Module 3_ Macromolecules_ Fall_2023

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Feb 20, 2024

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BISC 1111 Fall 2023 - Module 3: Macromolecules *Adapted from Professor Tara Scully’s “Chemical Building Blocks Lab 2022”* Objectives & Purpose: Students will be able to explain the significance of the four macromolecules of life: nucleic acids, carbohydrates, proteins, and lipids. Additionally, students will use different qualitative tests to detect the presence of carbohydrates, proteins, and lipids in a variety of substances. These techniques will then be leveraged to identify the types of macromolecules present in an unknown food sample. During the minilecture at the start of lab, students will take their first foray into the vital calculations which will serve them well throughout their STEM careers; namely, the math behind unit conversions, how to calculate dilutions of various solutions given certain information, and the construction and use of standard curves in analytical chemistry. These last two topics will be explored further in Module 4: Quantification of Proteins. Introduction: Organisms require particular chemical “building blocks” in order to grow and function (these are often referred to as nutrients ). Many of the chemicals an organism needs to consume in relatively large amounts are macromolecules . These large molecules are formed from the covalent bonding of small organic molecules (each of which, by definition, contains at least one carbon atom covalently bonded to one or more hydrogen atoms). A covalent bond is the strongest chemical bond in which at least two atoms are linked by electron sharing. Macromolecules are too large to cross the plasma membrane of a cell. Therefore, when you ingest food, these macromolecules must be broken down so that your body can absorb their components. Confoundingly, even if an organism’s body requires the exact macromolecule found in the food it just consumed, nearly all macromolecules are broken down into their smallest components to aid in the digestive process of absorption, and are then reconstructed into the necessary macromolecule by the organism’s metabolism. Four main macromolecules are common to all life-forms: nucleic acids , carbohydrates , proteins , and lipids . In today’s lab, we will be concentrating on the latter three, leaving nucleic acids for another time. Some nucleic acids, such as DNA and RNA, store and transport genetic information; others— most important, the molecule ATP—provide energy to cellular reactions. Carbohydrates are used primarily for direct or short-term energy storage, whereas lipids provide long-term energy storage. Proteins assist the body in many different ways; for example, they form physical structures such as collagen in skin, they enable muscle contraction, and as enzymes they hasten chemical reactions vital to the functioning of cells. Macromolecules are often polymers , repetitive arrangements of small organic molecules called monomers . Covalent bonds link the smaller repeating units of monomers to form polymers. The properties of organic polymers are influenced by attached groups of atoms called functional groups . A functional group is a distinct arrangement of specific atoms. The number and types of functional groups present in the macromolecule dictate the overall chemical properties and the roles that these molecules play within cells. Functional groups are especially important in protein structure and function. Food ingested by all organisms is composed of different chemical components, not necessarily containing all essential nutrients. For this reason, it is important to understand what you are consuming and the importance of all nutrients on a chemical and cellular level. An understanding of the chemical processes of ingestion, digestion, absorption, and elimination (the subject of further study in the spring semester), can greatly inform how we interact with food in our daily lives. For example, there is a common misconception and fear among the lay public that DNA & RNA in the genetically modified foods we eat can somehow alter or change our own genetic make-up. However, this is patently false; DNA & RNA are broken down into nucleic acid monomers, which are in turn further broken down into nitrogenous bases and sugars by enzymes in the small intestine, where they then are absorbed. The other macromolecules are similarly digested, absorbed, and then manufactured into macromolecules needed by your body, though each type goes through a different metabolic process to achieve this outcome. Vitamins and minerals—dietary nutrients required in smaller amounts—are also essential for the thousands of chemical reactions necessary to life. In this lab you will explore the 1

composition and nutritional value of carbohydrates, lipids, and proteins, and how all these nutrients play a role in an organism’s diet. In the next week’s Module 4 protocol, we will be using a spectrophotometer to determine the amount of protein in a sample. This technique does not identify or provide a list of the various proteins in a given sample, but yields quantitative data, i.e. the amount of protein in the sample. An important application of today’s lab can be seen in the pharmaceutical industry, for example in the protein concentration of insulin, human growth hormone, tissue plasminogen activator, erythropoietin, and blood clotting factor VIII. It is important to know the proper amounts of proteins in pharmaceuticals in order to administer safe and effective doses. Quantifying proteins can also have industrial applications such as the production of silk for textiles, barnacle adhesion chemicals in the production of glue, or the enzymes used to make detergents and Vitamin C. In order to effectively purify the protein you are interested in you must first find out how much of it you have. Once you know how much pure protein you have you can use this information to determine the various economic values for the sample, such as production cost and the profit margin of your synthesis techniques. Part 1: Carbohydrates The common term “sugar” describes small carbohydrates such as glucose, fructose, and sucrose. These sweet molecules are very important to all life. Cells break down these sugars to harness the energy stored in the covalent bonds linking their atoms. Sugars (in strict biochemical terms) are just one of the many types of carbohydrates your body uses, both for nutrition and as components of cellular structures. The smallest component (monomer) of carbohydrates (polymer) is a monosaccharide , which means “one sugar” (Figure 5.1). A sugar formed by linking two monosaccharides is a disaccharide (“two sugars”), and any molecule larger than two is a polysaccharide (“many sugars”). All sugars are a specific combination of three elements— carbon, hydrogen, and oxygen—whose atoms are always in the ratio of 1:2:1, or CH 2 O. The composition of carbohydrates gives this group of molecules its name: carbo- refers to the carbon; hydrate refers to water, which is made of oxygen and hydrogen. Glucose not only provides a quick source of energy but also can be fashioned into a polymer to provide structural support or short-term energy storage. Chitin and cellulose are both polysaccharides of glucose, found in the cell walls of fungi and plants, respectively. These polysaccharides are generally too difficult for most organisms to digest, which is what makes them such great structural elements to the cell walls of plants & fungi, so they are considered dietary fiber (passing through most digestive systems without being chemically altered). In plants, other glucose molecules are linked to form the energy storage polymer called starch . Animals use glycogen , another polymer of glucose, to store energy. Some large polysaccharides, such as chitin and cellulose, cannot be broken down in our digestive system, but they add insoluble fiber to our diet, promoting intestinal health. The body must use energy to break down other polysaccharides (such as starch) into smaller monosaccharides. These monosaccharides are easily and quickly absorbed to be used right away, or stored for later use. In this activity you will use two different tests to examine the presence and amount of monosaccharides in different substances and determine whether any of the substances contain the polysaccharide starch. Protocol 1.1: Testing for Monosaccharides Here you will detect the presence of monosaccharides in a variety of solutions by using a chemical called Benedict’s solution, which reacts with monosaccharides but not with disaccharides or polysaccharides. 1. Obtain a hot plate, test tubes, test tube rack, a beaker, transfer pipettes, a marker, Benedict’s solution, water, glucose solution, the unknown solution, and a variety of substances, as specified by your instructor. 2. Before you start, formulate a hypothesis indicating which substance you expect to have the greatest concentration of monosaccharides. Clearly record your hypothesis in your lab notebook, wherein you must also answer the following questions.Which substance is the positive control (the solution that should react with Benedict’s solution, indicating that the test works) and which substance is the negative 2

control (the solution that shouldn’t react with Benedict’s solution, indicating that the test is specific for monosaccharides)? Which solutions are you unsure of? 3. Fill the beaker halfway with water and place it on the hot plate. Set the temperature as directed by your instructor. 4. Prepare a series of test tubes. To the first test tube add 1 ml of distilled water, to the second test tube add 1 ml of the glucose solution, and to each successive tube add 1 ml of each additional substance. Label each test tube #1-6 with a sharpie and record the contents of each test tube in the second column of Table 1 to keep track of which number is which substance. For all qualitative tests (protocols 1.1, 1.2, 2.1, 3.1), use a transfer pipette NOT micropipette. 5. Get beaker to add a small amount of benedict’s solution to and use transfer pipette to add 5 ml of Benedict’s solution to each tube and mix, using Parafilm to cover the tube for inversion. Leave the Parafilm on the tube. What is the starting color of Benedict's solution? Record this in your observations section of your lab notebook. 6. Place the test tubes in the beaker of water. Boil the tubes for 3 minutes to allow Benedict's solution to react with the sugars. USE TUBE TONGS TO TRANSFER YOUR TEST TUBES BETWEEN THE BOILING BEAKER AND YOUR TEST TUBE RACK. 7. The color indicates the amount of monosaccharide product—with green representing the least and red representing the most. Construct Table 1 in your lab notebook, and under “Monosaccharide test results, rate your product on a scale of 0 to 4, depending on its final color: Blue = 0, Green = 1, Yellow = 2, Orange = 3, Red/Brown = 4. Data table 1: Test tube number Substance name Starting color Monosaccharide test result Starch test result 1 Water 2 10% Glucose solution 3 Unknown solution 4 5 6 8. Which fluid contains the highest amount of monosaccharides? Record in your “Results & analyses” section of your lab notebook. 9. Which fluid contains the lowest amount of monosaccharides? Record in your “Results and analyses” section of your lab notebook. 10. Which fluid would you drink for a source of quick energy? Note: NEVER drink or consume any solutions prepared in this lab. Record in your “Results and analyses” section of your lab notebook. 11. TURN OFF & UNPLUG THE HOTPLATE!!! 12. Dump test tube waste into the liquid waste beaker on your lab bench & put used test tubes in blue and white cardboard container for glass waste. Protocol 1.2: Testing for starch Now you will use an iodine solution to detect the presence of starch, a polysaccharide. 3

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1. Obtain iodine, a dropper, and the same types of substances provided by your instructor that your group used for the monosaccharides test. Using 6 new test tubes, prepare the test tubes the same way as step 4 (1mL of each substance to their respective test tubes) of the monosaccharides test. What color is the iodine? Record in your observations section of your lab notebook. 2. Before you start, formulate a hypothesis indicating which substance you expect to have the greatest concentration of starch. Clearly record your hypothesis in your lab notebook, wherein you must also answer the following questions. Which substance is the positive control (the solution that should react with iodine, indicating the presence of starch) and which substance is the negative control (the solution that shouldn’t react with iodine)? 3. For each substance, place one drop of iodine in the test tube. If the drop turns black, the test is positive for starch. Record the results of the test in your “Table 1” in your lab notebook, under “Starch test results.” Under “Starch test results”, indicate color of solution after addition of iodine (dark green/black = positive & orange/red = negative). 4. Which substances contain starch, a polysaccharide? Which substances contain monosaccharides? Note: you will record your results for monosaccharides AND starch in the same table in your lab notebook. 5. Dump test tube waste into the liquid waste beaker on your lab bench & out used test tubes in blue and white cardboard container for glass waste. 6. Do you think it makes any difference to your digestive system whether you ingest a monosaccharide or a polysaccharide? If so, postulate how? Record in your answers in the “Results and analyses” section of your lab notebook. Part 2: Lipids Lipids are unlike the other three types of macromolecules in that they are hydrophobic (repelling water), while the other macromolecules are hydrophilic (attracting water). This characteristic is crucial to the many functional roles of lipids in a cell, such as providing a structural barrier that separates the inside of the cell from the environment outside. Lipids are three different groups of molecules: fatty acids , phospholipids , and sterols . In this activity we will focus on fatty acids—primarily long chains of carbon atoms. These carbon chains are bonded to hydrogen atoms, creating molecules called hydrocarbons, which can be arranged in many patterns, such as rings and chains. A hydrocarbon chain binds to a carboxyl group to form a fatty acid. The type of fatty acid is determined by its length and the bonds within it, as illustrated in Figure 1. Fatty acids containing only single bonds between all of the carbon atoms are saturated fatty acids , which are straight hydrocarbon chains. This structural characteristic allows these molecules to pack together tightly, forming semi- solids or solids at room temperature. If a fatty acid contains any double bond between two or more carbon atoms in the hydrocarbon chain, the chain will bend and is then called unsaturated . The presence of one or more double bonds reduces the number of hydrogen atoms in the molecule. Unsaturated fatty acids are bent and cannot lie tightly together in a solution at room temperature. Unsaturated fatty acids are typically produced in plants; animals form saturated fatty acids. There are exceptions to both generalizations. Trans fatty acids , commonly referred to as “trans fats,” are a human invention—straight fatty acids created by the modification of unsaturated fats. In the nutritional labels on food products, the term “ partially hydrogenated ” indicates the presence of trans fats. Research shows that this form of fat is extremely unhealthy, increasing the risk of heart disease. 4

Figure 1. Common fatty acid chains (one component of many lipids) are depicted as both molecular space- filling and skeletal models, along with common foods which contain them. Note the differences in chemical structure between a saturated (a) and unsaturated (b) fatty acid chain. Protocol 2.1: Testing for lipids In humans, lipids are taken up and fashioned into triglycerides, compounds with three fatty acids bound to a glycerol molecule (Figure 2). Triglycerides are used as a long-term energy storage molecule in your fat cells. It is more efficient to store energy as fats instead of as carbohydrates or proteins because each gram of fat provides more than twice as much energy as these other molecules do. In this activity you will examine foods to determine whether they contain fats. Figure 2 . Constituent molecules in a triglyceride, represented in skeletal molecular structures and a space- filling module. 1. Obtain pipettes, a pencil, a test tube to use as a circular stencil, one filter (Whatman) paper, Sudan stain, water, vegetable oil, the unknown substance, and a variety of substances provided by your instructor. 2. On the Whatman paper, use the pencil to trace the outer edge of the opening of a test tube. Trace one circle for each of the substances. Label the circles #1-6 next to each circle for each substance that you 5

are instructed to test. Indicate which number correlates to which substance under “Name of substance” in Data Table 2. 3. Before you start the test, formulate a hypothesis indicating which substance you expect to have the greatest concentration of lipids. Clearly record your hypothesis in your lab notebook, wherein you must also answer the following questions. Which substance is the positive control (the solution that should appear oily and react with the Sudan stain, indicating that the test works) and which substance is the negative control (the solution that shouldn’t appear oily and react with the Sudan stain, indicating that the test is specific for fats). Which solutions are you unsure of? 4. Using a separate, clean pipette for each substance, apply one drop of the substance to the appropriate circle on the paper. Let the paper absorb the substance. Record the name of each substance in the second column of Table 2 in your lab notebook. 5. Wait 5 minutes for the spots to dry and then examine the spots on your paper. During this time, you may gently pick up the Whatman paper and move it through the air to encourage drying. Under the “Whatman results” column, indicate which ones look oily (appear shiny and don’t dry) like the positive control. Also Record the results for each substance—either positive or negative for lipids - in your lab notebook under “Whatman results” column of table 2. 6. Use Sudan stain to confirm your results. Using the same type of substances as the Whatman paper test, label and fill test tubes with 1 mL of each substance. Then, add 2-3 drops of Sudan stain to each tube and gently stir. Sudan stain turns deep red when a fat is present or remains pink if no fat is present. 7. Examine your positive and negative controls. What colors are they? Record these results in Table 2 in your lab notebook, under “Sudan stain test results.” 8. Do the Sudan stain test results corroborate the paper test results? Record in your “results and analyses” section of your lab notebook. 9. Placeused Whatman paper in your solid waste beaker, pour contents of the test tubes into your liquid waste beaker, and place used test tubes in blue and white cardboard container for glass waste. 10. Which substances, if any, differ in results between the two tests? What do you think causes these differences? Data table 2: Stenciled Filter Paper Name of substance Whatman results Sudan stain results 1 Water 2 Vegetable oil 3 Unknown solution 4 5 6 Part 3: Proteins Proteins are important macromolecules and have a range of diverse functions, such as providing structure, transporting other molecules, serving as enzymes and hormones, and enabling movement. The proteins found in the vast majority of organisms are composed of the same 20 amino acids, which are 6

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chemically linked together, one by one, sometimes forming very long chains or polymers. These chemical links are called peptide bonds , and sometimes proteins are also referred to as polypeptides . Each protein, or polypeptide, is marked by its unique arrangement (sequence) of these 20 amino acids. Different sequences of amino acids lead to different proteins. The number of possible proteins is endless: consider all the proteins which are 100 amino acids long. Applying rules from permutation mathematics to this example, in theory it is possible to envision 20,100 different proteins, each with a unique sequence of amino acids. Each of these 20 different types of amino acids has a central carbon-atom which bonds to 4 groups: a lone hydrogen-atom , a carboxyl group (-COOH), an amino group (-NH2) and a side chain (often denoted generally as the “R” group). Only this side chain is different in each of the 20 amino acids, determining their different properties (e.g. solubility, acidity, charge). Check out the list of amino acids, their structures, and their abbreviations in your Urry et al. 2021 Campbell Biology textbook (figure 5.14 on page 77). The linear sequence of amino acids is called the primary structure of the protein, and it simply lists the order of the amino acids in the protein. Hydrogen bonds then form between the amino and carboxyl groups of the different amino acids giving the protein its secondary structure - either an α-helix or a β-pleated sheet. In addition, interactions and bonds form between the side chains of the amino acids that fold the protein into its final three-dimensional shape or tertiary structure . Important to the tertiary structure are a variety of features, such as the presence of disulfide bonds, the arrangement of hydrophobic and hydrophilic side chains, and the incidence of ionic bonds. In some proteins there is an even higher level or organization called a quaternary structure . This structure results from the bonding of 2 or more polypeptides, essentially forming a “super- protein”, e.g. hemoglobin. Note, this means fully functional proteins consisting of only one polypeptide DO NOT HAVE quaternary structure. Please note that a major, recurring theme in biology is that structure determines function. Proteins are a perfect example of this theme at the molecular level. While the linear, primary structure is important to study and to understand, it is ultimately the three-dimensional structure, i.e. the shape of the proteins, which determines their functions. Protocol 3.1: Qualitative test for proteins 1. Here you will determine whether proteins are present in various different substances. Obtain test tubes, a marker, Parafilm, Biuret solution, water, egg white solution, unknown solution, and a variety of substances provided by your instructor. 2. Before you start, formulate a hypothesis indicating which substance you expect to have the greatest concentration of proteins. Clearly record your hypothesis in your lab notebook, wherein you must also answer the following questions. Which substance is the positive control (the solution that should react with Biuret solution, indicating the presence of protein), and which substance is the negative control (the solution that should not react with Biuret solution)? 3. Label one test tube for each substance you are instructed to test. Record the name of each substance in the first column of Table 3 in your lab notebook. 4. To each test tube, add 1 ml of the appropriate substance using a transfer pipette. 5. What is the starting color of the Biuret solution? Record in your observations section of your lab notebook. 6. Add 2 ml of Biuret solution to each test tube. Cover the tube with Parafilm and immediately invert with thumb also over the opening to mix the contents, then set in the rack upright. 7. If protein is present, the Biuret solution changes to a deep violet color. For each test tube, record any color change in Table 3 in your lab notebook, under “Results.” 8. Which substances contain proteins? 9. Dump test tube waste into the liquid waste beaker on your lab bench & out used test tubes in blue and white cardboard container for glass waste. 10. Which substance would you consume to get the greatest amount of protein? The least amount? Record in your “results and analyses” section of your lab notebook. Table 3. Qualitative test for protein presence 7

Test tube number Name of substance Biuret test results 1 Water 2 10% Egg white solution 3 Unknown solution 4 5 6 Wrapping up: Now that you have tested the unknown solution for several different types of macromolecules, record the types of macromolecules you are certain the solution contains in your “Results & analyses” section of your lab notebook. Do you have any guesses as to the identity of the substance? What types of macromolecules might be in the sample that our tests could not detect? Do you think the substance would make a decent meal for a person seeking to maximize their nutritional intake? Why or why not? Part 4: Cleaning up 1. Pour all liquid waste into the labeled liquid waste containers on your lab bench. If full, alert your lab instructor. 2. Discard all used plastic tubes, soiled parafilm, and micropipette tips into the labeled waste containers on your lab bench. Once full, dump that container into the classroom biohazard bin (red bin). Do so at the end of class regardless. 3. Wash all glassware in the sink with hot water, and Alconox detergent. Scrub well with appropriate implements (brush, sponge, etc…). Rinse thoroughly with hot water, and hand dry with paper towels. 4. Return all washed and dried glassware to your lab bench for the next section. 5. Spray bench tops with 70% alcohol solutions, wash, and dry with paper towels. 6. Once your lab space is completely clean (all student benches and surrounding areas), remove your PPE and wash your hands in the sink with soap and water before exiting the lab space. 8

Module 3_ Macromolecules_ Fall_2023

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