Characteristics of Enzyme Function: Liver Lab Introduction: When inside our body, hydrogen peroxide is a poisonous chemical that is continually formed as a byproduct of reactions in living cells. Since it is poisonous, the cells must either get rid of it or change it into something nonpoisonous. This detoxification occurs in the liver. If the liver cannot do this, you will die. In order for us to survive, chemical reactions in our body must take place quickly and at optimal temperatures and pH. Enzymes are proteins which mediate all such chemical reactions. Liver contains the enzyme catalase, which breaks down hydrogen peroxide (H2O2) formed in cells to water (H2O) and oxygen gas (O2). When hydrogen peroxide is broken down by catalase, bubbles of oxygen gas are released. The chemical equation for the process is described below: 2 H2O2 → 2 H2O + O2 In this investigation, you will observe some characteristics of enzyme function using hydrogen peroxide as the substrate. You will use liver (which contains the enzyme catalase) to determine what happens when hydrogen peroxide is added to it. You will also investigate some conditions that might affect the rate of the reaction. Materials: 3% hydrogen peroxide 10 mL graduated cylinder fresh liver forceps
test tubes test tube rack glass stirring rod thermometers
hot plate ice 250 mL beakers
Procedure: As you go through each step, estimate the reaction rates using this scale and record your numbers in Data Table 1. 0 = no bubbling 1 = slow 2 = fast 3 = very fast
1. Will a reaction occur when a small piece of liver is added to hydrogen peroxide? PREDICTION: Will there be bubbles upon adding a piece of liver to hydrogen peroxide? Why or why not? ________________________________________________________________________ 1
DIRECTIONS: Measure out 2 mL of hydrogen peroxide in a graduated cylinder. Pour the hydrogen peroxide into a clean test tube. Obtain a small piece of liver from the teacher. Carefully drop the liver into the test tube. QUESTIONS: What did you observe? __________________________________________________________________ __________________________________________________________________ Propose a hypothesis as to why the reaction stopped. Was all of the hydrogen peroxide changed into product or was the catalase (enzyme) used up? __________________________________________________________________ __________________________________________________________________
2. Test your hypothesis. DIRECTIONS: a. Pour the used hydrogen peroxide from step 1 into a clean test tube. Add a fresh piece of liver to the used hydrogen peroxide. b. Measure out 2 mL of fresh hydrogen peroxide. Add it to the used liver from step 1 (in the first test tube). c. Record your results in Table 1.
QUESTIONS: What did you observe in these two test tubes from steps 2(a) and 2(b)? (a)_______________________________________________________________ (b)_______________________________________________________________ Propose a hypothesis as to why the reaction stopped. Was all of the hydrogen peroxide changed into product or was the catalase (enzyme) used up? __________________________________________________________________ __________________________________________________________________
3. Does temperature have any effect on the rate of reaction? DIRECTIONS: a. Add a piece of liver sitting in boiling water to a clean test tube. b. Add a piece of liver sitting in warm water to a second clean test tube. c. Add a piece of liver sitting in ice cold water to a third clean test tube. d. Add 2 mL of hydrogen peroxide to each of the three test tubes. e. Record your results in Table 1. QUESTIONS: Did the temperature affect the rate of the reaction? How? __________________________________________________________________ __________________________________________________________________
RINSE AND CLEAN THE TEST TUBES THOROUGHLY!!!
4. Does pH have any effect on the rate of reaction? DIRECTIONS: f. Add a piece of liver sitting in hydrochloric acid (HCl) to a clean test tube. g. Add a piece of liver sitting in sodium hydroxide (NaOH) to a second clean test tube. h. Add a piece of liver sitting in tap water to a third clean test tube. i. Add 2 mL of hydrogen peroxide to each of the three test tubes. j. Record your results in Table 1. QUESTIONS: Did the pH affect the rate of the reaction? How? __________________________________________________________________ __________________________________________________________________
Data & Results: Table 1. Procedure Step and Liver Treatment Everyone does steps 1 and 2: 1. fresh liver and fresh hydrogen peroxide 2. (a) new liver and old hydrogen peroxide 2. (b) old liver and new hydrogen peroxide Effect of Temperature on Enzyme Reaction Rate: 3. (a) boiled liver and hydrogen peroxide (temp. = _______) 3. (b) warm liver and hydrogen peroxide (temp. = _______) 3. (c) ice cold liver and hydrogen peroxide (temp. = _______) Effect of pH on Enzyme Reaction Rate: 4. (a) liver in HCl and hydrogen peroxide (pH = _______) 4. (b) liver in NaOH and hydrogen peroxide (pH = _______) 4. (c) liver in tap water and hydrogen peroxide (pH = _______)
Graphing: 1. Make a bar graph of normal enzyme function by plotting your data (steps 1 and 2) from Table 1. Put the type of liver and hydrogen peroxide combination (new or old) on the xaxis and reaction rate (05; # bubbles produced) on the yaxis. Be sure to give the graph a title, label the axes, and provide a key/legend.
2. Make a second bar graph showing the effect of temperature on enzyme activity by plotting your temperature data (steps 3ac) from Table 1. Put temperature (°C) on the xaxis and reaction rate (05; # bubbles produced) on the yaxis. Be sure to give the graph a title, label the axes, and provide a key/legend.
3. Make a third bar graph showing the effect of pH on enzyme activity by plotting your data (steps 4ac) from Table 1. Put pH (014) on the xaxis and reaction rate (05; # bubbles produced) on the yaxis. Be sure to give the graph a title, label the axes, and provide a key/legend.
Conclusion: 1. Why does the catalase reaction eventually stop?
2. How did temperature affect catalase activity?
3. How did pH affect catalase activity?
4. Many mammals have a body temperature close to 37°C. Does your data indicate that catalase is most active close to the temperature at which it exists in a living organism? Explain.
5. Mammals have a blood pH of 7.4. Does your data indicate that catalase is most active close to the pH at which it exists in a living organism? Explain.
