Lab 2 Enzymes
external image c8.8x17.enzyme.cycle.jpg

Objectives
  • Observe the reaction of catalase and hydrogen peroxide
  • Demonstrate the effects of extreme temperatures on catalase activity
  • Learn how to establish a baseline for the amount of peroxide in a 1.5% solution
  • Use titration techniques to determine the rate of hydrogen peroxide decomposition by enzyme catalysis
  • Investigate spontaneous decomposition of hydrogen peroxide to oxygen and water
MATERIALS
Part A. Testing Enzyme Activity
Hydrogen peroxide, 1.5%, 30 ml
Fresh, boiled and acid potato

Part B. Establishing a Baseline — Determining the Amount of Hydrogen Peroxide in a 1.5% Solution
Hydrogen peroxide, 1.5%, 10 ml
Sulfuric acid, 1 M, 10 ml
Potassium permanganate, 2%


C. Rate of Hydrogen Peroxide Spontaneous Decomposition
Hydrogen peroxide, 1.5%, 25 ml
Catalase working solution, 1 ml
Sulfuric acid, 1 M, 10 ml
Potassium permanganate, 2%

D. Rate of Hydrogen Peroxide Decomposition by Enzyme Catalysis
Hydrogen peroxide, 1.5%, 60 ml
Catalase working solution, 6 ml
Sulfuric acid, 1M, 60 ml
Potassium permanganate, 2%


PROCEDURE
Part A. Testing Enzyme Activity
1. Obtain a 10 ml syringe, remove the tip, and label the syringe ‘H’ for hydrogen peroxide (H2O2).
2. Using the syringe, add 10 ml of hydrogen peroxide to a provided 2 oz. plastic cup.
3. Add a fresh piece of potato to the hydrogen peroxide.
4. Mix the contents by swirling and observe for approximately 30-60 seconds. Record any observations.

Effect of Extreme Temperature on Enzyme Activity
1. Using your syringe, dispense 10 ml of hydrogen peroxide solution into a 2 oz. cup.
2. Add a boiled piece of potato to the hydrogen peroxide.
3. Mix the contents by swirling and observe for approximately 30-60 seconds. Record any observations.

Effect of Extreme pH on Enzyme Activity
1. Using your syringe, dispense 10 ml of hydrogen peroxide solution into a 2 oz. cup.
2. Add a acid piece of potato to the hydrogen peroxide.
3. Mix the contents by swirling and observe for approximately 30-60 seconds. Record any observations.

Part B. Establishing a Baseline — Determining the Amount of Hydrogen Peroxide in a 1.5% Solution
1. Obtain two more 10 ml syringes, remove the tips, and label one of the syringes ‘S’ for sulfuric acid. Label the other syringe ‘T’ for transfer.
2. Dispense 10 ml of hydrogen peroxide into a 2 oz. cup using the properly labeled syringe.
3. Add 1 ml of distilled water to the cup using a pipet.
4. Using the syringe labeled ‘S’, carefully add 10 ml of sulfuric acid to the cup. Mix the contents by gently swirling.
5. Using the syringe labeled ‘T’, transfer 10 ml of the mixture into a new 2 oz. plastic cup.
6. Fill a titration syringe to the 10 ml marking with potassium permanganate (KMnO4). Note the initial reading.

7. Slowly add one drop of potassium permanganate and swirl the solution to mix. Continue to add potassium permanganate, one drop at a time and swirl after each addition, until the solution permanently turns pink or brown. The amount of KMnO4 added is proportional to the amount of H2O2 that was present in the solution.
If you use all of the potassium permanganate in the syringe, refill to the 10 ml mark and continue your titration.
8. Record the final volume in the titration syringe.
C. Rate of Hydrogen Peroxide Spontaneous Decomposition
1. Dispense 25 ml of hydrogen peroxide in a 2 oz. plastic cup. Let the beaker sit, uncovered, for 24 hours at room temperature.
2. After 24 hours, dispense 10 ml hydrogen peroxide into a new 2 oz. cup using the properly labeled syringe.
3. Add 1 ml of distilled water to the cup using a pipet.
4. Using the syringe labeled ‘S’, carefully add 10 ml of sulfuric acid to the cup. Mix the contents by gently swirling.
5. Using the syringe labeled ‘T’, transfer 10 ml of the mixture into a new 2 oz. plastic cup.
6. Fill a titration syringe to the 10 ml marking with potassium permanganate. Note the initial reading in Table 3 in the Analysis section of the lab.
7. Slowly add one drop of potassium permanganate and swirl the solution to mix. Continue to add potassium permanganate, one drop at a time and swirling after each addition, until the solution permanently turns pink or brown. The amount of KMnO4 added is proportional to the amount of H2O2 that was present in the solution.

If you use all of the potassium permanganate in the syringe, refill to the 10 ml mark and continue your titration.
8. Record the final volume in the titration syringe .
D. Rate of Hydrogen Peroxide Decomposition by Enzyme Catalysis
1. Dispense 10 ml of hydrogen peroxide in a 2 oz. plastic cup using the 10 ml syringe labeled‘H’.
2. Using a pipet, add 1 ml of catalase solution and swirl gently for 10 seconds to mix.
3. Using the syringe labeled ‘S’, add 10 ml of sulfuric acid to stop the reaction.
4. Using the syringe labeled ‘T’, transfer 10 ml of the mixture into a new 2 oz. plastic cup.
5. Fill a titration syringe to the 10 ml marking with potassium permanganate. Note the initial reading in Table 4 in the Analysis section of the lab.
6. Slowly add one drop of potassium permanganate and swirl the solution to mix. Continue to add potassium permanganate, one drop at a time and swirling after each addition, until the solution permanently turns pink or brown. The amount of KMnO4 added is proportional to the amount of H2O2 that was present in the solution.

If you use all of the potassium permanganate in the syringe, refill to the 10 ml mark and continue your titration.
7. Record the final volume in the titration syringe in Table 4.
8. Repeat the procedure for 30, 60, 120, and 180 seconds of allowing the enzyme to react.

Remember to reestablish your baseline, as in part B, if more than 24 hours have passed since your last assay.

Results

THIS IS WHAT MISS SCOTT POSTED! FEEL FREE TO MODIFIY IT IF YOU THINK YOU CAN MAKE IT BETTER!
Part A - Observations (someone can create a chart if they want to!!!)




Type of Potato


Physical Change


Presence of Reaction
Fresh potato
Fizz/ bubbles were released
yes
Boiled potato
none
no
Acidified potato
none
no





I made this table for my report...i mean it isn't much but it is still a set up for the results of part A

Visible Reaction
Fresh
Immediatly began producing bubbles (O2) when submurged into H2O2
Boiled
No reaction- No change
Acid Soaked
No reaction- No change

Part B - Establishing a Baseline


Team Name
Minoriteam
APALA Interns
The Wolfpack
Alpha & Omega
Team Safety Violations
Team Osmosis Jones
Amount of KMnO4 Titrated
7
5
5
6
7
8

C. Rate of Hydrogen Peroxide Spontaneous Decomposition
Team Name
Minoriteam
APALA Interns
The Wolfpack
Alpha & Omega
Team Safety Violations
Team Osmosis Jones
Amount of KMnO4 Titrated
7
6
6
5
6
6

D. Rate of Hydrogen Peroxide Decomposition by Enzyme Catalysis
Team Name
Minoriteam
APALA Interns
The Wolfpack
Alpha & Omega
Team Safety Violations
Team Osmosis Jones
Amount of KMnO4 Titrated
10 seconds
5
5.5
5
6
6
5.5
Amount of KMnO4 Titrated
30 seconds
3.5
4.75
4
3.5
5
2
Amount of KMnO4 Titrated
60 seconds
2.3
3.5
2.8
2
3
2
Amount of KMnO4 Titrated
120 seconds
1
1.5
.2
1
1
.5
Amount of KMnO4 Titrated
180 seconds
.7
.9
.1
.2
.5
.25








Good Questions to think about…..

1. What is the function of enzymes in a living system?

2. Different enzymes work better under different conditions. Where in a human body might it be beneficial to have enzymes that work well in very acidic environments?

3. There is a large amount of catalase found in a human liver. Does the liver break down more hydrogen peroxide in the summer or winter? Explain your answer.

4. Many enzymes end with “ase”. Come up with your own enzyme, then name and explain what this enzyme does. Draw the enzyme and substrate in the space provided below along with the enzyme substrate complex.

5. Recent advances have allowed humans to mass-produce certain enzymes. Research one such enzyme and explain how this enzyme has been used to benefit society.

6. Amylase is an enzyme that aids in the digestion of starches and has an ideal temperature range of 35-40°C (approximately human body temperature). Predict what would happen to the rate of amylase activity at 25°C.

7. In part A of the experiment you observed the formation of gas bubbles in the reaction mixture. What gas do you think was forming? What test do you think you could employ to see if you were correct?

8. Identify different steps in this procedure that may lead to erroneous results.

9. When you added the catalase to hydrogen peroxide, you observed a strong reaction in the mixture. Do you think this reaction will continue indefinitely? Why or why not?

10. In part D, you investigated the rate of hydrogen peroxide decomposition over time. What purpose did each of the following serve in the experiment?

Catalase –
Hydrogen peroxide –
Sulfuric acid –
Potassium permanganate –

11. Before performing part D of the experiment, why was it necessary to establish a baseline?

.