Hydrogen Peroxide and Enzymatic Breakdown by Catalase (John Austin)

Hydrogen Peroxide and Enzymatic Breakdown by Catalase (John Austin)

Principles Investigated:

Students will make hypotheses based on observed phenomenon.

Students will be able to see how the rate of reaction is related to surface area.

Students will see how cellular membranes can affect the rate of reaction in this experiment.

Students will come up with tests to determine what the reaction product(s) are.

Standards:

Cell Biology: The fundamental life processes of plants and animals depend on a variety of chemical reactions that occur in specialized areas of the organism's cells. As a basis for understanding this concept:

a. Students know cells are enclosed within semi permeable membranes that regulate their interaction with their surroundings.

a. Students know enzymes are proteins that catalyze biochemical reactions without altering the reaction equilibrium and the activities of enzymes depend on the temperature, ionic conditions, and the pH of the surroundings.

Materials:

- One fresh potato. (The market)

-1 lb. fresh beef or chicken livers. (The market)

-Fresh 3% hydrogen peroxide solution. Old stock may have broken down to water and O_2.(Costco, Drugstores, Markets)

-7 baby food jars per group. (Someone that has a baby or an old cat)

-2 clear1 l. glass bottles. (Recycling bin)

-2 clear .5 l ± plastic bottles per group. (Recycling bin)

-Helium balloons. (The Party Store, Walmart etc.)

-A short stick. (The back yard or garage)

-Tape. (Your desk)

-Flame source. (Lighter, matches, lit candle)

-Medium sized cooler. (The garage or possibly the trunk of your car)

-Ice. (A local glacier or possibly, in your freezer)

-Wood splints. Long fireplace matches will work. (The market)

-Small plastic bags. (A drawer in your kitchen)

Procedure:

Before class.

-Cut a fresh potato into enough 1 cm squares that each group can have two and store in a plastic bag in a cooler with ice.

-Finely chop around twice as much potato and store in a plastic bag or jar in the ice cooler.

-In your favorite margarita blender, add a small amount of water and blend about a third of a medium potato and store the product in a plastic bag or jar in the ice cooler.

-Clean the blender to prevent cross contamination and blend about one half the beef/chicken livers from the one pound sample that has been purchased. Store in a jar in the ice cooler.

-Transfer hydrogen peroxide into clear containers and label the containers A.

-Prepare similar containers with tap water and label the containers B.

At the beginning of class.

-The students group into their experiment groups that have been previously established.

-Have the students in each group prepare 7 baby food jars labeled 1, 2,3,4,5, 6 and 7.

For the experiment.

Part A.

-Into jar #1 have each group add about 20 ml of liquid A.

Note: 20 ml of liquid is about 20 mm of depth in a baby food jar.

-Into jar #2 have each group add about 20 ml of liquid B.

- Add an approximate 1 cm³ piece of freshly cut potato into each jar.

- Have each group write down their observations for each jar.

-After a suitable period of time have the groups share their observations and write them down on some form of projectable media so the entire class can see it.

Part B.

-After analyzing the observations the groups should come to the consensus that something in the liquid in Bottle B is causing a reaction with the potato cube that is producing a gas.

-Ask the class what they think can be done to speed up this reaction process and list the responses on the overhead projection. Hopefully increasing the surface area had been suggested. If not, you will creatively introduce into the list.

-You, the teacher have the minced potatoes ready after you pick “increasing the surface area” from the list.

-Have the groups each make hypotheses and have them test in jar #3. Minced potatoes and liquid A.

-Record the observations on the overhead. (There is a greater rate of reaction as evidenced by the amount of bubbles)

-Ask the students if they think a “pureed” potato substrate will increase the reaction.

-Have the students predict and test again with the “pureed” potatoes and Liquid A in jar #4.

-Record the results on the overhead. There should be a greater reaction observed.

-Ask the students why the blended/pureed potatoes produced a greater reaction. Have them speculate as to other reasons beyond surface area why the reaction might be greater.

Part C.

Lecture time.

-Tell the students that Liquid A contains hydrogen peroxide and that it is reacting with an enzyme called catalase that is in the potatoes.

-explain how catalase is specifically located in the peroxisomes of cell and remind the class that the cell has a phospholipid bilayer membrane and how the peroxisomes also have the phospholipid bilayer membrane.

-Tell the class that the shearing forces in the blender break apart the phospholipid bilayer membranes effectively increasing the amount of catalase available for reaction because the H₂O₂ does not have to cross the membranes to react.

-Explain how hydrogen peroxide is a powerful oxidizer that is produced during cellular respiration. Give some example why oxidizers are dangerous in the cell.

-Give the formula for hydrogen Peroxide H₂O₂ . H-O-O-H.

-Introduce the basic function of enzymes.

- Ask the class if they know of any other proteins that work with Oxygen that also contain heme groups. (hemoglobin).

- Show, using Google images or a transparency, how the catalase enzyme is made up of four protein chains and four heme groups. Also show how hemoglobin has a similar structure with four heme groups but that it functions differently.

-Give some example of other enzymes in the human body and show how they have different roles. Digestion, cellular respiration, detoxification in the liver (hint for the next section) etc.

Part D.

- Re-explain that more catalase is found in cells that are undergoing cellular respiration and give the groups a list of different tissue types and have them list them in order of how much catalase they would expect to find. Liver, skin, fat, kidney, muscle, heart, brain, intestine etc. Like the crash landing on the moon example given in SED 525, there is no one correct answer.

-Ask the students which of the human tissues is most like the potato tissue. (Fat and Starch both are storage.)

-Tell the class that you have chicken/beef liver and have the groups predict which reaction will be greater. Blended/pureed Potatoes with H₂O₂ or 1 cm³ pieces of liver.

-In jar #5 combine about 1 cm³ blended/pureed potatoes with H₂O₂ and in jar #6 combine about 1 cm³ of chicken/beef liver with H₂O_{2 .}

- Record the results on the overhead and discuss.

-Ask the groups if they would like to try “pureed” chicken/beef livers with the H₂O_2. Of course they will want to because they should be expecting an even greater reaction and the need to know has been established. (I hope)

-In jar #7, combine around 1 cm³ of blended chicken liver with H₂O₂ and have them observe and record the reaction.

-Make sure to tell the students to keep the lid off of Jar #7! Paper towels should be available if the reaction gets overzealous.

Part E.

-Project on a screen some possible non balanced reactions.

H₂O₂ → H₂O + O₂

H₂O₂ + Carbon source like starch or liver → CO₂ + H₂O

H₂O₂ + Carbon source like starch or liver → CO₂ + H₂

H₂O₂ → H₂ + O₂

-Ask the groups to hypothesize which gas is being produced in the reaction.

-Have the groups devise a test to determine which gas is being produced.

Possibly buoyancy, Glowing splint.

-Have the groups explain their tests and help them perform the tests in front of the class.

Part F.

-For the class, demonstrate the large scale gas collection of the “pureed” liver and H₂O₂ gas collection technique. (Bottle with H₂O₂ with “blended” chicken liver added after a balloon has been attached)

-Ignite the balloon. Explain about misconceptions in science.

Students Prior Knowledge:

The students should know how to work in small groups. The students should have some experience in working with the scientific method, making hypotheses, designing tests and analyzing data. The students should know the parts of the cell and how they are bounded by the phospholipid bilayer membrane.

Explanation:

_· Under normal conditions H₂O₂ breaks down very slowly to H₂O and O_2. 2H₂O₂ → 2 H₂O + O₂

_· H₂O₂ is a toxic byproduct of cellular respiration in all eukaryotic cells.

_· H₂O₂ is a strong oxidizing agent that can cause great damage to cells if it is not removed from the system.

_· The enzyme catalase is present in the peroxisomes of cells to catalyze the reaction of H₂O₂ to H₂O and O_2.

· One molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second. (Wikipedia)

The lab allows the students to build on their scientific method skills. The lab is directed towards introducing the concepts of the importance of surface area for reactions and, the introduction of enzymes and enzymatic activity. Other directions such as the effect of temperature or ph on the reactivity of the catalase enzyme can also be investigated using this technique but, is too much for one lab. Possibly these directions could be used as an extra credit investigation. Also, it is possible to measure the amount of O₂ produced in a reaction. See references for link to simple experimental set up.

The lecture introduces enzymes as important proteins that change the rate of reaction without being consumed in the reaction. This is the main point to teach. Reintroduction of phospholipid bilayer construction of cellular membranes to the students provides some reinforcement of previously learned material and, the regulatory aspects of cellular membranes can be elaborated upon. The description and image of the structure of the catalase enzyme can build on prior teaching on protein structure and, the comparison to the hemoglobin protein can show how similar proteins can have completely different functions. (Transport vs. catalyst) The introduction of the danger of oxidants in our bodies can be used to bring relevance by describing the roles of antioxidants in our bodies and the importance of having antioxidants in our diet. Also, a comparison of hydrogen peroxide and ozone can be made as examples of strong oxidizing agents.

The igniting of the gas filled balloon can help correct a scientific misconception for some people.

Questions and Answers.

1. Why is the presence of catalase important in a cell?

The enzyme catalase catalyzes this reaction. 2H₂O₂ → H₂O + O_2. H₂O₂ is a strong oxidant that can damage most molecules found in a cell. This molecular damage can inhibit or stop the molecules functioning. The reaction also returns Molecular O₂ to the system for use in cellular respiration.

2. Why do you find more catalase in liver tissue than in potato sample?

More cellular respiration is taking place in the liver tissue than in potato storage cells. More catalase is needed in cells that have high metabolism rates because more H₂O₂ is being produced there and must be removed.

3. Are the “blended” samples an example of a reaction that limited by the surface area availability?

No. The cells in the blended samples have had their cell membranes mostly destroyed by the shearing forces in the blender. The cellular molecules are in solution and the reaction rate is not governed by the trans membrane movement of molecules.

Applications to Everyday Life:

Enzymes are essential in the breaking down and treatment of sewage in our sewage treatment plants. Without the enzymes in the bacteria used in the treatment plants, our waste would be dumped “as is” into our oceans, lakes and rivers.

The release of CO₂ by yeast while making bread causes the bread dough to expand. (rise)

Reduced levels of catalase in hair follicles as people age is believed to cause whitening of hair by excess hydrogen peroxide.

http://picasaweb.google.com/csun.science.education/525F10?locked=true#5544303586103098306

Photos

http://picasaweb.google.com/csun.science.education/525F10#5544298109706405330

http://picasaweb.google.com/csun.science.education/525F10#5544303544950745266

http://picasaweb.google.com/csun.science.education/525F10#5544303569623671986

http://picasaweb.google.com/csun.science.education/525F10#5544303586103098306

References

3D Catalase picture link

htpp://upload.wikimedia.org/wikipedia/commons/7/71/Catal http ase_Structure.png

3D Hemoglobin picture link.

http://animalscience.ucdavis.edu/hedricklab/ruben/assig2/heme.jpg

Catalase H₂O₂ Test Apparatus

http://www.practicalbiology.org/data/images/originals/investigating-an-enzyme-controlled-reaction-catalase-and-hydrogen-peroxide-concentration-164.jpg

Wikipedia Information on Catalase.

http://en.wikipedia.org/wiki/Catalase

Shields, Martin. (2006) Biology Inquiries. San Francisco, CA: John Wiley and Sons.