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  • Measure the effects of changes in temperature, pH, and enzyme concentration on reaction rates of an enzyme catalyzed reaction in a controlled experiment.
  • Explain how environmental factors affect the rate of enzyme-catalyzed reactions.

Enzyme Lab

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 (KAT-uh-LAYSS); it speeds up a reaction which breaks down hydrogen peroxide, a toxic chemical, into 2 harmless substances–water and oxygen.

The reaction is: 2 H2O2 —-> 2 H2O + O2

This reaction is important to cells because hydrogen peroxide (H2O2) 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: 13 test tubes, measuring pipette, acid, base, water, 10-ml graduated cylinder, 40 ml 3% Hydrogen peroxide solution, scissors and forceps (tweezers), fresh liver, apple, potato, yeast, ice bath, warm water bath, boiling water bath

PROCEDURE: Choose 1 team member to be the investigator and 1 to be the manager. The manager will be in charge of reading the directions and recording the data on the data table page. The investigator will follow the directions the manager reads. Both members are responsible for understanding what is happening and why.

There are questions in bold in the instructions. Be sure to answer those on your Data Table page.

PART A – Observe Normal Catalase Reaction

  1. You should have a test tube rack with test tubes facing down. Clean test tubes are down, dirty are facing up.
  2. Place 1 pipette (dropper) full of the 3% hydrogen peroxide (normal peroxide) solution into a clean test tube.
  3. Using forceps collect 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 produced in the reaction? (hint: look at the chemical equation above, which of the products is a gas)
    • Throughout this investigation you will estimate the rate of the reaction (how rapidly the solution produces bubbles) on a scale of 0-5 (0=no reaction, 1=slow, to 5= very fast).
      • This reaction from step 2 is the normal reaction, we say it reacted at a rate of 4. This has been filled in for you. You will compare all the other reactions to this one. If it is faster, it will be a 5. If it is slower, it will be below 4.
      • The rate is how fast or slow the reaction goes. Not how many bubbles it produces! That is based on how much liver or peroxide there is.
    • Recall that a reaction that absorbs heat is endothermic; a reaction that gives off heat is exothermic. Now, feel the temperature of the test tube with your hand.
    • Has it gotten warmer or colder? Is the reaction endothermic or exothermic?
  4. Pour the liquid leftover into a second test tube, so not pour the liver out. You should have the used liquid in one test tube and the used liver in a second test tube.
    • Assuming the reaction is complete and there is no hydrogen peroxide left.
      • What is this liquid composed of? (hint: it is not liver juice)
      • What do you think would happen if you added more liver to this liquid?
    • Add another piece of liver to the liquid from the first reaction and record the reaction rate. (1 – 5). What happened? Why do you think this happened?
    • Add another 1 pipette-full of hydrogen peroxide to the original liver remaining in the first test tube and record the reaction rate.
      • Is catalase reusable? Explain how you know. 
  5. Empty the contents of the test tubes into the “Waste” container. Put the test tubes facing up back in the test tube rack. Do not reuse the test tubes. We will wash all test tubes at the end of the lab.
  6. Make sure everything on your Data Table Page for Part A is filled out before you move on to Part B.

PART B – What Tissues Contain Catalase

You will now test for the presence of catalase in tissues other than liver.

  1. Get 3 clean test tubes, place 1 pipette-full of hydrogen peroxide in each.
  2. To the first tube, add a small piece of potato and record the reaction rate.
    • Does potato contain catalase?
  3. To the second tube, add 1 pipette-full of yeast and record the reaction rate.
    • Does yeast contain catalase?
  4. To the last tube, add a small piece of apple and record the reaction rate.
    • Does apple contain catalase?
  5. Do some contain more catalase than others? How can you tell?
  6. Empty the contents of all test tubes into the “Waste” container. Put the test tubes facing up back in the test tube rack. Do not reuse the test tubes. We will wash all test tubes at the end of the lab.
  7. Make sure everything on your Data Table Page for Part B is filled out before you move on to Part C.

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

  1. Obtain a piece of liver that has been boiled for 5 minutes. Record how the liver looks compared to your original liver.
    • Place the boiled liver in a test tube. Add 1 pipette-full of normal hydrogen peroxide. Record the reaction rate.
  2. Find the beakers with test tubes labeled like the picture.
    • Get one test tube of cold liver and one of cold peroxide.
    • Pour the cold peroxide on the cold liver. Record the reaction rate.
      • How did cold affect catalase function?
    • Get one test tube of warm liver and one of warm peroxide.
    • Pour the warm peroxide on the warm liver. Record the reaction rate.
      • How did heat affect catalase function?
  3. Empty the contents of all test tubes into the “Waste” container. Put the test tubes facing up back in the test tube rack. Do not reuse the test tubes. We will wash all test tubes at the end of the lab.
  4. Make sure everything on your Data Table Page for Part D filled out before you move on to Part E.

PART D – What is the Effect of pH on Catalase Activity?

  1. Place 1 pipette-full of normal hydrogen peroxide in a clean test tube. Collect a piece of liver from the beaker labeled PART D ACID.
    • Add the piece of liver to the test tube and record the reaction rate.
  2. Place 1 pipette-full of normal hydrogen peroxide in a clean test tube. Collect a piece of liver from the beaker labeled PART D BASE.
    • Add the piece of liver to the test tube and record the reaction rate.
  3. Place 1 pipette-full of normal hydrogen peroxide in a clean test tube. Collect a piece of liver from the beaker labeled PART D NEUTRAL.
    • Add the piece of liver to test tube and record the reaction rate.
  4. Based on your observations; answer the following questions:
    • Does there appear to be a pH that catalase works best at? What is it?
    • What is the effect of low pH (acid) on enzyme activity?
    • What is the effect of high pH (base) on enzyme activity?