Biol 107: Lab 1-Biologically Important Macromolecules

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1 Biol 107: Lab 1-Biologically Important Macromolecules All living organisms are composed of various types of organic molecules, such as carbohydrates, starches, proteins, lipids, and nucleic acids. These substances, contained in the foods we eat, provide us with the energy and cellular building blocks necessary for life (you really are what you eat!) Several of these molecules can be detected by simple chemical tests. In today's lab, you will perform tests to detect carbohydrates, proteins, and lipids, and then apply these tests to selected foods. Work in pairs or small groups. Exercise 1 - Benedict's Test for Simple Sugars Sugars are small carbohydrate molecules used as a source of energy by all organisms and manufactured in the chloroplasts of plants. There are many kinds of carbohydrate molecules, all of which are made up of various arrangements of carbon, hydrogen, and oxygen atoms. The simplest carbohydrates are monosaccharides, or simple sugars, such as glucose and fructose. Disaccharides consist of two bonded monosaccarides. Examples of disaccharides include lactose, the sugar contained in milk, maltose, used in brewing, and sucrose, common table sugar. Some sugars can be detected using the Benedict's Test. Benedict's solution contains blue cupric ions (Cu++). When heated, the cupric ions join with certain sugars and are reduced to insoluble, red cuprous ions (Cu+). The solution changes from blue to green to reddish brown as more cuprous ions are formed. Many disaccharides do not react with Benedict's reagent because the reactive groups are often not exposed. 1. Number four clean test tubes 1-4 (use grease pencil). 2. To tube #1 add a small piece of potato and a dropper full of Benedict's solution. 3. To tube #2 add a dropper full (about 1 ml) of sucrose solution and a dropper full of Benedict's solution. 4. To tube #3 add a dropper full of glucose solution and a dropper full of Benedict's solution. 5. To tube #4 add a dropper full of distilled water and a dropper full of Benedict's solution. 6. Place all the test tubes in a boiling water bath for 3 minutes. 7. After 3 minutes, remove the tubes, turn off the hot plate, and record your results. 1

2 Exercise 2 - Iodine Test for Starch Organisms store simple sugars in the form of large macromolecules comprised of hundreds or thousands of linked monosaccharides. Some of these polysaccharides are broken down as needed for energy. Other polysaccharides serve a structural function (the most abundant organic compound on earth is cellulose, a polysaccharide that is the main component in plant cell walls.) Plants store sugars for later energy needs in the form of a polysaccharide called starch. In this exercise, you will use the iodine test to detect the presence of starch. Starch interacts with iodine to produce a bluish-black color (a yellowish-brown color indicates that no starch is present.) 1. Wash your test tubes and if necessary, number them To tube #1 add a small piece of potato and 3-5 drops of iodine. 3. To tube #2 add a dropper full of sucrose solution and 3-5 drops of iodine. 4. To tube #3 add a dropper full of glucose solution and 3-5 drops of iodine. 5. To tube #4 add a dropper full of distilled water and 3-5 drops of iodine. Observe and record your results. Exercise 3 - Biuret Test for Proteins Proteins consist of linked subunits called amino acids, which are in turn composed of carbon, hydrogen, oxygen, and nitrogen. The Biuret Test detects the presence of a particular type of chemical bond in amino acids, called a peptide bond. When copper sulfate (CuSO4) reacts with peptide bonds in the presence of sodium hydroxide (NaOH), a violet color results. Only proteins with four to six linked amino acids will react in this way, thus free amino acids will not be detected. The intensity of the color is related to the number of amino acids linked together. 1. Wash your test tubes and if necessary, label To tube #1 add a dropper full of egg albumin solution. 2

3 3. To tube #2 add a dropper full of milk. 4. To tube #3 add a dropper full of amino acid solution. 5. To tube #4 add a dropper full of distilled water. 6. Carefully add one dropper full of 10% NaOH to each tube. NaOH is a strong base and can burn your skin. Use extreme care with this solution. 7. Add 4 drops of 0.5% CuSO 4 to each tube. Mix gently and let stand five minutes for the color to develop. Record your results. Exercise 4 - Sudan IV Test for Lipids There are three forms of lipids - triglycerides, sterols, and phospholipids - all of which contain primarily carbon, hydrogen, and oxygen. Lipids absorb pigments in fat-soluble dyes such as Sudan IV. A red color occurs at the interface of a lipid and Sudan IV dye. 1. Wash your tubes and if necessary, label To tube #1 add 1 ml of salad oil plus 5 drops of distilled water. Do not shake the tubes. 3. To tube #2 add 1 ml of salad oil plus 5 drops of Sudan IV. 4. To tube #3 add 1 ml of sucrose solution plus 5 drops of Sudan IV. 5. To tube #4 add 1 ml distilled water plus 5 drops of Sudan IV. Observe and record your results. (Look for a deep red color at the interface of the lipid with the dye; it may help to hold the tube over a piece of white paper.) Exercise 5 - Grease Spot Test for Lipids 3

4 A simpler (and perhaps easier to read) test for lipids is based on the ability of lipids to produce a translucent spot on paper. 1. Place one drop of salad oil on a piece of brown paper. 2. Place one drop of amino acid solution on a second piece of brown paper. 3. Wait a few minutes for the spots to dry. 4. Hold the papers up to the light and determine if each spot is translucent. Record your results. Exercise 6 - Testing Food Samples Wash your test tubes thoroughly. Perform the Benedict's Test, Iodine Test, Biuret Test, and Sudan IV or grease spot test on the food or foods assigned by your TA. Record your results. Add your findings to the class results on the front board. Before you leave, wash your tubes and leave them to drain in the rack at your table. Turn off the hot plate. Clean up your area and wipe your table. 4

5 Questions for Review 1. What subunits make up? Carbohydrates? Proteins? 2. You have been given an unknown solution; describe how you would test for Starch Lipids Sugars Proteins Fats Applying your knowledge: 1. Since potatoes have starch in them, why don t they taste sweet after they are boiled? 2. Why might a substance taste sweet, yet give a negative reaction with the Benedict s test? 3. Why might plant storage organ (such as a fruit or tuber) contain both starch and sugar? 4. You have tested an unknown sample with Biuret and Benedict s reagents. The solution mixed with biuret reagent is blue. The solution boiled with Benedict s reagent is also blue. What does this tell you about the sample? 5