ENZYMES

Enzymes are proteins that facilitate chemical reactions. Enzymes are not used up or changed by
the reaction. Therefore, theoretically, if you have 100,000,000 molecules to change and one
enzyme molecule, it will eventually be enough to complete the job for all 100,000,000
molecules. But if you had 2 enzyme molecules it would happen in ½ the time, of four enzyme
molecules it would happen in ¼ the time, etc.

As you will remember from your text, proteins are composed of chains of amino acids. The
shape of the amino acid is determined by the bonds between the side chains of the amino acids.
These bonds are mostly the weaker hydrogen bonds, van der Waals interactions, and ionic bonds.
These bonds are susceptible to changes in pH and increases in heat. So, protein shape could be
altered by changes in pH or increases in temperatures.

The shape of an enzyme is also important to its function. Enzymes have an active site where
substrate molecules bind. If you change the shape of the active site, you might make the enzyme
non-functional. This is called denaturation.

In this lab you will investigate the effects of concentration, temperature and pH on the
functionality of the enzyme catalase. Catalase is found in livers, where it converts hydrogen
peroxide to water and oxygen:

2 H2O2 2 H2O + O2

EXPERIMENTS
Since this reaction creates oxygen gas, one way to measure the rate of reaction is to capture the
oxygen that is generated by the reaction. Observe the demonstration set-up. The tube you see
inverted into to beaker of water is called a eudiometer. The small flask is called a reaction flask.
This is where the catalase will generate the oxygen. The hose from the reaction flask to the
eudiometer ensures that all of the oxygen generated will be captured.

In every run of these experiments you will have 10 ml of liquid in the reaction flask. You will
time how long it takes to generate 9 ml of oxygen. You will always add the same amount of
hydrogen peroxide (your instructor will tell you how much, the amount will depend upon how
active the enzyme is on the day of your lab). You will have a magnet in the reaction flask, and
the spin setting on the hot plate or spin plate will spin the magnet.
The things that can go wrong with this experiment:

1) Spinning the magnet at different speeds for different runs. Be sure that the spin setting is
always the same.
2) Allowing oxygen to escape. Be sure that the stopper is tight in the reaction flask and that
the needle is pressed tightly into the stopper. Also be sure that the hose connecting the
reaction flask to the eudiometer is undamaged.
3) Be sure that the heat element is NOT on.
4) Be sure all of your measurements are accurate.
5) Do not pull the hypodermic needle out of the stopper. To fill the needle, grasp it at the
plastic base next to the stopper and twist. Fill the needle and twist back on.

Experiment 1: Effects of Concentration on Enzyme Activity

In this experiment, you will examine the effects of concentration on catalase activity. You will
add the following mixtures into the reaction flask:

Concentration Catalase Ringer Total

Extract Solution volume
100% 10 ml 0 ml 10 ml
70% 7 ml 3 ml 10 ml
50% 5 ml 5 ml 10 ml
30% 3 ml 7 ml 10 ml

1. Fill the eudiometer with water and insert into the beaker. Hook the hose into the inverted
eudiometer and lower the eudiometer to the bottom of the beaker. This will trap the glass
hose connection so that it cannot slip out during the experiment.
2. Add the appropriate concentration into the reaction flask.
3. Add a magnet.
4. Set the spin setting at 700 rpm, or similar setting.
5. Add the stopper tightly into the flask.
6. Inject the hydrogen peroxide through the needle in one quick continuous stream. Start
timing as soon as you start inserting hydrogen peroxide.
7. Stop timing when 9 ml of oxygen is generated.
8. Refill the eudiometer after each run.
9. Be sure to clean the reaction flask after each run.
10. Record your information in Table 1.
Table 1

Concentration Time to generate 9 ml Oxygen

100%
70%
50%
30%
At which concentration was catalase most active? Which was least active? Explain your results.

Graph your results properly.

Experiment 2: Effects of Temperature on Enzyme Activity

In this experiment, you will examine the effects of temperate on catalase activity. You will add
the following mixtures into the reaction flask:
 10 ml room temperature catalase (for this, you can use the data collected above for
the 100% solution.
 10 ml warm catalase (approximately 37 Celsius).
 10 ml hot catalase (approximately 80 Celsius)

1. Fill the eudiometer with water and insert into the beaker. Hook the hose into the inverted
eudiometer, and lower the eudiometer to the bottom of the beaker. This will trap the hose
connection so that it cannot slip out during the experiment.
2. Add the appropriate temperature catalase into the reaction flask.
3. Add a magnet.
4. Set the spin setting at 700 rpm, or similar setting.
5. Add the stopper tightly into the flask.
6. Inject the hydrogen peroxide through the needle in one quick continuous stream. Start
timing as soon as you start inserting hydrogen peroxide.
7. Stop timing when 9 ml of oxygen is generated.
8. Refill the eudiometer after each run.
9. Be sure to clean the reaction flask after each run.
10. Record your information in Table 2.
Table 2

Temperature Time to generate 9 ml Oxygen

Room Celsius
Warm Celsius
Hot Celsius

At which temperature was catalase the least reactive? Explain why this is the result.

Experiment 3: Effect of pH on Enzyme Activity

In this experiment, you will examine the effects of pH on catalase activity. You will add the
following mixtures into the reaction flask*:
 10 ml pH 2 solution.
 10 ml pH 5 solution
 10 ml pH 7 solution
 10 ml pH 10 solution
 10 ml pH 12 solution

* pHs may differ from the list above depending on what we have in stock.

1. Fill the eudiometer with water and insert into the beaker. Hook the hose into the inverted
eudiometer, and lower the eudiometer to the bottom of the beaker. This will trap the hose
connection so that it cannot slip out during the experiment.
2. Add the appropriate pH catalase into the reaction flask.
3. Add a magnet.
4. Set the spin setting at 700 rpm, or similar setting.
5. Add the stopper tightly into the flask.
6. Inject the hydrogen peroxide through the needle in one quick continuous stream. Start
timing as soon as you start inserting hydrogen peroxide.
7. Stop timing when 9 ml of oxygen is generated.
8. Refill the eudiometer after each run.
9. Be sure to clean the reaction flask after each run.
10. Record your information in Table 3.
Table 3

Concentration Time to generate 9 ml Oxygen

pH 2
pH 5
pH 7
pH 10
pH 12

Explain the results for pH 2 and pH 12. Explain the results for these pH values.

At which pH was (or should have been) catalase most active? Why do you suppose that is?