Materials Included In Kit

Additional Materials Required

Prelab Preparation

Biochemistry Guided-Inquiry Lab Activity

1. To prepare the unknowns in Part A, use the following recipes. Note: The recipes below will provide enough of each solution for at least eight student groups.

   Unknown A: Use only distilled water.
   Unknown B: Mix 1 g of gelatin into 100 mL of hot distilled water.
   Unknown C: Mix 1 g of soluble starch into 100 mL of hot distilled water.
   Unknown D: Use mineral oil provided mixed with an equal volume of water.

2. Prepare the testing reagents and unknowns for dispensing without cross contamination. Label beakers and disposable pipets with iodine, biuret solution, Sudan III, and unknowns A–D. To label the pipets, use the included labels and a waterproof marker to make a flag. Write on only one-half of the label. Fold the label in half around the pipet barrel just below the bulb. Keep these stock solutions at dispensing stations to prevent cross contamination.

Safety Precautions

Iodine solution is an eye and skin irritant. Biuret test solution is corrosive especially to eyes. Sudan III solution is alcohol based and is flammable and toxic by ingestion and inhalation. Conduct activity in a well-ventilated room or in a chemical fume hood. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The casein solution may be stored at basic pH for several months. Alternatively, the solution may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b. The iodine byproduct may be reduced with sodium thiosulfate according to Flinn Suggested Disposal Method #12a. The leftover biuret solutions may be neutralized with hydrochloric acid according to Flinn Suggested Disposal Method #10. The leftover Sudan III solutions may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #18a.

Lab Hints

• Enough materials are provided in this kit for 8 groups of students to complete the guided-inquiry lab activity and for the demonstration to be completed four times. There are enough reagents to test for biological molecules in both Part A and Part B. Students will need to provide their own specimens for testing in Part B.
• This module can reasonably be completed in four, 50-minute class periods. Complete the POGIL^® activity on day one, the demonstration on day two, the introductory activity and guided-inquiry design on day three, and perform the guided-inquiry lab on day four.
• To improve student flow in the classroom, assign groups to start testing with different reagents during Part 3A.

Teacher Tips

• Students should have some background knowledge of ions, polarity and intermolecular forces before starting this activity.
• The POGIL^® activity is designed to be completed in class using the POGIL teaching method. This includes students working in groups with assigned roles to construct their own learning using modeling. For more information, visit www.pogil.org
• The demonstration activity itself is short, but student-produced models and class discussion may take more time depending on the students’ experience with this type of activity.
• Models are expressed as diagrams on paper, however using manipulatives to create three-dimensional models is a great extension. Student must be able to explain their models and how they fit with the observations they made during the demonstration.
• This module is a good topic to teach after students have learned the properties of water, pH and bonding. If students have already taken chemistry, this module will review what they learned in chemistry and apply it to biology.
• The following student laboratory kits can be used to further explore biochemistry: Help From Mom—Biochemistry of Antacids—Demonstration Kit (Flinn Catalog No. FB1857) and Enzymes—The Catalysts of Life—Student Laboratory Kit (Flinn Catalog No. FB0436).
• This learning module incorporates the following kits: pH and Protein Solubility—A Reversible Demonstration Kit (Flinn Catalog No. AP6692) and Chemicals of Life—A Super Value Laboratory Kit (Flinn Catalog No. FB1435).
• A video showing the chemistry of the demonstration experiment called, What If It’s an Acid and a Base, presented by John Little, is available for viewing at the Flinn website (www.flinnsci.com).

Answers to Prelab Questions

Part 2. pH and Protein Solubility Demonstration

1. Describe or draw the molecules and ions in an acidic solution (pH less than 7).

   The description or drawing must include more H⁺ ions than OH^– ions. It should also contain water molecules and may include negative ions that have dissociated from the H⁺ ion, such as Cl^–.

2. Describe or draw the molecules and ions in a basic solution (pH greater than 7).

   The description or drawing must include more OH^– ions than H⁺ ions. It should also contain water molecules and may include positive ions that have dissociated from the OH^– ion, such as Na⁺.

3. Water is a polar molecule. Explain how this is related to the solubility of substances in water.

   Substances that dissolve in water are ionic or polar because water is polar. A polar molecule has a positive end that attracts the negative end of other polar molecules and negative ions. It also has a negative end that attracts the positive end of the other polar molecules and positive ions. These attractions dissolve the substances by breaking up intermolecular forces between the molecules or ions. Nonpolar molecules are not attracted to water molecules and do not dissolve.

Sample Data

Part 2. pH and Protein Solubility Demonstration
Data Table

Modeling
In your group, construct three models that show the solubility of casein at different pH levels. The first model is a sample with a pH of 2. The second model is a sample with a pH of 4. The third model is a sample with a pH of 12.
Modeling the Demonstration
Student models should include some of the following ideas.

• Water’s polarity makes it so that ions and other polar molecules dissolve more readily in water than nonpolar molecules.
• Casein molecules are charged in the pH 2 and the pH 12 model since this is where they are most soluble in water.
• Casein molecules are as nonpolar in the pH 4 solution because this is where casein is least soluble in water. Depending on the prior understanding of your students, they may or may not be equipped to explain why the acid and base change the protein. Ionization and protonation of the casein molecule by the H⁺ and OH^– molecules explain this phenomenon.

Use these models as a formative way to see the students’ current level of understanding and to address misconceptions during a discussion before moving to the guided-inquiry activity.

Part 3A. Introductory Activity—Testing Unknowns
Data Table

Answers to Questions

Part 3A. Introductory Activity—Testing Unknowns  

2. Identify the major biochemical(s) in each unknown.

   Unknown A—Negative for all biochemicals
   Unknown B—Contains protein
   Unknown C—Contains carbohydrate
   Unknown D—Contains lipids

Part 3B. Guided-Inquiry Design and Procedure  

In your group, design an investigation that uses one or more of the tests above to answer a question concerning food and biochemical molecules.

1. Record the research question.

   The research questions may include those that test for one type of molecule across many different items or that test for several molecules. More advanced groups may test for more molecules.

2. Define the limitations of the tests including the types of compounds not detected by the test.

   Student answers will vary. All of these tests are qualitative, not quantitative, so students will not be able to measure the amount of any of the substances. The biuret test is not very sensitive, so a false negative is possible. Since these tests involve color changes or binding of dyes to molecules, if the original sample has a dark color, it may be difficult for students to determine the result, therefore students need to choose items for testing carefully.

3. Identify the control, independent variable, dependent variable and critical constants.

   Student answers will vary.

4. Record the working procedure. If changes must be made to the working procedure, record those changes here.

   Student answers will vary.

5. Explain the safety procedures needed to carry out the investigation safely.

   Student answers will vary. Conduct activity in a well-ventilated room or in a chemical fume hood. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

6. Carry out the investigation and record relevant data.

   Student answers will vary.

7. Display relevant data in a meaningful way to help communicate the results of the investigation.

   Student answers will vary.

Post-Lab Analysis

3. Using a claims, evidence and reasoning model, explain the results the experiment.
   1. Propose a claim based in scientific understanding.

      Student answers will vary. The claim is a short statement that connects the observed evidence to scientific understanding of a concept. In this case, the claim would connect the tests performed to the substances in the samples.

   2. Discuss specific evidence from the experiment.

      Student answers will vary. The evidence must match the observations the students made during data collection. For example, if the students claims their samples have starch, the evidence presented would include that the sample turned dark purple when iodine was added.

   3. Discuss the reasoning for the claim based on connections to the POGIL^® activity, the demonstration, and the introductory lab activity.

      Student answers will vary. This section should include information from researching the three types of tests and why they show the results they do.
      A positive starch test occurs because when iodide ions and starch interact, they form a complex. This complex absorbs different wavelengths of light than either substance on its own and turns purple. The POGIL^® activity shows that starch is a polysaccharide made from glucose molecules. This chain often forms a helix due to the interaction of the glucose monomers. Some of the iodide ions are attracted to the helix for starch and get trapped inside. This changes the behavior of the electrons, therefore changing the types of wavelengths of light that are absorbed and emitted.
      A positive biuret test occurs when the peptide bonds in a protein form a complex with copper ions (Cu²⁺). The positive charge of the Cu²⁺ ions attracts the nitrogen atoms in the peptide bonds. Each Cu²⁺ ion forms the complex with four nitrogens. This complex changes the behavior of the electrons causing the blue solution to turn purple.
      A positive Sudan III test occurs because Sudan III is nonpolar, and is therefore attracted to lipids in the sample that are also nonpolar. There is no color change, but when both lipids and water are present in the sample, the dyed lipids will separate from the water, leaving clear water and dyed lipids.
      Students may also discuss the components of the items they decided to test and what background information they discovered about the biochemistry of those substances. For instance, if they test a food that is an animal product, they should not find starch because starch is only present in plants.

Discussion

pH and Protein Solubility Demonstration

Casein is the principal protein in milk (80% of the total protein content). Casein is a phosphoprotein—it contains a large number of phosphate groups attached to the amino acid side chains in its polypeptide structure. The negatively-charged phosphate groups are partially balanced by positively charged calcium ions and are responsible for the high nutritional calcium content in milk.. The solubility of a protein is usually at a minimum at its isoelectric point. The isoelectric point is defined as the pH at which a protein has a net charge of zero. For casein, due to the attached phosphate groups, the isoelectric point is close to pH = 5.

Casein, like other proteins, is an ionic species containing amino groups and carboxyl groups on its terminal amino acids. It also contains a variety of other acidic and basic groups on the side chains of its non-terminal amino acids. The effect of pH on the solubility of casein reflects the ionization of the acidic and basic groups in its structure.

At high pH, casein will have a net negative charge due to ionization of all the acidic side chains (—CO₂^–) in its structure.

Because casein is ionized at high pH values, it is soluble in dilute sodium hydroxide solution.

At very low pH, casein will have a net positive charge due to protonation of all basic side chains (—NH₃) in its structure. Because casein is ionized at low pH values, casein is also soluble in strongly acidic solutions. At slightly acidic to intermediate pH values, casein will contain roughly equal numbers of positively and negatively charged groups and the protein will have a net charge of zero. Casein is insoluble in neutral solutions because it is not charged under these conditions.

References

Biochemistry. POGIL^® Activities for High School Biology. Trout, L., Editor; Flinn Scientific: Batavia, IL (2012).

The demonstration activity was adapted from Flinn ChemTopic^™ Labs, Volume 20, Biochemistry; Cesa, I., Editor; Flinn Scientific: Batavia IL (2002).

Introduction

One of the unifying aspects of life that supports the theory of evolution from a common ancestor is that all life is made of the same four groups of macromolecules. These carbon-based compounds are proteins, lipids, carbohydrates and nucleic acids. Explore the structure and function of these four types of molecules and investigate the biochemistry behind how the molecules are detected by standard methods.

Concepts

• Structure and function of macromolecules
• Polarity of water
• Chemical and physical changes

Experiment Overview

The purpose of this learning module is to facilitate understanding of the structure and function of macromolecules through collaborative modeling and experimentation. First, use models in the POGIL^® activity to discover the similarities and differences in the structure and bonding patterns of each group of macromolecules. Then observe a demonstration of proteins interacting with an aqueous (water-based) solution at changing pH levels. Collaborate to make a model of the interactions of the protein molecules and the water molecules at different pH levels. Finally, carry out a guided-inquiry investigation to explore the detection of different types of macromolecules in food.

Part 1. Establishing Background Knowledge 
In groups, complete the Biological Molecules POGIL^® activity. 

Part 2. Demonstration—pH and Protein Solubility 
Casein, the major protein in milk, is an ionic protein with many differently charged portions. This demonstration shows how casein interacts with the molecules around it as the pH of its environment changes. The sample contains a pH indicator that changes color as acid or base is added. Before the demonstration, complete the Pre-Demonstration Questions under Part 2 of the Biochemistry Worksheet. During the demonstration, complete the table and collect data to link the solubility of casein to pH. In the observations portion of the table, pay close attention to the clarity of the solution. 

Next, work with your group to complete the Modeling section of the Biochemistry Worksheet. Produce models in the form of drawings that show how the casein molecules interact with the H⁺ ions and OH^– ions present in pH 2, pH 4 and pH 12 solutions and also how solubility is affected.

Part 3. Biochemistry—Guided-Inquiry Lab Activity
The Biochemistry—Guided-Inquiry Lab Activity is divided into two parts. The purpose of the introductory activity in Part A is to practice tests that identify three of the four groups of macromolecules in various food samples. Since all living things have DNA, the food samples will not be tested for DNA. The tests are the iodine test for starches, the biuret test for proteins and the Sudan III test for lipids.

Materials

Safety Precautions

Iodine solution is an eye and skin irritant. Biuret test solution is corrosive especially to eyes. Sudan III solution is alcohol based and is flammable and toxic by ingestion and inhalation. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Conduct activity in a well-ventilated room or in a chemical fume hood. Follow all laboratory safety guidelines.

Procedure

Part 3. Biochemistry

Part A. Introductory Activity—Testing Unknowns

1. Label four microcentrifuge tubes A–D, respectively.
2. Add 12 drops of unknown A to tube A, 12 drops of unknown B to tube B, etc., through tube D.
3. Add 2–4 drops of iodine to each tube to test for the presence of carbohydrates. The sample will turn purple in the presence of starch.
4. Use a “+” to indicate a positive test and a “–” to indicate a negative test. Record the results on the Biochemistry Worksheet.
5. Remove the unknowns from the tubes, and thoroughly clean and rinse the microcentrifuge tubes.
6. Repeat steps 1–5 using 12 drops of biuret test solution instead of iodine to test for proteins. A lavender or purple color indicates there is protein in the sample, while a redish-pink color indicates there is no protein.
7. Repeat steps 1–5 using 5 drops of Sudan III. Mix thoroughly. The Sudan III is attracted to lipids and will stain them dark red.
8. Identify the major biochemical(s) in each unknown on the Biochemistry Worksheet.

Part B. Guided-Inquiry Design and Procedure 

1. With your group, discuss how the tests above could be used to identify the components of common food items. Choose a research question that involves the use of at least one of the tests above.
2. Research the limitations to the tests above and determine what types of compounds within a class of macromolecules may not be detected. Determine if additional techniques may better answer the research question.
3. Discuss safety procedures with your group.
4. Use the guide on the Biochemistry Worksheet to plan the investigation and write appropriate procedures.
5. Once you have obtained permission from your instructor, carry out your investigation.
6. Consult your instructor for appropriate disposal procedures. The resulting samples from each test in the Introductory Activity may be poured into the correctly labelled container.

pH and Protein Solubility Demonstration

1. Add 25 mL of 0.1 M sodium hydroxide and a stirring bar to a 600-mL beaker or flask and place the beaker on a magnetic stirrer. Add 225 mL of distilled or deionized water and stir at moderate speed. Add 1 g of casein and stir to dissolve. (The solution will be slightly cloudy or translucent.)
2. Add 1–2 mL of universal indicator to observe pH changes. Consult the universal indicator color chart for pH values.
3. With rapid stirring, add 2 M hydrochloric acid in 0.5-mL increments (10 drops) using a disposable pipet. (The solution will turn cloudy, but will clear up again as the hydrochloric acid is dispersed. After 1–2 mL of acid has been added, the cloudiness will reach a maximum—this is the isoelectric point. The pH at the isoelectric point is 4–5.)
4. Once the isoelectric point has been reached, pause just long enough to record observations (15–20 seconds.) Continue adding 2 M hydrochloric acid in 0.5-mL increments while stirring until the solution is clear again. (The cloudiness will fade and the precipitate will re-dissolve after the addition of another 2–3 mL of acid, when the pH drops below the isoelectric point, pH ≤ 2.)
5. Continue to stir the solution. Reverse the process by adding 2 M sodium hydroxide in 0.5-mL increments using a clean, disposable pipet. (After 2–3 mL of sodium hydroxide has been added, the protein will precipitate out again at the isoelectric point.)
6. Continue adding sodium hydroxide in 0.5-mL increments with stirring until the solution is clear again. (The solution will clear up after an additional 1–2 mL of sodium hydroxide has been added and the pH > 10–12.)
7. The process may be repeated using the same casein solution. This will give time for student groups to discuss what is happening.

Student Worksheet PDF

11359_Student1.pdf