Lab 3 MACROMOLECULES OBJECTIVES Define macromolecule, vitamin, mineral, carbohydrate, monosaccharide, disaccharide, polysaccharide, lipid, protein, amino acid, calorie; Describe the basic structures of carbohydrates, lipids, glycerides, and proteins; Recognize positive and negative tests for carbohydrates, lipids, and proteins; Identify the roles that carbohydrates, lipids, proteins, minerals, and vitamins play in the body s construction and metabolism; Test food substances to determine the presence of biologically important macromolecules; Identify common dietary sources of nutrients; INTRODUCTION Man needs a wide range of nutrients to perform various functions in the body and to lead a healthy life. The nutrients include proteins, fat, carbohydrates, vitamins and minerals. These nutrients are chemical substances which are present in the food we eat daily. The foods containing these nutrients which we consume daily are classified as cereals, legumes (pulses), nuts and oilseeds, vegetables, fruits, milk and milk products and flesh foods (fish, meat and poultry) Man needs all these nutrients, i.e. energy, proteins, vitamins, minerals in different amounts to grow, live and thrive. Since man derives all the nutrients he needs through the diets he eats, his diets must be well balanced to provide all the nutrients in proper proportions. In planning a diet for the community, foods have to be chosen in proper amounts to provide all the nutrients in required amounts and also keeping in view the dietary habits and availability of foods. In this exercise, you will test for the presence of carbohydrates, lipids, and proteins, and then use your knowledge to determine the macromolecular composition of various common food products. I. IDENTIFICATION OF MACROMOLECULES You will use some simple tests for carbohydrates, lipids, and proteins in a variety of substances, including food products. Most of the reagents used are not harmful; however, observe all precautions listed and perform the experiments only in the proper location as identified by your instructor. A. Carbohydrates General names for carbohydrates include sugars, starches, saccharides, and polysaccharides. The term saccharide is derived from the Latin word "saccharum" from the sweet taste of sugars. The name "carbohydrate" means a "hydrate of carbon." The name derives from the general formula of carbohydrate is C x (H 2 O) y - x and y may or may not be equal and range in value from 3 to 12 or more. For example glucose is: C 6 (H 2 O) 6 or is more commonly written, C 6 H 12 O 6. 1
The chemistry of carbohydrates most closely resembles that of alcohol, aldehyde, and ketone functional groups. As a result, the modern definition of a CARBOHYDRATE is that the compounds are polyhydroxy aldehydes or ketones. The chemistry of carbohydrates is complicated by the fact that there is a functional group (alcohol) on almost every carbon. In addition, the carbohydrate may exist in either a straight chain or a ring structure. A major part of the carbon cycle occurs as carbon dioxide is converted to carbohydrates through photosynthesis. Carbohydrates are utilized by animals and humans in metabolism to produce energy and other compounds. Carbohydrate Functions: Carbohydrates are initially synthesized in plants from a complex series of reactions involving photosynthesis. Store energy in the form of starch (photosynthesis in plants) or glycogen (in animals and humans). Provide energy through metabolism pathways and cycles. Supply carbon for synthesis of other compounds. Form structural components in cells and tissues. Photosynthesis is a complex series of reactions carried out by algae, phytoplankton, and the leaves in plants, which utilize the energy from the sun. The simplified version of this chemical reaction is to utilize carbon dioxide molecules from the air and water molecules and the energy from the sun to produce a simple sugar such as glucose and oxygen molecules as a by product. The simple sugars are then converted into other molecules such as starch, fats, proteins, enzymes, and DNA/RNA i.e. all of the other molecules in living plants. All of the "matter/stuff" of a plant ultimately is produced as a result of this photosynthesis reaction. A.1. Test for Sugars Using Benedict s Solution Benedict s reagent (a blue colored solution containing copper ions) is used to test for the presence of reducing sugars. When a solution containing Benedict s reagent and a reducing simple sugar is heated, the copper (II) ions in the Benedict s reagent are reduced to copper (I) ions and the solution changes from blue to green to orange to red-orange to brick-red. A brick-red precipitate (solid), copper (I) oxide (Cu 2 O), may appear in the bottom of the tube. The more reducing sugar present in the mixture, the more precipitate will form. The half-reaction for Benedict s Test for Reducing Sugars can be shown as: 2 Cu +2 + 2 e - 2 Cu +1 Each copper (II) ion, Cu +2, is reduced to a copper (I) ion, Cu +1, by an electron from the reducing sugar. The reducing sugar is oxidized as a result of giving up its electron. 1. Half-fill the beaker with tap water and apply heat with a hot plate or burner to bring the water to a gentle boil, then reduce the temperature dial to the half setting. 2. Using the china marker, number the test tubes 1-11. 2
3. Pipet 2.5 ml of the correct stock solutions into the test tubes as described in Table 3-1. Table 3-1 Benedict s Test for Sugars Tube Contents Initial Color 1 Deionized Water 2 Glucose 3 Maltose 4 Lactose 5 Sucrose 6 Starch 7 Fructose 8 Lemon Juice 9 Orange Juice 10 Regular Soda 11 Diet Soda Color After Heating Conclusion +/- 4. Add 10 drops of Benedict s solution to each test tube, agitate the mixture by shaking the tubes from side to side or with a vortex mixer. 5. Record the color of the mixture in Table 3-1 in the column Initial Color. 6. Heat the tubes in the hot water bath for 2 minutes. Remove the tubes with the test tube clamp. 7. Record any color changes that have taken place in the column Color After Heating. 8. Record your conclusions regarding the presence or absence of simple carbohydrates in the column Conclusion. A.2. Test for Starch Using Lugol s Iodine Solution 1. Using the china marker, number the test tubes 1-7. 2. Pipet 2.5 ml of the correct stock solutions into the test tubes as described in Table 3-2. 3. Record the color of each solution in the column Initial Color in Table 3-2. 4. Add 2 drops of Iodine solution to each test tube; agitate the mixture by shaking the tubes from side to side or with a vortex mixer. 5. Record the color of the mixture in the column Color After Adding Iodine Solution. 6. Record your conclusions regarding the presence (purple product) or absence of starch in each test solution in the column Conclusion. 3
Table 3-2 Lugol s Iodine Solution Test for Starch Tube Contents Initial Color Color After Iodine Solution Conclusion +/- 1 Deionized Water 2 Glucose 3 Maltose 4 Lactose 5 Sucrose 6 Starch 7 Cream B. Lipids Lipids are oily or greasy compounds insoluble in water, but dissolvable in non-polar solvents such as ether or chloroform. Those lipids having hydrocarbon chains with a carboxyl ( COOH) group at one end are called fatty acids and combine with glycerol to form glycerides, a rich source of stored energy. Lipids with three fatty acid chains attached to a glycerol backbone are called triglycerides. Lipids provide long-term energy storage in cells and are very diverse. Lipid digestion occurs primarily in the small intestine where bile produced by the liver breaks lipid globules into smaller droplets, and then pancreatic enzymes break large lipid molecules into smaller components for absorption. The lipid components are then transported throughout the body in lymph, the fluid that bathes the tissues. Substances we think of as fats and oils are examples of lipids. One of the simplest tests for lipids is to determine whether they leave a grease spot on a piece of uncoated paper, such as a grocery sack. A test commonly used to identify fats and oils in microscopic preparations (Sudan IV) can also be used to indicate their presence in a test tube. B.1. Test for Lipids using Uncoated Paper You perform this test every time you buy muffins or doughnuts in a paper bag. Lipids make grocery bag paper (brown paper) translucent. 1. Put a drop of each test substance on a piece of grocery bag paper. 2. Draw a circle around the spot with a soft pencil. 3. Write the name of the sample in pencil next to the spot. 4. Allow all spots to dry thoroughly. 4
5. Hold the paper in front of a light source and observe the spots. 6. After 10 minutes, describe the appearance of each spot on the grocery bag paper. Deionized water Vegetable oil Hamburger juice Onion juice Regular soda Cream 7. What can you conclude about the lipid presence and quantity of each substance? B.2. Test for Lipids using Sudan IV Sudan red, dissolved in alcohol, is a lipid soluble dye. When Sudan red is added to a mixture of lipids and water, the dye will move into the lipid layer coloring it red. 1. Add 9 drops of Sudan red dye to each tube containing 2.5 ml of the substances in the table below and 2.5 ml of water. Mix the contents of each tube using the vortex. 2. Wait 2 minutes. 3. Examine each tube carefully. Where is the red color found? 4. Record your results in Table 3-3. Table 3-3 Test for Lipids with Sudan IV Tube Contents Color After Sudan IV Conclusion +/- 1 Deionized Water 2 Vegetable Oil 3 Hamburger Juice 4 Onion Juice 5 Regular Soda 6 Cream 5
5. Which test, the uncoated paper test or the Sudan IV test, do you think is most sensitive to small quantities of lipids? Explain your answer. 6. What are the limitations of these tests, or what can t these tests (uncoated paper and Sudan IV) tell you? (Hint: think about what the tests can tell you and the information it can t tell you are its limitations.) C. Proteins Proteins are the building blocks that grow and repair your body. Proteins are needed not only for muscle but also for hair, skin and internal organs. Some proteins travel around your body in the blood as hormones, enzymes and red blood cells. Protein is unique because it is the only food source of nitrogen, which is essential to all plant and animal life. Proteins are made up of chains of amino acids. For each protein, there are specific amino acids in a specific amount, and they are joined in a unique order. This is what makes a chicken different from cheese or a fingernail different from a strand of hair. There are 22 amino acids. Eight of these are called the essential amino acids because they cannot be made by the body and must be provided by the diet. Protein digestion begins in the stomach lining and continues in the small intestine, where various enzymes break protein molecules first into protein fragments, and then into amino acids, which are absorbed across the small intestine wall. The amino acids are then transported throughout the body in the blood. C.1. Test for Proteins using Biuret Reagent Biuret reagent is a light blue solution which turns purple when mixed with a solution containing protein. When the copper ions of the Biuret Reagent react with peptide bonds in the polypeptide chains, a purple color complex is formed. 1. Using the china marker, number the test tubes 1-5. 2. Pipet 2.5 ml of the correct stock solutions into the test tubes as described in Table 3-4. 3. Add 10 drops of Biuret reagent to each test tube and agitate the mixture by shaking the tubes from side to side or with a vortex mixer. 4. Wait 2 minutes and then record the color of the mixture in Table 3-4, in the column Color After adding Biuret. 5. Record your conclusion about the presence of protein in the substance in the column Conclusion. 6
Table 3-4 Test for Protein with Biuret Reagent Tub e Contents 1 Distilled water 2 Starch 3 Egg Albumin 4 Glucose 5 Non-Diet Soda Color After adding Biuret Conclusion +/- D. Testing Unknown Food Substances Your group will be provided with four samples of unidentified food substances. 1. Following the previous procedures, perform tests for carbohydrates (Benedict s test and Lugol s Iodine solution test), lipids (uncoated paper and Sudan IV tests), and proteins (Biuret reagent) on each unknown food item. 2. Record your test results (+ or for each test) in Table 3-5. Table 3-5 Test of Unknowns Carbohydrates Lipids Proteins Unknown Benedict s Iodine Uncoated Paper Sudan IV Biuret 1 2 3 4 7
POST-LAB QUESTIONS 1. Let s suppose you are teaching science in a part of the world without ready access to a doctor and you re worried that you may have developed diabetes. (Diabetics are unable to regulate blood glucose levels, and glucose accumulates in blood and urine.) What test could you do to gain an indication of whether or not you have diabetes? 2. The test tubes contain Benedict s solution and two unknown substances that have been heated. One tube turned orange after heating, the other turned green. What do the results indicate? 3. How could you verify that a soft-drink contains diet soda rather than soda sweetened with fructose? 4. When you eat french fries, the potato material is broken down in your small intestine into what small subunits? 5. A test tube contains water at the bottom and another substance that has been stained with Sudan IV at the top. What is the macromolecular composition of this stained substance? 6. You are given a sample of an unknown food. Describe how you would test it for the presence of lipids. 7. You wish to test the same unknown food for the presence of sugars. Describe how you would do so. 8. What is the purpose of the distilled water sample in each of the chemical tests in this exercise? 9. Many health food stores carry enzyme preparations that are intended to be ingested orally (by mouth) to supplement existing enzymes in various organs like the liver, heart, and muscle. Explain why these preparations are unlikely to be effective as advertised. 8