylase is different for these species.

1
Femi-Ola, T. O.; Olowe, B. M. Characterization of Alpha Amylase from Bacillus subtilis BS5 Isolated from Ameritermes evuncifer
Silvestri. Research Journal of Microbiology 6 (2011): 140–146.
2
Dutta, T.K.; Jana, M; Pahari, P. R; Bhattacharya, T. The Effect of Temperature, PH, and Salt on Amylase in Heliodiaptomus viduus (Gurney)
(Crustacea: Copepoda: Calanoida). Turkish Journal of Zoology 30 (2006): 187–195.

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Design and Conduct an Experiment Worksheet

Many factors that affect the structure and function of enzymes and the reaction rate of enzyme-catalyzed reactions
can be easily manipulated in the lab. Identify one of these factors and design an experiment to determine how that
factor affects the rate of an enzyme-catalyzed reaction.

Develop and conduct your experiment using the following guide.

1. Based on your knowledge of enzymes and reactions, what environmental factors (abiotic or biotic) could affect
the rates of enzyme-catalyzed reactions?

______________________________________________________________________________________________

______________________________________________________________________________________________

2. Create a driving question: choose one of the factors you've identified that can be controlled in the lab and
develop a testable question for your experiment.

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3. What is the justification for your question? That is, why is it biologically significant, relevant, or interesting?

______________________________________________________________________________________________

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4. What will be the independent variable of the experiment? Describe how this variable will be manipulated in
your experiment.

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5. What is the dependent variable of the experiment? Describe how the data will be collected and processed in the
experiment.

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6. Write a testable hypothesis (If…then…).

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7. What conditions will need to be held constant in the experiment? Quantify these values where possible.

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8. How many trials will be run for each experimental group? Justify your choice.

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9. What will you compare or calculate? What analysis will you perform to evaluate your results and hypothesis?

______________________________________________________________________________________________

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10. Describe at least 3 potential sources of error that could affect the accuracy or reliability of data.

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11. Use the space below to create an outline of the experiment. In your lab notebook, write the steps for the
procedure of the lab. (Another student or group should be able to repeat the procedure and obtain similar
results.)

12. Have your teacher approve your answers to these questions and your plan before beginning the experiment.

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