BIO Lab 2

BIO 101 LAB 2: MACROMOLECULES AND ORGANIC MOLECULES NOTIFICATION: If you have a technology issue or disability that makes it difficult to complete this lab, please contact your instructor at least 2 days before the due date. For disabilities, please provide your instructor a copy of the Memorandum of Accommodation (MOA) from NVCC Disability Support Services. Objectives: ● Describe the basic structures and properties of monomers and polymers of carbohydrates, proteins, and lipids. ● Identify reagents and positive test results for simple sugar, starch, proteins, and lipids ● Explain how to determine nutritional components of foods based on biochemical analysis Background: Macromolecules are polymers (linked units) of smaller monomers (single molecules). For example, nucleic acids are monomers that make up DNA and RNA macromolecules. The macromolecules lab introduces tests for 3 of the 4 essential organic macromolecules found in all living organisms: carbohydrates (sugars), lipid (fats), and proteins (Figure 1). This activity does not include any reagents (chemicals used in reaction) to test for nucleic acids. Macromolecules are essential components for all cells and serve a wide array of functions inside of cells as structural building blocks. For example, cellulose (carbohydrate) is a building component of plant cell walls; phospholipids (lipids) are structural components of cell membranes and fats (lipids) store energy and provide insulation; proteins are structural components of cell membranes and some are enzymes that speed up chemical reactions. Organic molecules are composed of carbon atoms in the form of rings or long chains. Atoms of hydrogen (H), oxygen (O), and often nitrogen (N) are bound to the carbon structures (Figure 2) in a variety of configurations. As you have learned in lecture, elements such as carbon, hydrogen, oxygen, and nitrogen are important and necessary components of living beings. These elements must be acquired from our surrounding environment. Plants and other autotrophs (organisms that make their own food) are able to make their own organic macromolecules by using sunlight and atmospheric gases. Heterotrophs, such as humans and other animals, are not capable 1 BIO 101 LAB 2: MACROMOLECULES AND ORGANIC MOLECULES of making their own organic molecules this way and so they eat other organisms to acquire their carbon (organic) components. Through their diet, heterotrophs incorporate the macromolecules from those organisms that they eat. Understanding of these macromolecules in nutrition will assist with studying upcoming topics discussed in Biology. Carbohydrates Carbohydrates (sugars) are molecules made up of carbon, oxygen and hydrogen atoms bonded together. Carbohydrates are the fuel that our bodies burn for energy to maintain life. Sugars containing one glucose-like molecule are called monosaccharides (mono- “single”; saccharide“sugar”). Sugars containing two glucose-like molecules are called disaccharides (di- means “two”). Starches are composed of many of these small sugar molecules joined together and are technically called polysaccharides (poly- means “many”). Individual glucose molecules (a type of monosaccharide) that is used for making ATP are called monomers (“one part”). When monomers link together to form long chains of carbohydrates, they are referred to as polymers (“many parts”). The long chains can occur in different shapes called isomers. For example, the starch found in potatoes and cellulose in plant cell walls are both polymers of glucose, linked and arranged in different structures. Polysaccharides and other polymers are formed by dehydration synthesis or condensation synthesis (“putting together by removing water”). This can be visualized below (Figure 3). Testing for carbohydrates: The presence of carbohydrates can be tested using two separate chemical reagents: Benedict’s solution and Iodine. Benedict’s interacts with simple sugars (monosaccharides) and will turn green, yellow, orange or red in the presence of various concentrations of simpler sugars (Figure 4). Iodine will stain starch (complex carbohydrate or polysaccharide) a dark blue or black color. Proteins Proteins perform many functions in living cells. There are structural proteins, storage proteins, transport proteins, contractile proteins, proteins that speed up reactions, proteins that fight infection, proteins that regulate life processes and proteins that detect chemical signals. Proteins are polymers of amino acids. There are twenty different amino acids and each one is formed with an -NH2 group, a –COOH group, and an R group. There are 20 different R groups, and it is the R group that designates which type of amino acid it is. During dehydration synthesis the –OH group from the -COOH end and one of the hydrogens from the -NH2 are removed and form a water (H2O) molecule (Figure 4). Amino acids will bind to other amino acids through peptide bonds, and so are called peptides when bound. A protein is formed when strings of amino acids called polypeptide (“many” peptides) bind together. 2 BIO 101 LAB 2: MACROMOLECULES AND ORGANIC MOLECULES +H2O This process can continue indefinitely to form a polypeptide. This polypeptide chain is arranged in a complex three-dimensional form that is covered in your lecture text. Changes in this three-dimensional structure may cause the protein to not be able to perform its function. A protein that has been changed in this way is said to be denatured. Testing for proteins: The presence of peptide bonds or full proteins can be tested for using Biuret reagent. Biuret will turn light to dark purple in the presence of proteins or pink in the presence of smaller peptide fragments. Lipids Lipids are not a single type of molecule; rather, they are various types of organic molecules that are poorly soluble in water. The common feature of lipids is that they are hydrophobic, which means they do not mix with water easily. The lipids most familiar to the general public are the fats and oils. While also performing structural duties, their major function is to store energy for later use by the organism. While not really polymers, lipids are still formed via dehydration synthesis between their parts. The fats and oils are composed of glycerol and several fatty acids. Again, dehydration joins the fatty acids to the glycerol through the removal of water to form an ester linkage (an oxygen atom bonded between two carbons). Typical fats and oils have 3 fatty acids attached to the glycerol. Figure 6. Formation of a lipid molecule 3 BIO 101 LAB 2: MACROMOLECULES AND ORGANIC MOLECULES Testing for lipids: The presence of lipids can be tested for using the Sudan IV reagent or paper. Sudan IV dissolves more effectively in the presence of lipids or fats, forming a red color. The red solution will float to the top, above any water-based solution. If no lipids or fats are present, no red is found. Lipids will turn paper translucent which is visible when held up against light. Your instructor will perform this test and show you. For this lab, you will be working in groups of 4-5 students. Each student will turn in their work, in their own words for credit (do not just copy each other’s work even if you work together). Materials: Each tray will contain all the required materials. Let your professor know if you are missing any materials. Safety: Follow all standard laboratory safety procedures. You will be working with chemical and hot surfaces. Use caution when handling hot objects. Procedure: Experiment 1a. Positive and negative tests for Benedict’s reagent 1. Take two small test tubes and label them as “B-G” and “B-W” plus your initials. 2. Add 20-30 drops of glucose solution to the “B-G” tube. 3. Add 20-30 drops of water to the “B-W” tube. 4. Add 5 drops of Benedict’s solution to each tube. 5. Mix the solution well by shaking it or tapping. 6. Heat 250 ml of water in a 300ml beaker using a hot plate. 7. Carefully place tubes in boiling water and incubate for 3-4 mins. Make sure water does not enter experimental tubes. 8. Observe for any changes in the solutions. 9. Record your observations for each solution in each tube in Table 1 of the Lab Worksheet. Experiment 1b. Positive and negative tests for Iodine starch test 1. Take a porcelain spot plate and label two of the wells as “I-S” and “I-W” using a wax pencil. 2. Add 5 drops of starch solution to the “I-S” well. 3. Add 5 drops of water to the “I-W” well. 4. Add 5 drops of Iodine to each of the two wells. 4 BIO 101 LAB 2: MACROMOLECULES AND ORGANIC MOLECULES 5. A reaction between iodine and starch should occur almost immediately. 6. Record your observations of each solution in each well in Table 1 of the Lab Worksheet. Experiment 1c. Positive and negative tests for protein using Biuret solution 1. Take your porcelain spot plate and label two of the wells as “U-A” and “U-W” using a wax pencil. 2. Add 5 drops of Albumin solution to the “U-A” well. 3. Add 5 drops of water to the “U-W” well. 4. Add 5 drops of Biuret solution to each of the two wells. . 5. A reaction between the Biuret solution and the protein (albumin) should occur almost immediately. 6. Record your observations of each solution in each well in Table 1 of the Lab Worksheet. Experiment 1d. Positive and negative tests for lipids using Sudan IV 1. Take your porcelain spot plate and label two of the wells as “S-O” and “S-W” using a wax pencil. 2. Add 5 drops of water and 5 drops of vegetable oil to the “S-O” well. 3. Add 5 drops of water to the “S-W” well. 4. Add 5 drops of Sudan IV solution to both wells. 5. Mix the solutions well by using a clean toothpick for each (don’t use the same toothpick, cross contaminating your solutions!). 6. The Sudan IV dye will associate more strongly with oil than it will with water, and a reaction should occur relatively fast. 7. Record your observations of each solution in each tube in Table 1 of the Lab Worksheet. 5 BIO 101 LAB 2: MACROMOLECULES AND ORGANIC MOLECULES Experiment 1e. Positive and negative tests for lipids using the Grease Spot on paper Test 1. Take two pieces of brown paper, write O (for oil) on one paper and W for water on another. 2. Place a drop of oil on the piece of paper marked O and a drop of water on the piece of paper marked W. 3. Set them aside and wait 30-60 minutes 4. Hold up both pieces of paper to the light. 5. Record which piece of paper is allowing light to come through. (Lipids will turn paper translucent which is visible when held up against light.) Experiment 2. Testing macromolecules in different foods Fill in the table with the foods you will be using. These are set on the countertop in the back of the classroom. Sample foods for testing Solids Liquids For solid food samples: 1. Label 2 test tubes for each solid food sample. 2. Use a razor blade or mortar and pestle to grind a pea-sized portion of food. 3. Place the food paste sample into a test tube and add 5mL of water. 4. Place a clean, gloved thumb on top of the test tube and shake vigorously for one minute. 5. Pour the liquid solution into the second test tube labeled for that food. 6. Discard the solid portions in the trash can (and wash the test tube) 7. Use only the liquid (solution) for your tests. 8. Be sure you have enough solution (4mL) to complete 4 tests. If not, add more water. For liquid food samples, 1. Label one test tube for each liquid food sample. 2. obtain 5ml in labeled test tubes to use for testing. Test A. Benedicts test on each food sample 1. Label 5 small test tubes using a wax pencil with the name of each food sample. 2. Add approximately 1mL (or 20-30 drops) of each prepared solution to the appropriately labeled test tube. 3. Add 5 drops of Benedict’s reagent to each tube. 4. Mix the solution well by shaking it or tapping. 5. Incubate all the tubes in a hot water bath for 5 minutes. 6. Record whether or not each food sample contains simple sugars in Table 3 of the Lab Worksheet. Test B. Iodine test on each food sample 1. Label 5 wells on a spot plate using a wax pencil with the name of each food sample. 6 BIO 101 LAB 2: MACROMOLECULES AND ORGANIC MOLECULES 2. Add approximately 5 drops of each prepared solution to the appropriately labeled well on the spot plate. 3. Add 1-2 drops of Iodine solution to each well. 4. Mix the solution. 5. Record whether or not each food sample contains starch in Table 3 of the Lab Worksheet. Test C. Biuret test on each food sample 1. Label 5 wells on a spot plate using a wax pencil with the name of each food sample. 2. Add approximately 5 drops of each prepared solution to the appropriately well on the spot plate.. 3. Add 1-2 drops of Biuret solution to each well. 4. Mix the solution. 5. Record whether or not each food sample contains protein in Table 3 of the Lab Worksheet. Test D. Sudan IV test for food samples 1. Label 5 wells on a spot plate using a wax pencil with the name of each food sample. 2. Add approximately 5 drops of each prepared solution to the appropriately labeled well on the spot plate.. 3. Add 1-2 drops of Sudan IV solution to the appropriately labeled well. 4. Mix the solution, and then let it sit for at least 2 minutes. 5. Record whether or not each food sample tested contains lipids in Table 3 of the Lab Worksheet. 7 BIO 101 LAB 2: MACROMOLECULES AND ORGANIC MOLECULES Lab 2 Worksheet Questions and Analysis 1. In a chemical reaction, the chemicals on the left side are called:___________________ 2. The chemicals on the right side of the arrow in a chemical reaction are called:___________________ Data Analysis and Synthesis Questions: 1. TABLE 1. Results from positive and negative controls. Reagent Substance tested Benedict’s Glucose Observations/notable changes Water Iodine Starch Water Biuret Albumin Water Sudan IV Oil Water Grease Spot Oil Test Water 3. Explain why water is used as a negative control for the reagents in these experiments? 4. What is the result of a positive reaction for simple sugars using Benedicts (be specific)? 8 BIO 101 LAB 2: MACROMOLECULES AND ORGANIC MOLECULES 4. What is the result of a positive reaction for starch using iodine (be specific)? 5. What is the result of a positive reaction for proteins using Biuret (be specific)? 6. List the four macromolecules on the left column and their corresponding monomer to the right. ______________________ ________________________ ______________________ ________________________ ______________________ ________________________ ______________________ ________________________ 8. Table 2. Write your Predictions: Macromolecule Composition: Presence (+) or Absence (-) Food Simple Sugars Starch Protein Lipids 9. TABLE 3. Actual Results of food tests. Indicate positive (+) or negative (-) results Food Benedict’s Iodine 9 Biuret Sudan IV BIO 101 LAB 2: MACROMOLECULES AND ORGANIC MOLECULES 10. TABLE 4. List the macromolecules found in each food according to your results. Food Macromolecules present Scenarios – Application Questions 11. You buy a new protein bar that boasts high protein and zero fats. You decide to put the bar to the test. What results should you expect if this is true (list the reagents and specify the results)? 12. Some commercial cottage cheeses are made with additives such as corn starch for preservation and sweeteners. Examine the image to the right. (note: you must see this in color to answer correctly) Which reagent is being used to for test corn starch? Which of these cottage cheese brands contains more starch (A or B)? Explain your answer? *BE SURE TO CLEAN UP YOUR TEST TUBES, SPOT PLATES AND BENCH TOP, RETURN ALL MATERIALS TO YOUR TRAY. PLACE THE TRAY BACK WHERE YOU GOT IT. ANY MESS LEFT BEHIND BY YOUR GROUP WILL RESULT IN LOSS OF POINTS FOR EACH GROUP MEMBER. 10

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