Enzyme Catalase Lab

Catalase: Can the presence of enzymes in tissue be detected indirectly?

Objective: Demonstrate the activity of an enzyme in living tissues. Observe the effects of changes in temperature on the activity of catalase.

Process Skills: Performing analyses for the presence of an enzyme in tissues. Analyzing relationships between environmental conditions and enzyme activity.

Introduction:

Even when functioning normally, your cells make poisonous chemicals. Enzymes 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. The substrate is the molecule on which the enzyme acts, and the products are the molecules produced by the reaction. You have thousands 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 that breaks down hydrogen peroxide, a toxic chemical, into two harmless substances, water and oxygen. The reaction is as follows: 2H2O2 2H2O+O2

Materials:
Hydrogen Peroxide / Graduated Cylinder
Small Test Tubes 6 (or 7) / Test Tube Rack
Stirring Rod / Scissors or Scalpel
Thermometer / Test Tube Holder
Potato / Lab Apron

This reaction is important to cells because hydrogen peroxide (H2O2) is produced as a byproduct of many normal cellular reactions. If cells did not break down the hydrogen peroxide, they would be poisoned and die.

Under certain conditions, enzymes are denatured. An enzyme is denatured when it loses its proper shape and cannot function. Some things that can denature an enzyme are high temperatures, extremes of pH, heavy metals and alcohol.

In this lab, you will study the catalase found in potato cells. You will use a potato from the grocery store. It might see strange to use dead cells to study the function of enzymes by when a cell dies, the enzymes remain intact and active for several weeks, as long as the tissue is kept refrigerated.

Procedures:

PART A: NORMAL CATALASE ACTIVITY

Caution: Hydrogen peroxide is a mild skin irritant. It will bleach fabric. Put on a lab apron. Pour 2mL of the hydrogen peroxide solution into a clean test tube.Using forceps and scissors (scalpel), cut off a small piece of potato, and add it to the test tube. Push the potato into the hydrogen peroxide with a stirring rod. CAUTION: Use care when handling scissors (Scalpel) and glassware. (Note: Be sure to clean your stirring rod between steps.) Observe the bubbles. 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 this step proceeded at a rate of 3 and record the speed in Table 2 on the last page of this lab.

Control reaction rate: _____3_____ Observation:______

What gas is being released?______

A reaction that absorbs heat is endergonic and a reaction that gives off heat is exergonic. Feel the temperature of the test tube with your hand.

Has the test tube gotten warmer or colder? ______

What type of reaction does this indicate? ______

Describe what is happening in your test tube. ______

Pour off the liquid into another test tube. Do not allow the potato to go into the other test tube. Set aside potato.

What is the liquid composed of? ______

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

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

Pour half of the liquid into another test tube, and then do the following.

In one test tube with the liquid poured off from the first experiment add another piece of potato.

What happened in this test tube? ______

Why do you think this happened? ______

In the other test tube add another 2mL of hydrogen peroxide

What happened in this test tube? ______

Why do you think this happened? ______

Add another 2 mL of hydrogen peroxide to the potato remaining in the first test tube

Describe what you observe. ______

Are enzymes reusable? Why or why not? ______

PART B: Effect of Temperature on Catalase Activity

Put equal quantities of potato into three clean test tubes and 2 mL of hydrogen peroxide into three other test tubes. Two test tubes will go in each water bath. Put one test tube containing potato and one test tube containing hydrogen peroxide into each of the following baths: ice bath, room temperature, warm water (37 degrees Celsius). Allow this to sit for 3 minutes.

In your spare test tube, add a piece of the boiled potato provided by Mr. Markley. Add 2mL of hydrogen peroxide. Record your reaction rate in the table and describe what happened below.

Hot water (100 degrees C) bath reaction rate:______Observation:______

Predict what boiling will do to an enzyme. ______
After 3 minutes in the water bath (ice bath, room temperature, or warm water) pour each test tube of hydrogen peroxide into its corresponding test tube of potato and observe the reaction.

Ice water bath reaction rate:______Observation:______

Room temp. water bath reaction rate:______Observation:______

Warm water bath reaction rate:______Observation:______

Record the reaction rates (0-5) in the Table. Graph the estimated reaction rate as a function of temperature on the Graph paper on the next page. You should have five pints: 0 degrees Celsius (ice water), Room Temperature (in degrees Celsius), 37 degree Celsius (human body temperature), and 100 degrees Celsius (boiling).

Table 2 Relative Reaction Rates of Live Catalase as a Function of Temperature
Temperature / Rate of Enzyme Activity
Control reaction rate: / ______3______
(0 oCelsius) Ice water bath reaction rate: / ______
Room temp. water bath reaction rate: / ______
(37 oCelsius) Warm water bath reaction rate: / ______
(100 oCelsius )Hot water bath reaction rate: / ______

What is the “optimum” temperature for catalase? ______

Explain why the reaction proceeded as it did at 0 degrees Celsius and 100 degrees Celsius.______

Graph 1 Relative Reaction Rates of Potato Catalase as a Function of Temperature

Reaction Rate / 5
4
3
2
1
0
Control / O Degrees / Room Temp / 37 Degrees / 100 Degrees
Temperature (Degrees Celsius)