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Lab 2: Macromolecules Name: ____________________________________ Section: ___________________________________ Lab 2: Macromolecules THE MOLECULES OF LIFE Life on earth is based on the element carbon and compounds containing carbon. When carbon is combined with hydrogen and other atoms this makes organic molecules. Carbon chains form the skeletons of most organic molecules. The skeleton varies in length and arrangement. This variation allows for the molecular complexity and diversity seen in living matter. The distinct properties of these molecules depend not only on this complexity and variation, but also the chemical groups that are attached to the carbon skeleton. These chemical groups are called functional groups. Each functional group has a unique structure, and along with it, gives each molecule unique properties essential for life. Important Biological Functional Groups Lab 2: Macromolecules The large molecules important for all living things fall into four categories: carbohydrates, lipids, proteins, and nucleic acids. All of these, Proteins, Carbohydrates, Lipids and Nucleic Acids are very large molecules called macromolecules. In this lab, we will be conducting tests that reveal properties of carbohydrates, lipids, and proteins. Nucleic acids will be studied in a later lab. CARBOHYDRATES Carbohydrates are sugars and polymers of sugars. They occur in a wide variety of monomers (monosaccharides) and polymers of saccharides (disaccharides or polysaccharides). Monosaccharides, or simple sugars, are aldehydes or ketones with two or more hydroxyl groups attached. Examples include glucose, fructose, and ribose. A disaccharide is two joined monosaccharides and these are the simplest polysaccharides. Examples include sucrose and lactose. They are composed of two monosaccharide units bound together by a covalent bond known as a glycosidic linkage formed via a dehydration reaction, resulting in the loss of a hydrogen atom from one monosaccharide and a hydroxyl group from the other. A polysaccharide is a sugar made up of three or more sugar units attached by glycocidic linkages. Examples include cellulose found in the cell wall of plants and starch used by plants for storage. Carbohydrates are a major source of fuel for metabolism. Since no single test can be used as a marker for all of them, you will conduct two tests specific to two of the important classes of carbohydrates: The Benedict’s test, which tests for reducing sugars (any sugar that has an aldehyde or can form one) which includes all monosaccharides and some disaccharides and Lugol’s test which tests for the presence of starch. 1. BENEDICT’S TEST FOR REDUCING SUGARS Benedict's reagent is used as a test for the presence of reducing sugars. Benedict's test will detect the presence of aldehydes, and alpha-hydroxy-ketones. A positive test with Benedict's reagent is shown by a color change from clear blue to a brick-red precipitate. Benedict's reagent contains blue copper ions (Cu2+) which are reduced to copper (ions (Cu+). These are precipitated as red copper oxide which is insoluble in water. Benedict's Reagent provides a quantitative test for reducing sugars along with a qualitative test. The color of the obtained precipitate gives an idea about the quantity of sugar present in the solution. A greenish precipitate indicates about 0.5% concentration; yellow precipitate indicates 1% concentration; orange indicates 1.5% and red indicates 2% or higher concentration. Demonstration of Benedict’s test: a. Glucose + Benedict’s solution ______________________________________________ b. Glucose + Benedict’s solution + heat _________________________________________ Lab 2: Macromolecules Procedure for Benedict’s test Label five test tubes 1-5 and place 2 ml of the following solutions in the corresponding tubes: 1. 2. 3. 4. 5. 1% glucose 1% sucrose 1% starch 1% ribose 1% distilled water (DI H2O) Add 2 ml of Benedict’s solution to each tube and heat in a boiling water bath for 5 minutes; note the color change in the following chart, using the key below to indicate degree of change. – ++ +++ ++++ a. b. c. d. blue green orange red Under the conclusion column, note the reason why it did or did not change (ex. It is a polysaccharide). Use the figures on the next page to assist you with your conclusions. Circle the aldehyde or ketone on the diagrams (not the whole molecule) on the next page for the sugars that reacted with Benedict’s solution. Tube Contents 1 Glucose 2 Sucrose 3 Starch 4 Ribose 5 DI H2O Color (after heating) Results (+ or -) What is the purpose of using the distilled water in this test? Conclusions Lab 2: Macromolecules Carbohydrate Structures Sucrose Starch DI Water Lab 2: Macromolecules 2. IODINE TEST FOR THE PRESENCE OF STARCH Starch is a long polymer consisting entirely of glucose (a monosaccharide). Starch is the major nutrient storage polysaccharide in plants and their seeds. Starch itself is composed of subunits of small polysaccharides (amylose and amylopectin) which in turn are composed of chains of glucose molecules. Iodine solution contains iodine and potassium iodide. The coiled structure of the polysaccharide starch produces a dark, blue-black color in the presence of iodine, which is usually yellow to brownish in color. Because this reaction does not occur with monosaccharides or disaccharides, the blue-black color is considered to be positive for starch. Demonstration of Iodine test: a. Iodine ___________________________________________________________ b. Iodine + Starch ____________________________________________________ Procedure for Iodine test Label four test tubes 1-4 and place 2 ml of the following solutions in the corresponding tubes: 1. 2. 3. 4. 1% glucose 1% sucrose 1% starch 1% distilled water (DI H2O) Add 2 ml of Iodine solution to each tube and mix; note the color change in the following chart. (Use + or -, note colors and explain conclusions) Tube Contents 1 Glucose 2 Sucrose 3 Starch 4 DI H2O Color Results (+ or -) Conclusions Lab 2: Macromolecules LIPIDS Lipids are a diverse group of large biological molecules that are not made up of true polymers and are not usually large enough to be considered macromolecules. Members of this class of molecules have one thing in common, they are hydrophobic. This property means they mix poorly or not at all. A typical fat molecule is made up of three fatty acids attached to a glycerol molecule (triglyceride). Lipids can also be in the form of phospholipids and steroids (such cholesterol). Lipids are used for energy, insulation, membrane structure and as chemical messengers. 3. VISUAL AND SUDAN TEST FOR THE PRESENCE OF LIPIDS Lipids are hydrophobic and will separate when itself when dropped in water. Sudan is a special type of dye called a lysochrome that is typically used by histologists to demonstrate the presence of triglycerides in frozen tissue sections. It has a distinct orange red color that makes it easy to identify lipids in an aqueous solution. In our lab, we will use Sudan to detect the presence of lipids in various aqueous solutions. Exercise caution when using this dye. It is hydrophobic, and will dye your tissues as well as your clothing. The contents of any test tube containing Sudan must be poured into a discard bottle, not into the sink! Always tightly cap Sudan dye when you have dispensed the amount you need. Lab 2: Macromolecules Procedure for Visual tests Add three drops of salad oil to 10 ml of water. Mix thoroughly. Immediately observe the mixture. Let it stand for three minutes and then answer the following questions. Does the oil separate from the water after standing a few minutes? Where is the oil located? What can you say about the relative densities of oil and water? In light of your answer to the previous question, what function (other than energy storage) does fatty blubber serve in whales? Procedure for Visual tests (continued) Fat and oil form a translucent spot when placed on unglazed paper, such as a paper towel or a paper bag. The translucent spot admits the passage of light, but not sufficiently to distinguish objects by looking through it. By contrast, water soluble substances will leave a water spot that will dry completely preventing the passage of light. Obtain a piece of paper bag. Use a pencil to draw four circles approximately the size of a silver dollar so that all of the circles are spatially separated. Label each circle as Di water, albumin, glucose, salad oil. Place a drop of each corresponding substance into the appropriate circle. Allow the paper towel to sit on the desk top undisturbed until the water spot is completely dry (approximately 30 minutes). Hold the paper towel up to the light and determine which sample is translucent. Record your data in the table on the next page, in the column titled “Translucent”. Lab 2: Macromolecules Demonstration of Sudan test: a. Sudan Solution _______________________________________________________ b. Sudan Solution + Lipid ________________________________________________ Procedure for Sudan’s test Label four test tubes 1-4 and place 10 ml of DI water. Then add 2 ml of the following solutions in the corresponding tubes: 1. 2. 3. 4. DI water 1% albumin 1% glucose salad oil Add 10 drops of Sudan to each test tube and mix. Record your results in the table below. Tube Contents 1 DI H2O 2 Albumin 3 Glucose 4 Salad Oil Bag Translucent (+ or -) Color (after Sudan) Tubes Results (+ or -) Conclusions PROTEINS Proteins include a diversity of structures and have a wide range of functions. Proteins are structural components of cell and organelle membranes as well as muscle and tendons. The can act as enzymes (biological catalysts), they function as chemical messengers (hormones) and have a central role in the functioning of the immune system. Proteins are large molecules composed of amino acids. All amino acids share a common structure. They are a molecule that has an amino group, a carboxyl group, a hydrogen atom, and an R (side) group which differs with each of the twenty side chains. The subunits are linked together with the carboxyl group of one amino acid forming a bond with the amino group of another creating a peptide bond. Lab 2: Macromolecules 4. THE BUILDING BLOCKS OF PROTEINS Pepsin is an enzyme that works in the stomach to break down proteins. The silver halide on the photographic film is held on by an emulsion of protein. If there is protein digestion, the clear cellulose backing will be exposed. Enzymes work under very specific conditions such as temperature and pH. Procedure for Protein Digestion Label four test tubes 1-4 and place the following solutions in the corresponding tubes: 1. 6 ml of 7.0 buffer 2. 3 ml of 5% pepsin (enzyme) + 3 ml of 0.2% HCL 3. 3 ml of 0.2% HCL + 3 ml of DI H2O 4. 3 ml of 5% pepsin (enzyme) + 3 ml of 7.0 buffer Add a strip of photographic film to each tube and incubate at 37 degrees Celsius for one hour. Record the results in the table below. Tube 1 2 3 4 Contents Results (+ or -) Conclusions Lab 2: Macromolecules What conditions do you think are necessary for pepsin to work? What are the building blocks of proteins? Practice writing a null and alternative hypothesis for this specific experiment Ho: _____________________________________________________________________ Ha: __________________________________________________________________________ 5. BIURET’S TEST FOR THE PRESENCE OF PROTEINS Biuret reagent is composed of CuSO4 (copper sulfate) and KOH (potassium hydroxide). It reacts only with amino acids that are bound together by peptide bonds to form proteins. The copper sulfate (CuSO4) used in the biuret test is normally light blue colored and turns violet in the presence of peptide bonds in a strong alkali solution. A negative test, therefore, remains light blue in color. Demonstration of Biuret’s test: a. Biuret’s Solution _________________________________________________________ b. Biuret’s Solution + protein _________________________________________________ Procedure for Biuret’s test Label four test tubes with the numbers 1-4 and add 3 ml of 10% NaOH to each test tube. Add the following solutions in the corresponding tubes: 1. 2. 3. 4. 3ml of water 3ml of 1% albumin 3ml of 1% glucose 3 ml of 1% starch Add 1% CuSO4 to each test tube one drop at a time until a violet or pink color appears. NOTE: Adding CuSO4 too rapidly can result in a false negative! Start with tube #1 and add one drop to each tube, then repeat the process until one of the tubes turns pink or violet. Record your results below. Lab 2: Macromolecules Tube Contents Color (Before) Color (after) Results (+ or -) Conclusions 1 2 3 4 7. Unknown Now it is time to apply what you learned. Your instructor will provide you with an unknown. Each unknown has a unique number. Your unknown may contain: protein, starch, reducing sugar, fats or distilled water. Remember that your unknown may be a combination of these things, so be certain you complete all tests before reporting your results. For each test performed, use 1ml of unknown. YOU MUST GET YOUR INSTRUCTORS INITIALS WHEN YOU HAVE DETERMINED THE RESULTS TO GET CREDIT! Record your unknown number here: _______________________ Test Benedict’s Lugol’s Sudan III Biuret’s Tests for Results (+ or -) Conclusions