I’ve Got That Sinking Feeling
An Investigation of Enzymes
Teacher Materials
Learning Goals, Objectives, and Skills…………………………………………………………………………...2
Standards Alignments 3
Laboratory Set-Up Manual 5
Instructor Laboratory Guide 7
Answers to Student Questions 8
Post-Lab Extension Activities 10
*Please consider adapting this lab to include some student-centered investigation.
Some suggestions, ideas, and tips can be found in a separate document called "Student-Centered Investigation”.
Last updated: September 21st, 2017
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Copyright © MassBioEd 2013
I’ve Got That Sinking Feeling
Learning Goals, Objectives, and Skills
Students Learning Goals:
· Students will understand the role of enzymes in chemical reactions.
· Students will understand the basic steps of enzyme catalyzed reactions.
· Students will understand some of the factors that can influence the rate of enzyme-catalyzed reactions.
Student Learning Objectives:
· Students will articulate the function of catalase in breaking down hydrogen peroxide and identify the reactants and products of this chemical reaction.
· Students will explain the role of enzymes as catalysts that lower the activation energy of biochemical reactions.
· Students will measure the effect of enzyme concentration and other factors on the rate of catalase mediated chemical reactions.
Scientific Inquiry Skills:
· Students will pose questions and form hypotheses.
· Students will design and conduct scientific investigations.
· Students will make measurements and record data.
· Students will use mathematical operations to analyze and interpret data.
· Students will generate tables and graphs to present their data.
· Students will use experimental data to make conclusions about the initial question and to support or refute the stated hypothesis.
· Students will follow laboratory safety rules and regulations.
Laboratory Technical Skills:
· Students will demonstrate proper use of micropipettors.
I’ve Got That Sinking Feeling
Standards Alignments
MA Science and Technology/Engineering Curriculum Framework (2006)
Biology
· 1.3 Explain the role of enzymes as catalysts that lower the activation energy of biochemical reactions. Identify factors, such as pH and temperature that have an effect on enzymes.
Chemistry
· 7.2. Calculate concentration in terms of molarity. Use molarity to perform solution dilution and solution stoichiometry.
· 7.5 Identify the factors that affect the rate of a chemical reaction (temperature, mixing, concentration, particle size, surface area, catalyst).
Scientific Inquiry Skills
· SIS1. Make observations, raise questions, and formulate hypotheses.
· SIS2. Design and conduct scientific investigations.
· SIS3. Analyze and interpret results of scientific investigations.
· SIS4. Communicate and apply the results of scientific investigations.
Mathematical Skills
· Construct and use tables and graphs to interpret data sets.
· Perform basic statistical procedures to analyze the center and spread of data.
· Measure with accuracy and precision (e.g., length, volume, mass, temperature, time)
· Use common prefixes such as milli-, centi-, and kilo-.
DRAFT REVISED MA Science and Technology/Engineering Standards (2013)
*Please note that these are DRAFT standards that have not yet been submitted for formal review or adoption.
Chemistry
· HS-PS1-5. Construct an explanation based on collision theory for why varying conditions influence the rate of a chemical reaction or a dissolving process. Design and test ways to alter various conditions to influence (slow down or accelerate) rates of processes (chemical reactions or dissolving) as they occur.
[Clarification Statement: Explanations should be based on three variables in collision theory: quantity of collisions per unit time, molecular orientation on collision, and energy input needed to induce atomic rearrangements. Conditions that affect these three variables include temperature, pressure, concentrations of reactants, mixing, particle size, surface area, and addition of a catalyst.]
[Assessment Boundary: Assessment is limited to simple reactions in which there are only two reactants and to specifying the change in only one variable at a time.]
NRC Practices
· Asking questions and defining problems
· Planning and carrying out investigations
· Analyzing data
· Mathematical and computational thinking
· Constructing explanations and designing solutions
· Engaging in argument from evidence
· Obtaining, evaluating, and communicating information
I’ve Got That Sinking Feeling
Laboratory Set-Up Manual
Supply List:
For lab preparation:
· 300 mL 3 % hydrogen peroxide solution
· 100 mg catalase (e.g., Fisher Scientific catalog # S75082)
· 250 mL dH2O
· 1 ´ 4-L flask
For each group:
· 1 ´ p1000 micropipettors and pipette tips OR graduated 1-mL transfer pipette
· 1 ´ p20 micropipettors and pipette tips
· 1 ´ sheets of filter paper (Fisher Scientific catalog # 09-825B)
· 15 ´ single-hole paper punches
· 1 ´ non-treated 24-well plates (e.g., Fisher Scientific catalog # 07-201-590)
· 1 ´ forceps
· 2 ´ 20-mL clear vials (such as fruit fly vials or clear scintillation vials (e.g., Fisher Scientific catalog # 03-337-14))
· paper towels (about 2 sheets)
· calculator
· permanent marker (such as Sharpie)
· timer
· 1 ´ 500-mL bottles of distilled water
· 1 ´ 15-mL conical tubes with caps
· 1 ´ 500-mL opaque containers (such as Nalgene rectangular amber bottles (e.g., Fisher Scientific catalog # 03-313-412)) *Hydrogen peroxide is light-sensitive, which is why it’s important to store the solution in opaque containers.
Tips for supplies:
· Graduated 1-mL transfer pipettes can be substituted for the p1000 micropipettors and tips.
· Microcentrifuge tubes or small containers (such as bottle caps) can be substituted for the 24-well plate. Each group will need 6 containers.
Set-up Calendar:
2 weeks before lab:
¨ Check supplies and order any needed materials.
¨ If making any substitutions to the supply list, edit the student protocol accordingly.
1 day before lab
¨ Set up student lab stations with all durable materials.
Morning of lab:
¨ Prepare 3 L of a 0.3% hydrogen peroxide
1. Mix 300 mL of 3% hydrogen peroxide with 2700 mL of water in a 4-L flask.
2. Aliquot 150 mL of the 0.3% solution into 500-mL opaque bottles, 1/group. Put one bottle at each station. Tip! Aliquot a few extra in the event of spills.
¨ Prepare 250 mL of 0.4 mg/mL catalase stock solution by dissolving 100 mg of catalase into 250 mL of dH2O.
¨ Test the solutions:
1. Place 10 mL of the catalase (0.4 mg/mL) stock solution on a filter paper disk and dropping it into a vial of 20 mL of 0.3% hydrogen peroxide solution. The disk should sink and then rise to the surface of the solution in 20 seconds or less.
2. If the disk does not sink, dilute the catalase solution (1:4) and test again. Alternatively you can dilute the hydrogen peroxide solution incrementally by 50% until you reach the desire reaction rate. Important! It is critical to test the solutions prior to the lab, because students will obtain much better results if the stock solutions aren’t too strong.
¨ Aliquot 5 mL of the 0.4 mg/mL catalase stock solution into conical tubes, 1/group. Put one tube at each station. Tip! Aliquot a few extra in the event of spills.
I’ve Got That Sinking Feeling
Instructor Laboratory Guide
Laboratory Procedure Tips:
1. Before starting the I’ve Got That Sinking Feeling experiment, ask students to check their materials list to make sure they have everything.
2. Demonstrate how to set up the 24-well plates, the procedure for applying catalase to the disks, and how the disk will sink and float during the assay. In order to get the disk off of the forceps when inserting it into the vial, students should deposit the disk just under the surface of the hydrogen peroxide solution.
3. Remind students to make all dilutions using the 0.4 mg/mL catalase stock solution.
4. To avoid a buildup of catalase, students should use fresh hydrogen peroxide after performing 3 trials at each experimental condition.
5. To prevent contamination, students must wipe the forceps clean between each trial to remove any catalase.
6. During the assay it is fine if the disk does not sink all the way to bottom of the vial before it begins to rise to the top of the solution.
7. At the 0% catalase condition, the disk will not rise to the surface of the solution during the course of the experiment. However, since hydrogen peroxide breaks down when exposed to light, students may see the disk rise over an extended period of time (overnight).
8. Students can dispose of the paper disks in the trash and all solutions can be flushed down the drain.
9. Step 8: If a p20 micropipettor is not available, dip the disk into the solution and then touch it to a paper towel to remove the excess liquid. (You will want to modify the student hand out to reflect this procedural change.)
Lab Data Analysis Tip:
While students are working on their lab generate a table on the board or projector to collect class data.
Record the average time (sec) each group obtained for each experimental condition and calculate a class average. You could also have students perform a t-test and calculate a p-value.
Average Time (sec) for Disk to Sink and RiseCatalase Concentration (mg/mL) / Group Number
1 / 2 / 3 / 4 / 5 / ...... / 15 / Class Average
0.4
0.32
0.24
0.16
0.08
I’ve Got That Sinking Feeling
Answers to Student Questions
Protocol-Embedded:
p. 3: The tube with the catalase solution would contain bubbles, the other tube would not.
Completed dilution table:
Final quantity needed (mL) / Final catalase solution concentration / Microliters (mL) of0.4 mg/mL
catalase stock solution / Microliters (mL) of deionized water
500 / 0.4 mg/mL / 500 / 0
500 / 0.32 mg/mL / 400 / 100
500 / 0.24 mg/mL / 300 / 200
500 / 0.16 mg/mL / 200 / 300
500 / 0.08 mg/mL / 100 / 400
500 / 0.0 mg/mL / 0 / 500
Step 4 (p. 5): About half full
Step 17 (p. 6): The disk should not have moved
Pre-Lab:
1. Enzymes catalyze (lower the potential energy of) chemical reactions that occur within living things and are necessary for life processes.
2. Enzymes tend to be substrate-specific—they only work on specific substrates.
3. Because enzymes are not consumed by the reactions they catalyze, so they can be used again and again.
4. Catalase catalyzes the reaction to break down hydrogen peroxide.
5. Dependent: time that it takes the disk to sink and rise; Independent: catalase concentration; Control: distilled water set-up
6. Sample answer: The more concentrated the catalase, the faster the reaction rate (the less time it will take for the disk to sink and rise).
Post-Lab and Analysis
1. Because the oxygen produced during the reaction makes the disk more rise
2. Sample data table and graph:
Time (s) for Disk to Sink and Rise to SurfaceCatalase concentration / Trial 1 / Trial 2 / Trial 3 / Average Time
0.4 mg/mL / 6.6 / 7.7 / 7.3 / 7.2
0.32 mg/mL / 7.4 / 7.9 / 8.4 / 7.9
0.24 mg/mL / 8.7 / 9.3 / 8.9 / 9.0
0.16 mg/mL / 12.3 / 10.7 / 11.2 / 11.4
0.08 mg/mL / 19.9 / 16.6 / 17.5 / 18.0
3. The more concentrated the enzyme, the faster the reaction rate. This agrees with my hypothesis.
4. Because more enzyme was available to facilitate the hydrogen peroxide reaction.
5. I’d predict the graph on the right, in which the reaction rate levels off. I’d make this prediction because at some point, the amount of enzyme will surpass the available substrate, making additional enzyme have no further effect on the rate. In other words, some other factor will become limiting.
6. 1. There could have been human timing errors (e.g., failing to start and stop the timer with accuracy)
2. There could have been some kind of effect from “old” hydrogen peroxide. If it contained catalase from previous trials that might speed up the reaction
3. If using the “dip” method for transferring solution to the disks, there could have been variation in the amount of solution transferred. Some disks might have more solution on them than others
7. Yes, because the reaction releases oxygen, which makes the disks float. The faster the reaction, the more bubbles are released, and the faster the disk will rise to the surface.
8. If catalase were kept constant, then there would be an increase in the rate with increased concentration of hydrogen peroxide, assuming that enough catalase was present. The amount of catalase could be a limiting factor if the ratio of the number of molecules of hydrogen peroxide to molecules of catalase is high.
I’ve Got That Sinking Feeling
Post-Lab Extension Activities
Student Oral Presentation:
Students can report the findings of their student-centered investigations to the class using a PowerPoint presentation that includes the following information:
· Experimental question—what you hope to learn from performing the experiment.
· Hypothesis—a testable, proposed answer to the experimental question based on prior knowledge.
· Experimental system and data collection methods—flowchart of how the experiment was performed and how data was collected. This should NOT include a detailed summary.
· Results—observations, data tables, figures, etc.
· Conclusions—should the hypothesis be accepted or rejected as supported by key data.
Online resource for effective PowerPoint presentations:
http://office.microsoft.com/en-us/powerpoint-help/tips-for-creating-and-delivering-an-effective-presentation-HA010207864.aspx
Student Lab Report:
Students can report the findings of their student-centered investigations through a written lab report. Your school may have its own lab report format, but generally lab reports include the following information:
· Title—brief summary reflecting the factual content of the investigation.
· Introduction—includes questions being answered, hypothesis and background information.
· Materials—list of supplies needed to perform the lab.
· Procedure—step-by-step procedure (with enough detail so someone could repeat the experiment).
· Results—observations, data tables, figures, etc. and a brief narrative summary of results.