4: Enzyme Lab

Last updated

Jun 28, 2025
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- 3: Macromolecules
- 5: Osmosis Lab

Brad Basehore, Michelle A. Bucks, & Christine M. Mummert
Harrisburg Area Community College

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Purpose:

This lab investigates enzyme activity and the factors that influence it, including temperature, concentration, and pH. Students will gain an understanding of enzyme structure and function and apply the scientific method to study catalase activity.

Tasks:

Perform experiments to test the effect of temperature, concentration, and pH on enzyme activity.
Record and analyze bubble column heights as a measure of catalase function.
Use collected data to determine the optimal conditions for catalase activity.

Criteria for Success:

Accurately measure and record bubble column heights for all experiments.
Create clear, labeled graphs to visualize results.
Formulate conclusions supported by experimental data.

Enzyme Function

In every living cell, many chemical reactions are performed. In chemical reactions, the reactants are molecules that undergo a change, which results in the products (Figure 1). The arrow indicates the direction of the reaction as the reactants proceed to be transformed into the product(s). As you can see in the figure below, the number of reactants and products can vary, but the number of atoms is the same on both sides of the arrow. During synthesis reactions (Example 1), the reactants are joined to form a product. During decomposition reactions (Example 2), the reactant is broken down into products. During exchange reactions (Example 3) both decomposition and synthesis take place. The decomposition of two reactants is followed by an exchange and synthesis of two new compounds from them.

Example 1 (Synthesis Reaction):

$\ce{A + B → AB}$

$\ce{2H2 + O2 → 2 H2O}$

Example 2 (Decomposition Reaction): $\ce{AB → A + B}$

$\ce{2H2O → 2 H2 + O2}$

Example 3 (Exchange Reaction): $\ce{A + BC → AC + B}$

$\ce{2 Na + 2 H2O → 2 NaOH + H2}$

Figure

$\PageIndex{1}$ : In a chemical reaction, reactants are converted to products

Enzymes are very efficient catalysts for biochemical reactions. These catalysts speed up reactions by providing an alternative reaction pathway that requires a lower activation energy needed for a reaction to get started. The lower the activation energy for a reaction, the faster the rate at which it will proceed. Enzymes only lower the activation energy, but do not change the difference in energy levels between the reactants and the products. They work by binding to the reactants and converting them to a different compound (the product). Enzymes are specific to a type of reactant, and therefore can catalyze only one type of reaction. This enzyme specificity is the result of the particular shape of the enzyme that only permits binding to one type of reactant, much like a key fits into a lock (Figure 2). The reactants in an enzymatic chemical reaction are called substrates. Notice how the shape of the enzyme fits its substrate. The location where the enzyme binds a substrate is called the active site because the reaction occurs here. At the end of the reaction, the product is released, and the enzyme can then bind to more substrate. Thus, an enzyme can perform the reaction over and over again, as long as the substrate is present.

J2ZWkwVbscLPAdfZN-3_BbJqaLdZEp40Pbev_QSSwbPtlPseOud3VdWHFghWDr6PjW7HANJNTXcLaPXh72ZLfSppVcNlMrY17NydosnIWkD3eZ4BN3kPQ1NkMdVxGlRCzKTSOflw — Figure 2: Enzymes bind a specific substrate and convert it into the products. The products are released, and the enzyme is unchanged. (CC By 4.0; OpenStax)

Most enzymes are complex proteins that function most efficiently within a specific range of temperature and pH. Extremes in temperature or pH will denature the enzyme by permanently altering its chemical structure. Even a small change in the protein’s structure will change the enzyme’s shape enough to prevent the substrate from binding, and thus keep the reaction from occurring.

In this laboratory using readily available materials, you will demonstrate how altering the temperature, enzyme concentration, and pH will affect an enzyme’s ability to function. Specifically, you will be studying the actions of the enzyme catalase. Bananas contain the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen.

The reaction that breaks down hydrogen peroxide to water and oxygen with the addition of catalase is:

Hydrogen peroxide → water + oxygen gas

2 H₂O₂ → 2 H₂O + O₂

What is the reactant in this reaction? _______________________________
What is the substrate for catalase? ________________________________
What are the products for this reaction? _____________________________
Bubbling occurs as the reaction proceeds. Explain why. _____________________________________________________________

Lab Safety

Safety Guidelines for Working with Enzymes and Chemicals:

Wear safety goggles and gloves at all times.
Handle 0.1 M HCl and NaOH with caution, as they are corrosive.
Dispose of all chemical waste as instructed by the instructor.
Wash hands thoroughly after completing the lab.

Suggested Student Roles and Group Size

For groups of 2–3 students:

Experiment Leader: Conducts experiments and ensures accurate measurements.
Data Recorder: Records observations, measurements, and completes the worksheet.
Materials Manager (optional): Prepares solutions and cleans up after the lab.

Simplified Timeline for Enzyme Lab

Introduction and Setup (10 minutes): Overview of enzymes, safety guidelines, and lab objectives.
Catalase Activity Experiment (15 minutes): Test basic enzyme activity with controls.
Temperature Experiment (20 minutes): Test catalase activity at various temperatures.
Concentration Experiment (20 minutes): Test how enzyme concentration affects activity.
pH Experiment (20 minutes): Test catalase activity under acidic, neutral, and basic conditions.
Data Compilation and Discussion (15 minutes): Complete tables, analyze data, and discuss results.

Materials

Equipment	Materials
Disposable pipets	Distilled Water
Large test tubes (20 mL)- approximately 25 total per group	Hydrogen peroxide (3% solution)
Ruler	Sugar solution (5%)
Sharpie	Ripe bananas
Stopwatch or phone timer

For Temperature experiment:

Equipment
4 test tubes (10mL)
4 Celcius thermometers
Ice bath
Boiling water bath
Refrigerator
Safety glasses

For the Concentration experiment:

Equipment	Materials
5 test tubes (10 mL) for dilutions	3% hydrogen peroxide
5 test tubes (10 mL) for reactions
Graduated pipettes or 10 mL graduated cylinder
Ruler
Marker
Timer

For pH experiment:

Equipment	Materials
5 test tubes (10 mL)	pH buffers (pH 4, 6, 7, 8, 10)
	Or household alternatives: Vinegar (pH 4) Baking soda solution (pH 10) Distilled water (pH 7)
	pH strips (optional)

Equipment

Materials

5 test tubes (10 mL)

pH buffers (pH 4, 6, 7, 8, 10)

Or household alternatives:

Vinegar (pH 4)

Baking soda solution (pH 10)

Distilled water (pH 7)

pH strips (optional)

Exercise 1: Catalase Activity (Controls)

Banana Extract Preparation (may be prepared before lab)

Mash one small piece of ripe banana (approximately 3 cm³) with 30 mL of cold water
1. This amount is approximately what will be needed for each lab group
Filter through a coffee filter, cheesecloth, or fine strainer
Use this filtered banana extract as your catalase source

Procedure

Label three small test tubes 1 through 3 with the Sharpie marker
Mark each tube at 1 cm and 5 cm from the bottom
For tube #1: Add banana extract to the first mark, then hydrogen peroxide to the second mark
For tube #2: Add water to the first mark, then hydrogen peroxide to the second mark
For tube #3: Add banana extract to the first mark, then sugar solution to the second mark
Swirl each tube and measure bubble column height after 20 seconds
1. To measure the column, hold the ruler vertically against the tube
2. Measure from the bottom of the bubble column to the top of the foam in millimeters
3. If bubbles are uneven, measure to the highest consistent foam level

Data Collection

Table 1: Catalase Activity
Tube	Contents	Bubble Column Height (mm)
1	banana extract, hydrogen peroxide
2	water, hydrogen peroxide
3	banana extract, sugar solution

Which tube showed the most bubbling? Explain why. ________________________________________________________________

________________________________________________________________

Which tube was the negative control? __________________________________

Which tube was the positive control? __________________________________

Describe what you observe in test tube #3. What was the purpose of this tube? ________________________________________________________________

________________________________________________________________

Exercise 2: Effect of Temperature on Enzyme Activity

In general, cold temperatures slow chemical reactions, and warm temperatures speed up chemical reactions. Every enzyme, however, has an optimal temperature at which it works best. Some enzymes prefer cooler temperatures, and others prefer warm temperatures. The main factor that influences what temperature an enzyme works best in is related to the internal conditions of the organism in which it evolved. Usually, extreme changes in temperature (i.e. boiling) will denature enzymes, making them inactive.

In the following exercise, you will test enzyme function at four different temperatures (on ice, in a refrigerator, in a warm incubator, and in a boiling water bath). Before setting up this experiment, formulate a hypothesis regarding the effect on enzyme activity. For example, do you think the enzyme will perform better at a warmer temperature? A cooler temperature? On ice? At boiling temperatures?

Employing Steps in the Scientific Method:

Record the Question that is being investigated in this experiment. ____________________________________________________________________

Record a Hypothesis for the question stated above. _______________________________________________________________________________

Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

Procedure

Label four test tubes 1 through 4 and mark at 1 cm and 5 cm
Add banana extract to the first mark in each tube
Place tubes in different temperature environments:

○ Tube #1: Ice bath (0°C)

○ Tube #2: Room temperature (20-25°C)

○ Tube #3: Warm water bath (37°C)

○ Tube #4: Boiling water bath (100°C)

Place a thermometer in each temperature environment container
Wait 15 minutes
Record temperature readings in Table 2
Add hydrogen peroxide to the second mark
Measure bubble column height after 20 seconds using the measurement instructions from part 1.
Data Collection

○ Tube #1: Ice bath (0°C)

○ Tube #2: Room temperature (20-25°C)

○ Tube #3: Warm water bath (37°C)

○ Tube #4: Boiling water bath (100°C)
Place a thermometer in each temperature environment container
Wait 15 minutes
Record temperature readings in Table 2
Add hydrogen peroxide to the second mark
Measure bubble column height after 20 seconds using the measurement instructions from part 1

Data Collection

Table 2: Temperature Effects

Table 2: Temperature Effects
Tube	Actual Temperature Reading (°C)	Bubble Column Height (mm)
1 - ice bath
2 - room temp
3 - warm bath
4 - boiling

Which tube showed the most enzyme activity? Explain. ________________________________________________________________

What is the optimal temperature for catalase activity? Explain ________________________________________________________________

Was your hypothesis supported? ________________________________________________________________

What is your conclusion concerning the effect of temperature on enzyme activity? ________________________________________________________________

What is usually the optimal temperature for enzymes in the human body? ________________________________________________________________

What effect could a fever have on enzymatic activity in the human body? ________________________________________________________________

Exercise 3: Effect of Enzyme Concentration

In general, the amount of product (in this case, O₂) produced in a given amount of time should increase if you increase the enzyme concentration. This is not always the case because of the complex nature of chemistry and feedback mechanisms within a living cell. Here we will attempt to find the optimum concentration of catalase.

Employing Steps in the Scientific Method:

Record the question that is being investigated in this experiment. ________________________________________________________________

Record a Hypothesis for the question stated above. ___________________________________________________________________________
Predict the results of the experiment based on your hypothesis (if/then statement). __________________________________________________

Banana Extract Dilutions

Label five 10 mL test tubes with the concentrations found in the table below and prepare the following solutions

Concentration	Banana Extract	Distilled water	Total Volume
100%	4 mL	0mL	4 mL
75%	3 mL	1mL	4 mL
50%	2 mL	2mL	4 mL
25%	1 mL	3mL	4 mL
0%	0 mL	4mL	4 mL

Use a clean 10mL graduated cylinder to add the required water volume first
1. You can use a graduated pipette to make these measurements instead of a graduated cylinder
Use the same graduated cylinder (or pipette) to add banana extract volume second
Cap and invert each tube 3 times to mix
Use these dilutions immediately in the concentration experiment
Clean the graduated cylinder

Procedure for concentration experiment

Label five new test tubes 1 through 5
Mark reaction tubes at 1 cm and 5 cm
Add diluted extract to the first mark
Add hydrogen peroxide to the second mark
Measure bubble column height after 20 seconds

Data Collection

Table 3: Enzyme Concentration Effects

Table 3: Enzyme Concentration Effects
Tube	Banana Extract Concentration	Bubble Column Height (mm)
1	100%
2	75%
3	50%
4	25%
5	0%

Which tube showed the most activity? Explain. ________________________________________________________________

________________________________________________________________

If we waited for an unlimited amount of time, would the results be the same in all tubes? Explain. ________________________________________________________________

________________________________________________________________

In this experiment, was the amount of substrate the same in all three tubes? ________________________________________________________________

Generate a hypothesis about what you might observe if the amount of substrate concentration were increased while the enzyme concentration remained the same. Explain.

________________________________________________________________

Exercise 4: Effect of pH on catalase activity

Each enzyme has an optimal pH, or level of acidity or alkalinity at which it functions best. A higher or lower pH affects hydrogen bonding and can alter the structure of the enzyme, leading to reduced activity. An enzyme’s optimal pH activity level evolves for similar reasons to those in optimal temperature.

Procedure

Label five test tubes 1 through 5.
Prepare pH solutions using graduated pipettes:

○ Tube #1: 1 mL banana extract + 1 mL pH 4 buffer (vinegar)

○ Tube #2: 1 mL banana extract + 1 mL pH 6 buffer

○ Tube #3: 1 mL banana extract + 1 mL pH 7 buffer (distilled water)

○ Tube #4: 1 mL banana extract + 1 mL pH 8 buffer

○ Tube #5: 1 mL banana extract + 1 mL pH 10 buffer (baking soda solution)

Wait 5 minutes
Add hydrogen peroxide to the 5 cm mark
Measure bubble column height

Data Collection

Table 4: pH Effects
Tube	pH	Bubble Column Height (mm)
1	4
2	6
3	7
4	8
5	10

Note: Skip pHs 6 and 8 if using household products instead of buffered solutions

Which tube showed the most activity? ________________________________________________________________
What is the optimal pH for catalase? ________________________________________________________________

Graphing Extension Activity: (Optional)

The results of these experiments can be presented graphically using a computer program like Microsoft Excel or Google Sheets. The presentation of your data in a graph will assist you in interpreting your results. Based on your results, you can complete the next step of scientific investigation, in which you must be able to propose a logical argument that either allows you to support or reject your initial hypothesis.

Graph your results using the data from Tables 2, 3, and 4.

What are the dependent variables? Which axis is used to graph this data? ______________________________________________________________________

What are your independent variables? Which axis is used to graph this data? ______________________________________________________________________

When preparing graphs for scientific publications, APA style provides a clear framework focused on readability and precision. Your graph should use clean, minimalist design elements - think black and white or grayscale rather than colorful embellishments. Each axis needs a clear label showing both what you're measuring and its units, and these labels should run parallel to their axes.

Use a simple sans-serif font that's easy to read, keeping the size between 8 and 14 points. When you're showing means in your data, include error bars to indicate variability. The overall presentation should be straightforward - avoid decorative elements like gridlines, backgrounds, or three-dimensional effects that might distract from your data.

Below the graph, place a concise caption starting with "Figure X." followed by a descriptive title. Size your figure to fit comfortably within standard journal margins, typically 5-7 inches wide.

Keep in mind that these are general APA guidelines - individual journals often have their own specific requirements that may differ. For instance, some journals might prefer color figures or have strict requirements about file formats and resolution. Always check your target journal's author guidelines before finalizing your graphs.

Rubric for Graphing Extension Activity

Criteria	Excellent (5)	Proficient (4)	Needs Improvement (2–3)	Not Complete (0)
Accurate Data Representation	All data points are correct and well-labeled	Minor errors in data or labels	Multiple errors in data or labels	No data presented
Graph Design	Neat, clear axes, proper scaling	Mostly clear with minor issues	Axes or scaling unclear	No graph provided

Questions for Review

What is the function of an enzyme? What specific monomers (i.e. building blocks) make up enzymes?

What does it mean when an enzyme becomes denatured?

Describe at least 2 ways in which an enzyme can become denatured.

3. What types of bonds or interactions are broken when an enzyme becomes denatured (name at least 3)?

4. Explain the difference between substrate and active site.

5. If an enzyme functions well at a pH of 4, would you expect it to also work well at a pH of 7? Explain.

Practical Challenge

Eggs can contain bacteria such as Salmonella. Considering what you’ve learned in this laboratory exercise, explain how cooking eggs makes them safe to eat.

What can happen to an enzyme when the pH of the environment becomes too acidic or too alkaline? Use your observations from the lab to support your answer.

If you don’t want a cut potato to get brown, what could you do to prevent it assuming that the ‘browning’ occurs because of an enzymatic reaction? Explain your answer.

What would happen to enzyme activity if the pH of your blood decreased (became more acidic) or increased (became more basic)? Explain.

Alignment with SLOs and Program Competencies

Lab Task	SLO/Competency Alignment
Investigating enzyme activity	Develops technical lab skills
Measuring and analyzing data	Applies critical thinking and quantitative reasoning
Understanding enzyme specificity	Connects biological principles to experimental data

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Text Color

Text Size

Margin Size

Font Type

Data Collection

Data Collection

Purpose:

Enzyme Function

Lab Safety

Suggested Student Roles and Group Size

Simplified Timeline for Enzyme Lab

Materials

Exercise 1: Catalase Activity (Controls)

Banana Extract Preparation (may be prepared before lab)

Procedure

Data Collection

Exercise 2: Effect of Temperature on Enzyme Activity

Procedure

Data Collection

Data Collection

Exercise 3: Effect of Enzyme Concentration

Employing Steps in the Scientific Method:

Banana Extract Dilutions

Procedure for concentration experiment

Data Collection

Exercise 4: Effect of pH on catalase activity

Procedure

Data Collection

Graphing Extension Activity: (Optional)

Rubric for Graphing Extension Activity

Questions for Review

Alignment with SLOs and Program Competencies

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