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Catalase Lab

A Bio ENZYME ACTIVITY LABORATORY
Gen Nelson

INTRODUCTION

Hydrogen peroxide (H2O2) is a poisonous byproduct of metabolism that can damage cells if it is not removed. Catalase is an enzyme that speeds up the breakdown of hydrogen peroxide into water (H2O) and oxygen gas (O2).

    2H2O2--------catalase--------------> 2H2O + O2
REMEMBER: A CATALYST is a substance that lowers the activation energy required for a chemical reaction, and therefore increases the rate of the reaction without being used up in the process. CATALASE is an enzyme, a biological (organic) catalyst. Hydrogen peroxide is the substrate for catalase.
****YOU WILL BE WORKING WITH HOT WATER, ACIDS AND BASES IN THIS LABORATORY. USE EXTREME CAUTION AND WEAR GOGGLES AT ALL TIMES!!!

The general procedure for the lab is outlined below, and specific details for each variable follow.

GENERAL DIRECTIONS:
The assay system used in this lab consists of a filter paper disc which is coated with the enzyme and then dropped into a vial of substrate (hydrogen peroxide). As the catalyst breaks down the hydrogen peroxide into water and oxygen gas, the bubbles of oxygen collect underneath the filter and make it rise to the surface of the hydrogen peroxide. The time it takes for the filter to rise is an indication of the rate of enzyme activity.
RATE:
Rate of enzyme activity = distance (depth of hydrogen peroxide in mm)/time (in sec). We will assume that each filter is coated with the same amount of catalase (except in the investigation of the effect of enzyme concentration on enzyme activity).
The enzyme has been prepared for you as follows: 50g of peeled potato was mixed with 50 ml cold distilled water and crushed ice and homogenized in a blender for 30 seconds. This extract was filtered through cheesecloth and cold distilled water was added to a total volume of 100 ml. Extract concentration is arbitrarily set at 100 units/ml. ENZYME SHOULD BE KEPT ON ICE AT ALL TIMES!!

MATERIALS:
catalase, hydrogen peroxide 3% and 1.0%, forceps, filter paper discs, water, ice, water baths, vials, marking pencils, stopwatch or timer

PLAN:
Each group will investigate and report on two of the following questions. Suggestions for each question are given below. EVERY STUDENT IS RESPONSIBLE FOR RECORDING THE RESULTS OF ALL FOUR EXPERIMENTS in his/her lab report.
  1. What is the effect of enzyme concentration on enzyme activity?
  2. What is the effect of substrate concentration on enzyme activity?
  3. What is the effect of pH on enzyme activity?
  4. What is the effect of temperature on enzyme activity?

1. What is the effect of enzyme concentration on enzyme activity?
  • Set up five vials containing 40 ml of 3% hydrogen peroxide each. Measure and record the depth of the hydrogen peroxide in the vials.
  • Dilute the enzyme as follows. Make each dilution in a separate cup.
    100 units/ml = 20 ml 100 units/ml
    80 units/ml = 12 ml 100 units/ml + 3 ml cold dH2O
    50 units/ml = 10 ml 100 units/ml + 10 ml cold dH2O
    20 units/ml = 3 ml 100 units/ml + 12 ml cold dH2O
    0 units/ml = 20 ml cold dH2O

Using forceps, dip a filter into the enzyme solution at 100 units/ml, then remove it and drain it on a paper towel. Drop the disc into the vial of hydrogen peroxide labeled 100 units/ml and time how long it takes the filter to rise to the surface. Repeat this procedure for each of the other enzyme dilutions, and be sure to use a FRESH vial of substrate for each filter (WHY?) Record your results in the appropriate data chart.

2. What is the effect of substrate concentration on enzyme activity?
  • Obtain 1 vial of catalase at 100 units/ml
  • Dilute the substrate (hydrogen peroxide) as described below. Each dilution should be made in a separate labeled vial. Measure and record the depth of the hydrogen peroxide.

    3.0% H2O2: 40 ml 3% H2O2
    1.5% H2O2: 20 ml 3% H2O2 + 20 ml distilled water
    0.75% H2O2: 10 ml 3% H2O2 + 30 ml distilled water
    0.38% H2O2: 5 ml 3% H2O2 + 35 ml distilled water
    0.0% H2O2 : 40 ml distilled water
  • Dip a filter into the catalase, drain on a paper towel and then drop the filter into the 3% H2O2. Time how long it takes the filter to rise to the top. Repeat this procedure for each of the substrate dilutions. Record your results in the appropriate data chart.

3. What is the effect of pH on enzyme activity?
  • Obtain 1 vial of 40 ml 1% H2O2. Measure and record the depth of the hydrogen peroxide.
  • Label 5 small vials as follows: pH3, pH5, pH7, pH9, pH11 and dilute catalase into the appropriate vial as directed below:
    pH 3: 5 mL catalase + 5 mL pH 3 Buffer
    pH 5: 5 mL catalase + 5 mL pH 5 Buffer
    pH 7: 5 mL catalase + 5 mL pH 7 Buffer
    pH 9: 5 mL catalase + 5 mL pH 9 Buffer
    pH 11: 5 mL catalase + 5 mL pH 11 Buffer
  • Dip a filter into the catalase at pH 3, drain on a paper towel and drop it into the 1% H2O2. Time how long it takes the filter to rise to the top. Remove the filter and repeat this procedure for each of the pH's. Record your results in the appropriate data chart.

4. What is the effect of temperature on enzyme activity?
  • Set up an ice bath (0oC), a room temp water bath, a 37 oC bath and a boiling water bath
  • Place 5 ml of catalase at 100 units/ml in each of 4 test tubes. Place 1 test tube in each of the water baths.
  • Place 40 ml 1% H2O2 in each of 4 vials. Measure and record the depth of the H2O2. Place 1 vial in the 0oC bath and leave 3 at room temperature. This is necessary because heat will destroy the hydrogen peroxide.
  • Allow the catalase and substrate to incubate at each temperature for about 5 minutes, then test the reaction time at each temperature by dipping a filter into enzyme at that temperature, draining it, and then dropping it into substrate at the same temp. Use the second room temp. vial of hydrogen peroxide for the boiled catalase. DO NOT BOIL HYDROGEN PEROXIDE!!! Time how long it takes the filter to rise at each temperature. Record your results in the appropriate data chart.

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CATALASE LAB NAME_______________
DATA CHARTS

1. What is the effect of enzyme concentration on enzyme activity?



2. What is the effect of substrate concentration on enzyme activity?










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CATALASE LAB
DATA CHARTS
p. 2

3. What is the effect of pH on enzyme activity?




4. What is the effect of temperature on enzyme activity?








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ANALYSIS:
For EACH variable, use the AVERAGE rates to construct a graph of the independent variable vs. the dependent variable. This means you will have four graphs:
  1. Enzyme concentration (x axis) vs. Rate (y axis)
  2. Substrate concentration (x axis) vs. Rate (y axis)
  3. pH (x axis) vs. Rate ( y axis)
  4. Temperature (x axis) vs. Rate (y axis)

CONCLUSION:
Answer the following questions in complete sentences:
  1. What is the effect of enzyme concentration on enzyme activity? Explain how enzyme activity changes as enzyme concentration decreases, and discuss why this occurs (on a molecular level).
  2. What is the effect of substrate concentration on enzyme activity? How does enzyme activity change as substrate concentration decreases? Explain your observations by discussing this reaction on a molecular level.
  3. How does temperature affect the activity of catalase? Explain your observations by discussing the effect of temperature on protein structure. Discuss both high and low temperature effects.
  4. How does pH affect the activity of catalase? Consider both high and low pH, and explain your observations by discussing the effect of pH on protein structure.
  5. Ectothermic organisms have body temperatures that vary with the temperature of their surroundings. Discuss the effect this variation might have on the functioning of enzymes in these organisms. Suggest some ways ectothermic organisms might cope with this problem.

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