Biology Assignment Lab

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Enzymes:

Experiment Instructions (Lab Report)

LEARNING GOALS

By the end of this unit, you should be able to do the following:

1. Identify the substrates and products of the enzyme catalase.

2. Explain how factors such as concentration, pH, and temperature affect

the speed of an enzyme reaction, including the concept of denaturing.

3. Read and interpret a graph of results from a catalase experiment.

4. Identify the Independent, Dependent, and Control Variables in your

experiment.

MATERIALS NEEDED

3 see-through blank pill vials (ask to purchase blank ones from your

pharmacist; they’ll usually give them to you for free if you ask to buy them)

Tablespoon and cup measures

Knife and cutting board

3 small plates or bowls

Blender or grater (small holes)

Mesh strainer or colander

Ruler (with millimeter markings or "mm")

Kitchen timer, stopwatch, or regular watch

1 pot for boiling

2 regular potatoes

1/4 cup apple juice, tea, lemonade, or other non-carbonated liquid

1/4 cup lemon juice or white vinegar

1/4 cup ammonia

1 cup or bottle of hydrogen peroxide (from the first aid section of the

pharmacy)

Don't forget to fill out the Experiment Tracking Sheet for each section of

the exercise. Remember to take pictures for the lab report. You will need

four (4) pictures; one for each major experiment. You don't need pictures

of every single test.

ENZYMES

You will be blending or grating potatoes and straining the juice out to create

a source of catalase enzyme. When this extract is combined with hydrogen

peroxide, the catalase converts it into water and oxygen bubbles. Because of

the starch in our potato extract, the bubbles stay around and make a nice

foam. You will be using this reaction to test the specificity of the enzyme

for just one substrate (hydrogen peroxide), for the effects of increasing

enzyme concentration, for the effects of pH, and the effects of temperature.

Preparing Catalase Extract

(Preparation Note: Potato juice must be prepared fresh. It is good for up to

three hours after preparation.)

1. Cut 1/2 a potato into 6-8 chunks. Add to 1/2 cup tap water in a

blender. Blend on high until finely ground (about 1 minute).

2. (Alternate) If you do not have a blender, shred 1/2 a potato using the

side of the grater with small holes (usually the 2nd largest

holes). Add to 1/2 cup tap water and stir for 1 minute.

3. Pour potato blend through strainer or colander and collect the liquid

in a glass. Throw away potato mash.

Experiment #1 - Enzyme-Substrate Specificity (Catalase)

1. (Identify Control Variables) Add 1/2 tablespoon of potato extract,

1 tablespoon water, and 1/2 tablespoon of hydrogen peroxide to a pill

vial. Stir for 1 minute and leave uncapped! (It is critical that you

stir, never shake, for a full minute every time you do this

experiment.)

2. Use a millimeter ruler to measure the bubbles from the start of the

bubbles to the top of the bubbles. Don't measure from the bottom of

the tube; measure from the bottom of the bubbles (see picture

below). This is the positive control experiment.

3. (Negative Control Experiments) Are you sure the bubbles aren't

coming from the potato juice or hydrogen peroxide alone? Create a

couple of negative control experiments to confirm that this is a really

a reaction between both components.

4. (Hypothesis) Repeat the reaction, replacing the 1/2 tablespoon of

hydrogen peroxide with another substrate such as fruit juice, tea,

coffee, or any other non-carbonated beverage. Measure the bubbles.

5. (Hypothesis) Repeat the reaction and choose another alternate

substrate from the list above.

Conclusions - Was the catalase enzyme specific to one substrate?

Example of catalase

experiment. In this picture,

the bubbles start at the line

marked with the red arrow

and go up to the yellow

arrow, for a total of 10

lines. Since the first line

counts as the "0" mark on

any ruler, this means the

bubbles are 9 millimeters

tall.

Experiment #2 - Enzyme Concentration (Catalase)

(Preparation note: Place some of your potato extract, water, and hydrogen

peroxide in the refrigerator. You will use this in experiment #4, but it needs

to cool for at least half an hour.)

1. (Identify Control Variables) Add 1/2 tablespoon of potato extract,

1 tablespoon water, and 1/2 tablespoon of hydrogen peroxide to a pill

vial. Stir for 1 minute. (It is critical that you stir, never shake, for a

full minute every time you do this experiment.)

Measure the bubbles.

2. Repeat step 1. (You will be averaging the two results.)

3. (Hypothesis) Repeat the reaction with twice as much potato extract

(1 tablespoon) and less water (1/2 tablespoon). Do this reaction

twice as well.

4. (Hypothesis) Repeat the reaction with three times as much potato

extract (1 1/2 tablespoons) and no water. Do this reaction twice as

well.

5. Average your results for each concentration of enzyme.

Conclusions - What is the effect of adding more enzyme to your reaction?

Experiment #3 - Enzyme Denaturation by pH (Catalase)

Instead of repeating the reaction with 1/2 tablespoon of potato extract yet

again, you may use your average results from the 1/2 tablespoon reaction in

Experiment #2 for the next two experiments. For reference, ammonia is pH

12 (basic) and lemon juice is pH 3 (acidic).

1. (Hypothesis) Add 1/2 tablespoon of potato extract, 1 tablespoon of

base (ammonia), and 1/2 tablespoon of hydrogen peroxide. Stir for 1

minute and measure the bubble height.

2. Repeat step 1. (You will be averaging the two results.)

3. (Hypothesis) Make the same vial as in step 1, but use an acid (lemon

juice or white vinegar). Do this experiment twice as well.

4. Average your results for basic and acidic reactions.

Conclusions - What is the effect of changing the pH on your reaction?

Experiment #4 - Enzyme Denaturation by Temperature (Catalase)

As above, you may use your average results from the 1/2 tablespoon

reaction in Experiment #2 for this experiment. Cold temperatures do not

actually "denature" or unfold enzymes. However, a cold enzyme sometimes

can't make the necessary motions to catalyze a reaction.

1. (Hypothesis) Using the chemicals you placed in the refrigerator, add

1/2 tablespoon of cold potato extract, 1 tablespoon cold water, and

1/2 tablespoon of cold hydrogen peroxide to a pill vial. Stir for 1

minute and measure the bubble height.

2. Repeat step 1. (You will be averaging the two results.)

3. (Hypothesis) Add 1/2 tablespoon of room temperature potato extract

to 1 tablespoon of steaming hot water. Stir for one minute to allow

potato juice to heat up. Add 1/2 tablespoon of room temperature

hydrogen peroxide to a pill vial. Stir for 1 minute again and

measure the bubble height. Do this experiment twice as well.

4. Average your results for hot and cold reactions.

Conclusions - What is the effect of changing the temperature on your

reaction?

DISCUSSION

In this module is a link to the Discussion Group. Contribute to a discussion

of at least one of the questions there. It is important that you post by the

First Post date, that you post at least three times, and that at least one of your