liver and hydrogen peroxide experiment results

March 8, 2012

The Liver: Helping Enzymes Help You!

A fun chemistry challenge from Science Buddies

By Science Buddies

Key concepts Chemistry Acids Bases Temperature Physiology Molecular biology

Introduction Your liver is important for cleaning up any potentially dangerous substances you consume. But how does it do it?—With a little help from some complex chemistry. Within your liver, as within every tissue in the body, many chemical reactions occur. Often these reactions require "help" to happen at a faster speed, and this can be supplied by enzymes—tiny types of proteins.

The liver uses specialized enzymes to help it break down toxic substances and make them safer for the body to process. But an enzyme, just like the chemical reactions it modifies, needs certain conditions to do its work. So, some environments can make a liver enzyme effective, whereas others can prevent it from working at all.

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Background A chemical reaction occurs when compounds come together and their molecules interact to form new compounds. Sometimes these reactions happen by themselves, are usually very fast and spontaneous, and give off energy. Other chemical reactions need energy, without which they would proceed very slowly or not at all. Enzymes can help speed up these types of chemical reactions.

Enzymes are large proteins that speed up the rate of a chemical reaction by acting as a catalyst. A catalyst provides the necessary environment for the reaction to occur, thereby quickening it. Certain catalysts work for certain kinds of reactions; in other words, each enzyme has a particular type of reaction that it can activate. Enzymes can be very fussy and sometimes need to be in certain environments or conditions to work well—or at all. Some enzymes can even be damaged, such as when exposed to too much heat. A damaged enzyme may no longer work to catalyze a chemical reaction.

Catalase is an enzyme in the liver that breaks down harmful hydrogen peroxide into oxygen and water. When this reaction occurs, oxygen gas bubbles escape and create foam. Materials •    Raw liver (fresh or frozen, thawed; one quarter pound) •    Knife •    Cutting board •    Blender •    Water •    Refrigerator •    Medicine dropper •    Large plate •    Hydrogen peroxide (new or recently purchased bottle works best) •    Measuring teaspoon •    Two bowls •    Vinegar •    Baking soda •    Microwave-safe bowl (with a cover) •    Microwave oven Preparation •    Completely disinfect any surface that the raw liver touches during this activity. •    On the cutting board, carefully cut the liver into little, cube-shaped pieces, about one to two centimeters long. Be careful using the sharp knife. (An adult may need to help with this.) •    Place the liver cubes into a blender and add an equal volume of water. Blend on high speed, pulsing when necessary, until the liver is smooth and no chunks are present. Be careful of the sharp blades in the blender. •    Keep the blended liver in the refrigerator. Procedure •    Put one drop of the blended liver on the large plate. To the blended liver drop, add one drop of hydrogen peroxide. You should see a lot of bubbles! What do you think the bubbles are made of? This shows that the liver enzyme catalase is working to start the chemical reaction that breaks down the hydrogen peroxide that would be harmful to the body into less dangerous compounds. •    To test the effect of an acid on the liver enzyme, put one teaspoon of the blended liver in a bowl and mix it well with one teaspoon vinegar. What is the color and consistency of this mixture? Put one drop of the mixture on a clean part of the large plate and add one drop of hydrogen peroxide to it. Compared with the untreated blended liver, did more, less or about the same amount of bubbles form? Did they form more slowly, more quickly or at about the same rate? •    To test the effect of a base, put one teaspoon of the blended liver in a bowl and mix it with one teaspoon baking soda. What is the color and consistency of this mixture? Put one drop of the mixture on a clean part of the large plate and add one drop of hydrogen peroxide to it. Did more, less or about the same amount of bubbles form? Did they form more slowly, more quickly or at about the same rate? •    To test the effect of heat, put one teaspoon of the blended liver into a microwave-safe bowl. Cover the bowl and microwave it on high for 20 seconds. How does the blended liver look after heating? Remove a drop-size amount of the heated liver and put it on a clean part of the large plate. Add one drop of hydrogen peroxide to it. Did more, less or about the same amount of bubbles form? Did they form more slowly, more quickly or at about the same rate? •     Based on your observations, under which condition(s) does it look like the enzyme works best? Which condition(s) makes it work the worst? Why do you think this is so? •     Extra: Try experimenting with other conditions. For example, try freezing some blended liver or mixing it with salt and then test the enzyme's activity. Or you could try adding more than one teaspoon of vinegar or baking soda and then test the enzyme. Under which conditions does the enzyme work well, and under which ones does it work poorly? •     Extra: You could try this activity again using another enzyme, called bromelain, which digests proteins and can be extracted from pineapples. One protein that is fun to digest using bromelain is gelatin, which is found in many puddings and gelatinous desserts. How do different conditions affect the ability of bromelain to digest proteins? Observations and results

When exposed to hydrogen peroxide, did the blended liver bubble less when mixed with either the vinegar or baking soda compared with when it was untreated? Did it bubble even less after it was microwaved?

An enzyme needs certain conditions to work, and the ideal environment can be a hint as to where the enzyme normally works in the body. And because different body tissues have distinct environments—acidic or warm—each enzyme is tuned to work best under specific conditions.

Different tissues in the body have different pHs (pH is a measure of how basic or acidic a solution is). The liver maintains a neutral pH (about pH 7), which is easiest for its enzymes, such as catalase, to work in. Consequently, when exposed to hydrogen peroxide the liver should have produced more bubbles (oxygen gas), and at a faster rate, when it was untreated than when exposed to vinegar or baking soda. (It may have bubbled more when treated with baking soda, compared with vinegar, because it might have been better able to return the pH to around 7.)

Similarly, enzymes in the liver are also used to functioning at body temperature (37 degrees Celsius), so microwaving the blended liver to a temperature hotter than that should have damaged the catalase enzyme and clearly decreased the amount of bubbles when it was exposed to hydrogen peroxide.

Cleanup Safely dispose of any raw liver meat used in this activity by putting it in the trash when you are done. Completely disinfect any surfaces that the raw liver meat touched during this activity, and be sure to thoroughly wash your hands with soap and warm water.

More to explore " Enzymes Make the World Go 'Round " from Rader's Chem4Kids.com " Your Liver " from KidsHealth " Catalase " from David Goodsell and RCSB Protein Data Bank " Liver Stinks! " from Science Buddies " Which Fruits Can Ruin Your Dessert? " from Science Buddies This activity brought to you in partnership with Science Buddies

Investigation: Enzymes

Enzyme Lab Teacher's Guide

Measure the effects of changes in temperature, pH, and enzyme concentration on reaction rates of an enzyme

Explain how environmental factors affect the rate of enzyme-catalyzed reactions.

INTRODUCTION: What would happen to your cells if they made a poisonous chemical? You might think that they would die. In fact, your cells are always making poisonous chemicals. They do not die because your cells use enzymes to break down these poisonous chemicals into harmless substances.

Enzymes are proteins that speed up the rate of reactions that would otherwise happen more slowly. The enzyme is not altered by the reaction. You have hundreds of different enzymes in each of your cells.

Each of these enzymes is responsible for one particular reaction that occurs in the cell. In this lab, you will study an enzyme that is found in the cells of many living tissues. The name of the enzyme is catalase; it speeds up a reaction which breaks down hydrogen peroxide, a toxic chemical, into 2 harmless substances--water and oxygen. Light can also break down H 2 O 2 which is why the chemical is sold in dark containers.

The reaction is: 2H 2 O 2 → 2H 2 O + O 2

This reaction is important to cells because hydrogen peroxide is produced as a byproduct of many normal cellular reactions. If the cells did not break down the hydrogen peroxide, they would be poisoned and die. In this lab, you will study the catalase found in liver cells. You will be using chicken or beef liver. It might seem strange to use dead cells to study the function of enzymes. This is possible because when a cell dies, the enzymes remain intact and active for several weeks, as long as the tissue is kept refrigerated.

MATERIALS: 6 Test tubes / Test tube holders 3% Hydrogen peroxide Ice / Hot water Straight-edged razor blade  Scissors and Forceps Measuring Pipettes Stirring rod 

Fresh liver, Apple, and Potato, Yeast Vinegar / Baking Soda HCL and NaOH pH paper (optional)

PART A - Observe Normal Catalase Reaction

1. Place about 2 ml of the 3% hydrogen peroxide solution into a clean test tube.

2. Using forceps and scissors cut a small piece of liver and add it to the test tube. Push it into the hydrogen peroxide with a stirring rod. Observe the bubbles.         What gas is being released? (consider the equation above) _____

Throughout this investigation you will estimate the rate of the reaction (how rapidly the solution bubbles) on a scale of 0-5 (0=no reaction, 1=slow, ..... 5= very fast). Assume that the reaction in step 2 proceeded at a rate of "4"

Recall that a reaction that absorbs heat is endothermic; a reaction that gives off heat is exothermic. Feel the temperature of the test tube with your hand.

Has it gotten warmer or colder? Is the reaction endothermic or exothermic?

3. Pour off the liquid into a second test tube.            Assuming the reaction is complete, what is this liquid composed of?

What do you think would happen if you added more liver to this liquid?

Test this and record the reaction rate.  Reaction Rate:

4.  Add another 2ml of hydrogen peroxide to the liver remaining in the first test tube.   What is the reaction rate?

Synthesis --  Answer the question:  Is catalase reusable?   

REASONING .  

Part B - What Tissues Contain Catalase?

You will now test for the presence of catalase in tissues other than liver. Place 2 ml of hydrogen peroxide in each of 3 clean test tubes and then add each of the three test substances to the tubes.  As you add each test substance, record the reaction rate (0-5) for each tube.

Synthesis -- Do all living tissues contain catalase?   Claim:                                         

Evidence: Reasoning:

PART C - What is the Effect of Temperature on Catalase Activity?

1. Put a piece of liver into the bottom of a clean test tube and cover it with a small amount of water. Place this test tube in a boiling water bath for 5 minutes.

2. Remove the test tube from the hot water bath, allow it to air cool, then pour out the water. Add 2 ml of hydrogen peroxide. CAUTION: Use a test-tube holder for hot test tubes. 

 What is the reaction rate for the boiled liver and peroxide? __________

3. Put equal quantities of liver into 2 clean test tubes and 1 ml H 2 O 2 into 2 other test tubes. Put one test tube of liver and one of H 2 O 2 into an ice bath. Place the other set in a warm water bath (not boiling).

After 3 minutes, pour each tube of H 2 O 2 into the corresponding tube of liver and observe the reaction

What is the reaction rate for the cold liver/peroxide? _____  What is the reaction rate for the warm liver/peroxide? ____

Synthesis -- How does temperature affect the catalase enzyme?

Claim:                             

PART D - What is the Effect of pH on Catalase Activity

1. Add 2 ml hydrogen peroxide to 4 clean test tubes, then add:

Tube 1 – add 3 drops of acetic acid (vinegar)  pH =_______  Tube 2 – add 3 drops of sodium bicarbonate (base) pH =______ Tube 3 – add 3 drops of water (neutral) pH =_____ Tube 4 -- add 3 drops of 1M NaOH   pH =  _____

Now add liver to each of the test tubes (try to do it all at about the same time, so you can easily compare)

Rate of Reaction for:

Strong Acid  (HCL)  ____     Acid _____     Neutral ______   Base_____     Strong base (NAOH) _____

1.  How does  pH affect the reaction rate of catalase? Propose a way to  refine  your experiment to find the  exact , or OPTIMAL pH and temperature of catalase.

2.  The following graph shows reaction rates of various enzymes in the body.  Pepsin is found in the stomach, amylase in the saliva, and phosphatase in the liver. 

Synthesis:  How does pH affect the activity of enzymes?

Claim:      

Part E - Design an Experiment

Lactaid is a product designed to help people who cannot digest milk sugar (lactose) because they are missing the enzyme lactase. Many people are lactose-intolerant, a condition that is mainly genetic. Lactase breaks down lactose into two subunits: glucose and galactose.

To test for the presence of monosaccharides and reducing disaccharide sugars in food, the food sample is dissolved in water, and a small amount of Benedict's reagent is added. The solution should progress in the colors of blue (with no glucose present), green, yellow, orange, red, and then brick red when there is a large amount of glucose present. (Google benedict's test to see the way this looks.)

Design an experiment where you would determine how quicly lactaid works to break down milk sugar at different temperatures.. Be specific in your description, use drawings if necessary.

Other Resources on Enzymes

Analyzing Graphics - Enzymes - shows substrates and enzyme interactions and explores competitive inhibition

Observe Catalase Activity in Yeast - create sodium alginate spheres to observe how catalase breaks down hydrogen peroxide

Enzyme Activity Using Toothpickase - simulate the activity of an enzyme by breaking toothpicks.