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Catalase

By

Birju Amin

A lab submitted in partial fulfillment of the requirement for

Biology Honors

Block 2

2003

Instructor          _________________________________________________

 

 

 

 

 

Date _________________________________________________________

 


 


Problem

How do enzymes work in living tissues?

This lab covers a “Normal Catalase Activity”, the answer to the matter “Is Catalase Reusable?”, “Occurrence of Catalase”, “Effects of pH on Catalase Activity”, and an activity on the “Inhibitors of Catalase”.  Briefly the main objectives of this lab are to display the activity of enzyme in living tissues and learn how the temperature and pH of the substrate or enzyme have an affect on the catalase activity. We are going to accomplish this by experimentally testing for presence of catalase in living tissues and analyze factors affecting enzyme activity.

Hypothesis

My hypothesis was that during the normal catalase activity, the rate of the enzyme would be 4 at liver, potato at 2, chicken at 2 and carrot at zero. I also guessed that when liver would be added to the liver, there will be bubbling but didn’t know what gas was being separated. I didn’t think that catalase would be useable although I did think that effect of the temperature would have a great affect on the rate of reaction.

Materials

Normal Materials for Whole Lab:

Group of 2-4

▫ 40 mL 3% Hydrogen peroxide solution

▫ 10-mL Graduated cylinder

▫ 10 Clean Test Tubes

▫ Test Tube Rack

▫ Stirring rod

▫ Forceps and Scissors or Scalpel

▫ Thermometer

Part I: “Normal Catalase Activity”, “Is Catalase Reusable?” & “Occurrence of Catalase

Group of 2-4

▫ 3 Pea-Sized pieces of fresh liver from chicken or beef

▫ Slice of fresh potato, chicken meat, apple or carrot

Part II: “Effects of Temperature on Catalase Activity”, “Effect of pH on Catalase Activity”, & Inhibitors of Catalase

ENTIRE CLASS

▫ Boiling water bath (100 Degree Celsius)

▫ Warn water bath (About 37 Degree Celsius)

▫ Ice water bath (Zero Degree Celsius)

▫ Room temperature water bath (About 22 Degree Celsius)

GROUP OF 2-4

▫ 1N HCI in dropper bottle

▫ 1N NaOH in dropper bottle

▫ pH paper

▫ Eight Pea-sized pieces of fresh liver from Chicken or Beef

▫ Five Drops of 5% hydroxylamine Solution

▫ Two pieces of Graph Paper

 

 

 

 

 

Procedure

Part 1:

A) Normal Catalase Activity

1. Obtain a clean test tube and place 2 mL of 3% hydrogen peroxide solution (use graduated cylinder to measure) into the test tube

2. Use forceps and sicissors and cut a pea-sized liver into the clean test tube, use force to push it down and then stir wit a stirring rod.

3.  Feel the temperature of the test tube with you hand and Record Data

B) Is Catalase Reusable?

4. Place a 2 mL of 3% hydrogen peroxide solution and a pea-sized liver into a clean test tube

5. Pour the liquid consisted of hydrogen peroxide solution and liquid parts from liver into 2nd clean test tube.

6. Finally pour 2 mL of 3% hydrogen peroxide into test tube 1 that currently has liver remaining.

7. Record Data

 

 

C) Occurrence of Catalase

8. Pour 2 mL into 4 clean test tubes. In the 1st test tube add a small piece of liver

9. Add a small pice of potato into the 2nd test tube

10. Add a piece of chicken into the 3rd test tube.

11. Add a piece of apple or carrot to the final or the 4th test tube.

12. Record Data

Part II:

A) Effect of Temperature on Catalase Activity

13. Put a piece of liver into a clean test tube; make sure it is at the bottom. After accomplishing that task, cover the liver with a small amount of distilled water then place this test tube in a boiling water bath for five minutes.

14. Remove test tube from the boiling water and allow it some cool air. Pour out the water and add 2 mL of hydrogen peroxide.

15. Put pea-sized liver into three more clean test tubes

16. Add 1 mL of H2O2 into three separate test tubes and then place each of them in the “Ice bath”, “Room temperature bath” and “Warm water bath”

17. Wait for three minutes and then put each test tube of H2O2 into the matching tube of liver and observe the reaction.

18. Record Data.

B) Effect of pH on Catalase Activity

19. Add 2 mL of hydrogen peroxide into three clean test tubes. Treat each tube as the following:

Tube 1 – add a drop of 1N HCI(acid)at a time until pH 3.

Tube 2 – add a drop of 1N NaOh(base) at a time until pH 10.

Tube 3 – adjust the pH to seven by adding single drops of 1N HCI or 1N NaOh as many times as needed.

20. Add a small piece of liver to each test tube and Record Data.

C) Inhibitors of Catalase

21. “To test the effect of hydroxylamine on living tissue, Place 2 mL of hydrogen peroxide into a clean test tube. Add 5 drops of 5% hydroxylamine solution. Add a small piece of liver of this mixture and observe the reaction.”

22. Record Data.

 

 

Ovservation & Data

A) Questions and Answers

1. In the reaction that you will be studying in this lab, what is the enzyme? What is the substrate? What are the products?

A. In the reaction in this lab, the enzyme is the liver, the substrate is the hydrogen peroxide and the products of this reaction are water and oxygen.

 

2. Is the hydrogen peroxide bubbling?

A. The hydrogen peroxide is not bubbling.

 

3. Observe the bubbles; what gas is being released?

A. Oxygen is being released in this reaction

 

4. 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 is step B proceeded at the rate of “4” and record the speed on your chart II as the rate at room temperature.

A. The information or data is on Chart II

 

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

A. The test tube has gotten warmer and therefore the reaction of this is exothermic.

 

6. What is happening in your test tube?

A. The hydrogen peroxide is bubbling in the test tube.

 

7. What is this liquid composed of? What do you think would happen if you added more liver to this liquid? Why?

A.I believe that nothing would happen because the oxygen was already released and the hydrogen peroxide has no more oxygen to release therefore there will be no reaction.

 

8. Can you observe any reaction? What do you think would happen if you poured off this liquid and added more hydrogen peroxide to the remaining liver?

A. Yes I can observe reaction occurring and if we pour more liquid there will still be reaction because the oxygen from the new hydrogen peroxide still has to re separated and released.

 

9. Are enzymes reusable?

A. Yes, Enzymes are reusable.

 

10. As you add each test substance, record the reaction rate (0-5) for each tube in your chart I.

A. The data is on Chart II.

 

11. What tissues contained catalase?

A. The chicken, liver and the potato contains catalase and the apple or the carrot contains petite or none.

 

12. What will boiling do to an enzyme?

A. Boiling the enzyme would cause it not to attach with the substrate and there will be no reaction. The atoms will move at a different rate.

 

13. What is happen in the test tube? Explain you results and record the reaction rate (0-5) on your Chart II.

A. The data is on Chart II.

 

14. Record the reaction rates (0-5) in your chart II

A. The data is contained in chart II.

 

15. Make graph of estimated reaction rate as a function of temperature. You should have 4 points: 0˚C (ice water); room temperature (in degrees Celsius); 37˚C (human body temperatre and 100˚C (boiling).

A. The graph is attached at the end of this lab and is labeled Graph I.

 

16. What is the optimum temperature of catalase. (This is the temperature at which the reaction proceeds fastest.)

A. The optimum temperature of the catalase is 37˚C which is the body temperature.

 

17. Why did the reaction proceed slowly at 0˚C?

A. The extreme temperature made the molecules move much slower and reaction was slowed.

 

18. Why did the reaction not proceed at all at 100˚C?

A. The enzyme was denatured.

 

19. Record results in your chart III.

A. The data is on Chart III.

 

20. Record rates on Chart III.

A. The data is on Chart III.

 

21. Make a graph of the estimated reaction rate as a function of pH on a separate piece of graph paper. You should have 3 points on your graph.

A. The graph is attached at the end of the lab and is labeled Graph II.

 

22. Does there appear to be a pH “optimum”? At what pH?

A. The pH optimum was at pH 7.

 

23. What is the effect of low and high pH on enzyme activity?

A. The lower the rate, the pH level is low as well, it’s beginning to denature.

 

24. Record the rate of reaction (0-5) in your chart IV.

A. The rate of reaction is recorded on Chart IV.

 

25. Explain what happened in the test tube.

A. It filled the active site so the substrate couldn’t get in.

 

B) Charts

Chart I:

Sample

Rate of Enzyme Activity (0-5)

Liver

The rate of the reaction was 4.

Potato

The rate of reaction was 2.

Chicken

The rate of reaction was 3.

Apple or Carrot

The rate of reaction was 1.

 

 

 

Chart II:

Temperature

Rate of Enzyme Activity (0-5)

0˚ Celsius

The rate of the reaction was very slow therefore we gave it a rate of 1.

Room Temperature 22˚Celsius

The rate of the reaction was fast and we decided the rate was 4.

37˚ Celsius

The rate of the reaction was Very Fast, the rate was 5.

100˚ Celsius

There was no reaction at 100˚Celsius therefore the rate of the enzyme activity is 0.

 

Chart III:

pH of Sample

Rate of Enzyme Activity (0-5)

1

The rate of the enzyme activity was 1.

12

The rate of the enzyme activity was 2.

7

The rate of the reaction was fastest among all of them, it was three.

 

Chart IV:

Sample

Rate of Enzyme Activity (0-5)

Step B-without hydroxylamine

The rate of reaction was 5, very fast.

Step N-with hydroxylamine

There was no reaction, 0.

 

 

C) Graphs

The graphs are attached on a separate piece of paper at the end of this lab.

 

 

 

 

 

 

 

 

 

Analysis/Conclusions

Postlab Analysis

 

26. Make an inference about what happens to the heat when the reaction described in Step C occurs in living cells?

A. I think the cell internal temperature will change and it will absorb all the heat which might be transformed into energy.

 

27. Name several conditions that slowed down or stopped the action of enzyme catalase.

A. An extreme change in the temperate whether to freezing or boiling slowed down the action of enzyme catalase, actually boiling denatured it. The change of pH also slowed down or stopped the action of enzyme catalase and finally hydroxylamine slowed down the reaction.

 

28. Compare optimal temperature and optimal pH for catalase to physiological conditions of a cell.

A. The optimal temperature caused the reaction to stop or slow down and the pH did the same to the catalase.

 

30. Did you find catalase to be present in many different kinds of tissue? Explain why.

A. I found catalase to be present in many different kinds of tissue, catalase is in many places because our body creates many poisonous materials that catalase needs to be there to speed the rate of reaction.