Science in MotionWilkes University Revised 2010

Catalase Enzyme Lab- “Potato Power”

What Effect Will Different Concentrations of a Substrate Have On an Enzyme (and other variables)?

Standards:

PA ST & E:

3.1.10.A2. Explain cell processes in terms of chemical reactions and energy changes.

3.1.B.A2. Explain the importance of enzymes as catalysts in cell reactions.

3,1,12,A7. Evaluate metabolic activities using experimental knowledge of enzymes.

3.1.B.A9. Formulate and revise explanations and models using logic and evidence.

Introduction and Background:

Enzymes are biological catalysts. They are generally large proteins made up of several hundred amino acids. Enzymes catalyze thousands of chemical reactions that occur in living cells. Enzymes are highly specific so each one speeds up only one particular chemical reaction. Many kinds of enzymes are found in each cell but because they are used over and over there may be only a small amount of each enzyme present.

In this lab activity, you will study the catalase enzyme and its substrate, hydrogen peroxide. Catalase accelerates the breakdown of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2). The chemical equation for this reaction is:

2 H2O2 ----Catalase------> 2H2O + O2

Catalase is found in both plant and animal tissues. It is especially abundant in plant storage organs such as potatoes and the fleshy parts of fruits. Catalase is extremely important in cells because it prevents the accumulation of hydrogen peroxide. Hydrogen peroxide is a strong oxidizing agent, which tends to disrupt the delicate balance of cell chemistry. If too much hydrogen peroxide accumulates, it will kill the cell.

Several factors affect the action of enzymes: salt concentration, pH, temperature, enzyme poisons, radiation, the concentration of enzymes, and the concentration of the substrate.

This lab deals only with how an enzyme is affected by different concentrations of substrate - specifically, what effect will different concentrations of a substrate (H2O2) have on an enzyme (catalase in potato juice). For further study, the teacher or research student (with teacher’s permission and assistance) may change the independent variables of this experiment.

Guiding Questions:

  1. What function do enzymes perform in living cells?
  2. What are the independent and dependent variables in this experiment?
  3. What causes the paper disc to rise in the peroxide solution?
  4. What is your hypothesis concerning the concentration your group was assigned as

compared to other concentrations?

5. How do different concentrations of a substrate affect the enzymatic activity of the

catalase enzyme?

6. Describe the graph that will be produced from the data from this experiment.

7. What other factors could be studied?

Vocabulary:

Catalase is an enzyme found in plant and animal tissue that speeds up the break down of hydrogen peroxide, a product of cellular reactions into water and oxygen.

An Enzyme is a protein that catalyzesa specific chemical reaction within a living cell.

A Substrate is thesubstance upon which a specific enzyme acts.

Materials:(per group)

10 ml graduated cylinder

50 ml hydrogen peroxide ( the instructor will assign a specific concentration- 3%, 1%, 0.5%, 0.1%, or 0.0%)

forceps

5-10 paper circle disks (#1 filter-1.0cm in diameter- produced with a paper punch)

graph paper

small (30-50ml) beaker with20 ml filtered potato extract (prepared prior to class) or commercially prepared catalase

slightly larger beaker (100-150 ml) to hold ice

glass stirring rod

small funnel

stop watch

1 piece paper toweling

Safety Notes:

1.Hydrogen peroxide can damage your clothes. Rinse any spills with water

immediately.

2.Keep hydrogen peroxide out of your eyes. Wear safety glasses!

3. Take care the glass stirring rod does not roll off the table and also be careful the graduated cylinder does not break.

4. Report any accidents of spills to your instructor.

5. Wipe table and wash hands before leaving the lab.

Procedure:

*Keep the potato extract (catalase) in an ice bath throughout the experiment.

1. Obtain your assigned sample of H2O2 concentration along with the other materials

needed.

2. Record the percent of H2O2 in Table I.

3. Pour 10ml. of the hydrogen peroxide into the graduate cylinder. You may use the

small funnel is assist not spilling the peroxide.

4. Use forceps to obtain one paper circle disk and immerse the paper in the potato juice

holding with the forceps for 5seconds.

5.Drain excess juice from the paper circle disk by touching on a paper towel for 10

seconds.

6. Place the paper circle disk onto one rounded end of the glass stirring rod and carefully

but quickly guideinto the hydrogen peroxide cylinder (to the bottom). The oxygen

produced willget trapped in the fibers of the paper circle and cause the circle to rise to

the surface.

7. Use a stopwatch or the second hand on a clockto calculate the time it takes for the

paper toreach the surface. Begin timing (to the nearest .1 seconds) the instant the

paper touches the peroxide. To zero stop watch, (if not already on zero), set Mode

(center button) to _ _ _ _ _ , press left button to zero, and right button to start and

stop. In the event your paper disk does not rise after 300 seconds, you may stop

and try again. Record as 300+.

8. Record the time in seconds in Table I.

9. Repeat steps 3-8 at least 4 times using new hydrogen peroxide and a new paper

disk for each trial. You have time to redo any trials with questionable methods.

10. Calculate the average time and record in Table I.

11. Obtain class data for each concentration of hydrogen peroxide used by all groups in

the class and record this data in Table II.

12. Graph the results (using Portrait layout) with the Substrate (Hydrogen Peroxide) (%)

labeled onthe X- axis and Reaction Time (Seconds) on the Y-axis. (Label the X-axis

in equal units from0.0% to 3.0% and the Y-axis in equal units from 0.0 seconds to

300.0 seconds.

Optional activities:

1.Use a constant hydrogen peroxide concentration and vary the catalase

concentration.

2. Test various plant and animal tissues: i.e.- muscle, liver, heart, potato, fruits, roots,

and leaves. Samples may be chunks or ground, raw or cooked. Use 3%H2O2.

References:

Catalase: What effect will different concentrations of a substrate have on an enzyme? (1999).

Huntingdon, PA: Juniata College- Science in Motion.

Student Evaluation (Data Sheet)

What effect will different concentrations of a substrate have on an enzyme?

Name ______Date ______

Observations:

Table I - Group Data (_____%H2O2)

Trial / Time ( sec.)
1
2
3
4
5
Avg. Time

Table II - Class Data

Conc. H2O2 / Avg. time (sec.)
0%
0.1%
0.5%
1%
3%

Using the graph paper on the back of this page or provided by your teacher or a computer graphing program, write a Title, using correct units and titles, label the x and y axes of your graph and graph the Class Data.

Analysis/Conclusions:

  1. Choose the word in parentheses that completes the sentence.

The greater the concentration of H2O2, the (greater, less) time required to raise the

paper circle.

  1. What is the independent variable in this experiment? What is the dependent variable?
  1. Write a brief paragraph summarizing what you have learned about the effect of an enzyme on different concentrations of a substrate. Include the terms: ENZYME, SUBSTRATE, HYDROGEN PEROXIDE, and CONCENTRATION. Refer to your graph to help in your explanation. Be sure to explain why the results you observed occurred.

Teacher Notes:

1.Time needed to complete lab: 45 minute period

  1. Target grade level:General biology (9-11)
  1. Objectives: To determine that the higher the concentration of substrate (hydrogen peroxide), the faster the reaction will occur.
  1. Major concepts: Enzyme/substrate actions and the importance of enzymes in living cells.
  1. Preparation: Produce the potato extract (containing the catalase) by placing chunks of a large peeled potato (in 2 cm pieces) into a blender along with 4 ice cubes and 250ml of cold water, grind, then filter through cheese cloth taped over a large funnel into a large beaker - store on ice in the dark until class time.

Divide students into groups of 3-4 per group and depending on class size assign two groups to each variable. Enough materials have been provided for 8 groups. You can further give each student a job using the first letter of last names: Student 1 measures peroxide into graduate; Student 2 places the paper disk onto the stirring rod and into the peroxide; Student 3 works the stop watch; Student 4 (if used) records results (for others to copy before leaving the lab), finds average, and writes this on board or Excel program. If 3 per group- all students record results & Student 1 records on board.

Answers to Analysis Questions:

  1. Less
  1. The Independent (Manipulated) variable is the Peroxide %, the dependent

(responding) variable is the Time it takes the paper disk to rise to the top. The

dependent variable it what you are measuring.

  1. At 0% concentration of the substrate (H2O2) there is nothing for the enzyme to act upon so the disk will not rise. As the concentration of the substrate increases, more peroxide is present to come into contact with the catalase on the paper disk to increase the speed of the reaction and produce more oxygen bubbles. This will cause the disk to rise faster with increasing peroxide concentrations.

Catalase Enzyme LabPage 1