You Are What You Eat

Transcription

1 You Are What You Eat An Investigation of Macromolecules Student Materials Introduction....2 Pre-Lab Questions.6 Lab Protocol..7 Post-Lab Questions and Analysis 11 Last updated: 10/15/18 1

2 You Are What You Eat Introduction When deciding what to eat for lunch, do you ever base your choices on the amount of protein, carbohydrates (sugars), or fats (lipids) that are in the foods? Or do you think about the food pyramid or plate: grains, vegetables and fruits making of much of a healthy diet and protein, dairy and fats making up only a small portion? Figure 1. USDA food pyramids from 1992 and 2005 and the new MyPlate campaign showing the ideal servings of food from the basic food groups. If you don t often think about food groups when choosing lunch, maybe you should. The foods you eat provide the building blocks your body needs to survive proteins, carbohydrate and lipids. You probably already know that grains like bread contain carbohydrates, but did you know that bread is also a source of protein? You also probably know that meat such as chicken or fish contain protein, but did you know that they also contain fat? Even desserts such as chocolate aren t as simple as just containing sugar (carbohydrates), they are also made of fats and even proteins. To top it all off, almost every time you have a meal, you also eat DNA and RNA! How is it possible to eat DNA? Let s think about it. Much of our food comes directly from plants and animals and we all know that plants and animals are composed of cells. All cells are composed of large, complex molecules called macromolecules; macromolecules are just big molecules composed of multiple subunits. The four main macromolecules are proteins, carbohydrates, lipids, and nucleic acids. In this lab, you will be learning more about the macromolecules that make up the food you eat and the roles they play in your body. Most macromolecules are made of repeating subunits called monomers. Since macromolecules are made of several monomers they can be called polymers. When two monomers are joined together the chemical reaction is called dehydration synthesis. The reaction involves the removal of an OH from one monomer and an H + from another, which come together to make a water molecule. For every two monomers that are linked together, one water molecule is released (see Figure 2). 2

3 Image Source: Figure 2: Illustration of dehydration synthesis and hydrolysis reactions. In addition to forming new polymers, cells also need to break down polymers; this reaction is called hydrolysis. Hydrolysis requires the addition of a water molecule and breaks the bond between the two monomers (see Figure 2). Dairy products contain the carbohydrate lactose, which is made up of two sugars galactose (C 6H 12O 6) and glucose (C 6H 12O 6). When lactose forms, a molecule of water (H 2O) is released. What is the chemical formula for lactose? Hint! Think of the overall reaction: glucose + galactose lactose + water. Testing for macromolecules In this lab, you will use simple methods to test for the presence of proteins, carbohydrates, lipids, and nucleic acids. Proteins Proteins are used in cells in various ways; they are used as enzymes, as antibodies, as structural components, for cell signaling and more. There are, in fact, more than 10,000 different proteins found in a typical human cell 1. Proteins are so diverse because they made up of chains of amino acids. Figure 3. Simple chain of amino acids called a polypeptide. Each color represents a different amino acid. 1 Wilhelm, M., Schlegl, J., Hahne, H., Gholami, A.M., Lieberenz, M., Savitski, M.M., Ziegler, E., Butzmann, L., Gessulat, S., Marx, H., Mathieson, T., Lemeer, S., Schnatbaum, K., Reimer, U., Wenschuh, H., Mollenhauer, M., Slotta-Huspenina, J., Boese, J.-H., Bantscheff, M., Gerstmair, A., Faerber, F., and Kuster, B. (2014) Mass-spectrometry-based draft of the human proteome. Nature, 509: [doi: /nature13319] 3

4 The 20 different amino acids can join together in any combination or number to form different structures. Some proteins are composed of a single long chain of amino acids called a polypeptide. Other proteins are composed of two or more polypeptides. The order of amino acids in a polypeptide dictates the structure and function of the protein. In this lab, you will use a chemical called Biuret to test foods for the presence of proteins. Biuret is a blue liquid that will turn purple when mixed with proteins. What are two functions of proteins in a cell? Carbohydrates Carbohydrates, often called sugars, are primarily used as a source of energy for the cell but can also be used as structural components to give the cell shape. Carbohydrates are macromolecules that come in different forms and sizes but all have the same ratio of carbon to hydrogen to oxygen; 1 carbon to 2 hydrogens to 1 oxygen, or CH 2O (see Figure 4). Fructose C 6H 12O 6 Sucrose C 12H 22O 11 Figure 4. The monosaccharide, fructose, and the disaccharide, sucrose. Monosaccharides are known as simple sugars, they only contain only 3-7 carbons. Disaccharides are molecules composed of two simple sugars linked together. Polysaccharides, known as complex sugars, are made up of long chains of monosaccharides. The varying number of sugars in different carbohydrates makes it possible to test foods for different carbohydrates. In this lab, you will use Benedict s and iodine to test foods for simple and complex sugars. Benedict s will change from blue to a red or rusty orange when monosaccharides are present. You will also use Lugol s (iodine) that will change from dark red to blue-black when it is mixed with the polysaccharide starch. Strawberries are not only sweet due to the sugars glucose and fructose, but are also a great source of fiber. The fiber in strawberries is primarily cellulose. What type of macromolecule is cellulose? 4

5 Lipids Lipids, often called fats, are used for energy storage and are also important components of membranes. Lipids are a large and diverse group of molecules that are primarily composed of long chains of hydrogen and carbon (see Figure 5). Figure 5. Lauric acid, a fatty acid commonly found in coconut oil. They are nonpolar (hydrophobic) molecules, so they do not mix with water. Lipids are not composed of repeating subunits, so they are not actually polymers. Modified lipids called phospholipids are the major component of the plasma membrane and are critical to cells. In this lab, you will use Sudan IV to test for the presence of lipids. Like lipids, Sudan IV is hydrophobic, so if lipids are present, they will absorb the Sudan IV and form a noticeable bright-reddish orange layer. Given what you know about lipids, explain why some types of salad dressings separate. Nucleic acids Nucleic acids are primarily used for information storage and transmission. Their information is needed for cells to make proteins. Nucleic acids are polymers of nucleotides (see Figure 6). Each nucleotide is made up of a sugar, a phosphate group, and a base. The sugar found in DNA nucleotides is deoxyribose, while the sugar in RNA nucleotides is ribose. The bases adenine, cytosine, guanine, and thymine (A, C, G, T) are found in DNA, while RNA contains adenine, cytosine, guanine, and uracil (A, C, G, U). Figure 6. DNA is made up of double strands of nucleotides. Together, DNA and RNA contain the blueprints and instructions for all of life s processes. In this lab, you will use a DNA stain such as GelGreen to test for the presence of nucleic acids (or DNA). DNA stains contain a molecule that binds to DNA and causes it to glow green when viewed under blue light or UV light. 5

6 You Are What You Eat Pre-Lab Questions Directions: After reading through the introduction and protocol for the You Are What You Eat lab, answer the questions below. 1. What are the four primary types of macromolecules? 2. You read somewhere that having a protein-rich breakfast can help combat fatigue and improve test performance. Which of the following would provide a protein-rich breakfast on the morning of a big exam: an apple and toast OR a ham omelet? Explain why your choice has more protein. 3. Look at the diagram below. Count the number of carbons, hydrogens, and oxygens in the circled monomer. What type of molecule is this monomer? What type of macromolecule is formed by linking many of these monomers? Is the reaction below a dehydration reaction or a hydrolysis reaction? Adapted from 5. Match the following indicators with the type of macromolecule they will be used to test: 1. Benedict s a. Carbohydrates monosaccharides 2. Biuret b. Carbohydrate polysaccharides 3. Lugol s c. Lipids 4. Sudan IV d. Nucleic acids 5. GelGreen e. Proteins 6

7 You Are What You Eat Lab Protocol For each type of macromolecule, you will test a known sample as a standard. For example, you will test glucose, a monosaccharide, with Benedict s to see what a positive reaction looks like. Materials: Check your workstations to make sure all supplies are present before beginning the lab. Student Workstation: Common Workstation: 3 ml Biuret Microwave or hot plate 3 ml Benedict s UV or blue light 3 ml Lugol s 600 ml beaker of water 3 ml Sudan IV 60 μl GelGreen (1X) 3 ml BSA (2 mg/ml) 3 ml glucose (2%) 3 ml Starch (5%) 3 ml vegetable oil 30 μl lambda DNA (200 ng/ μl) 20 ml distilled H 2O 1 p200 micropipette and tips 10 1 ml graduate pipettes 2 microcentrifuge tubes (1.5 ml or 2.0 ml) 8 test tubes 1 test tube rack 1 microcentrifuge rack 1 test tube holder ml beaker 1 permanent marker 1 waste container Procedure: In this procedure, you will set up a test sample and control sample for one macromolecule at a time. Table 1 provides an overview of the testing samples. Cautions: Safety goggles should be worn throughout this lab. Handle glass test tubes with care. They can break easily. Use a clean transfer pipette for each. 7

8 Lipids 1. Use the permanent marker to label a transfer pipette water. You will use this pipette several times. Caution: Make sure you use this pipette for water only. 2. Use a permanent marker to label two test tubes L+ and L-. 3. Using the water transfer pipette, put approximately 2 ml of water into the L- test tube. 4. Using the water transfer pipette, put approximately 1 ml of water into the L+ test tube. 5. Using a clean transfer pipette, put approximately 1 ml of vegetable oil into the L+ test tube. 6. Add 3 drops of Sudan IV to both test tubes. Wait 8-10 minutes. Record the color in Table 1. Protein 7. Use a permanent marker to label two test tubes: P+ and P-. 8. Using the water pipette put approximately 2 ml of distilled water into the tube labeled P-. 9. Using a clean transfer pipette, put approximately 2 ml of BSA into the P+ test tube. 10. Add 5 drops of Biuret reagent to the P+ and P- tubes. Hold the tubes over white paper and examine. Record the color in Table 1. Carbohydrates: glucose and starch 11. Use a permanent marker to label two test tubes: G+ and G Using the water pipette, put approximately 2 ml of distilled water into the tube labeled G Using a clean transfer pipette, put approximately 2 ml of glucose in the tube labeled G Add 1 ml of Benedict s to the G+ and G- tubes. 15. Carefully place the test tubes in a hot water bath (at least 90 C). Wait 3-4 minutes. Using tongs or a test tube holder to protect your hand, remove the test tubes from the water bath and place them in a test tube rack. Record the color in Table 1. Caution: Be very careful when handling the test tubes, they will be hot. 16. Use a permanent marker to label two test tubes: S+ and S Using the water pipette put approximately 2 ml of distilled water into the tube labeled S Using a clean transfer pipette, put approximately 2 ml of starch into the S+ test tube. 19. Add 3 drops of Lugol s to the S+ and S- tubes. Record the color in Table 1. 8

9 Nucleic acids 20. Label two microcentrifuge tubes NA+ and NA Using a 200 μl micropipette, put approximately 25 μl of distilled water into the microcentrifuge tube NA Using a p200 micropipette and clean pipette tip, add 25 μl of Lambda DNA to the NA+ tube. 23. Using a clean pipette tip, add 25 μl of GelGreen to NA+ and NA- tubes. Important: Be sure to use a new tip for each test. 24. Gently mix the s and observe them under a UV light or blue LED light. Record the color in Table 1. Caution: Use the appropriate protective gear when using a UV light. 9

10 Nucleic acids Carbohydrates Proteins Lipids Table 1: Macromolecule Tests Tube Type Macromolecule Label Description Contents Test Reagent Color Change Observed L+ Lipids and vegetable oil Sudan IV L- Negative control, lipids Sudan IV P+ Protein BSA (protein) Biuret P- Negative control, protein Biuret G+ Glucose Glucose Benedict s G- Negative control, glucose and other monosaccharides Benedict s S+ Starch and other polysaccharides Starch Lugol s S- Negative control, starch and other polysaccharides Lugol s NA+ Nucleic acids (microcentrifuge tube) Lambda DNA GelGreen NA- Negative control, nucleic acids (microcentrifuge tube) GelGreen Images: 10

11 You Are What You Eat Post-Lab Questions and Analysis Directions: After completing the You Are What You Eat lab, answer the questions below. 1. A food sample is tested for all four macromolecules with the following results: Benedict s no color change Iodine orange to black BSA blue to purple GelGreen Safe no color change What macromolecules do these results suggest are in your food sample? Which macromolecules are likely not present? Explain your reasoning. 2. The nutritional information for fresh strawberries is below. Notice that there is no fat (Total Fat = 0g) in a serving of 8 medium berries. Your friend concludes that strawberries are fat free. Explain why that isn t true. Hint: What are strawberries made of? 11