Lab 4 - Macromolecules - updated 2_20_2023 (2)

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Texas A&M University, San Antonio *

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Apr 3, 2024

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Macromolecules: Carbs, Proteins, and Lipids – Good Eats Badge (Adapted from Biology Laboratory Manual 10 th Edition, by Darrell Vodopich and Randy Moore) Most organic compounds in living organisms are carbohydrates, proteins, lipids, or nucleic acids . Each of these macromolecules is a polymer made of smaller subunits, or monomers . These monomers are linked together in a reaction known as dehydration synthesis , which is an energy-requiring process in which a molecule of water is removed and the two subunits are bonded covalently. We can break the bonds between the subunits to release energy and separate the monomers gain by adding a water molecule in a process known as hydrolysis . Scientists have devised several biochemical tests to identify the major types of organic compounds in living organisms. Each of these tests involves two or more treatments: (1) an unknown solution to be identified, and (2) controls to provide standards for comparison. As its name implies, an unknown solution may or may not contain the substance that the investigator is trying to detect. Only a carefully conducted experiment will reveal its contents. In contrast, controls are known solutions. We use controls to validate that our procedure is detecting what we expect it to detect and nothing more. During the experiment, we compare the unknown solutions’ response to the experimental procedure with the control’s response to that same procedure. In our scientific method lab, we introduced the concept of a control group : a group that is subjected to the same conditions as the experimental group EXCEPT for the independent variable. This was to show that any differences observed were due to the independent variable alone and not to other factors within the procedure. But what if there were no differences detected between the groups? Could you be sure that it was due to the independent variable and not that there was a problem with the experimental procedure? A way to be sure is to introduce a new kind of control group: the positive control . A positive control contains the variable for which you are testing and should react in a predictable way in the procedure. For example, if you are testing a solution to see if it contains protein, an appropriate positive control is a solution that is known to contain protein and there should result in a positive reaction in your test. A positive reaction indicates that your test is working as expected and shows you what a positive result should look like. You can then compare the unknown and other samples with this known positive to check for the presence of the molecule. 1

Even with a positive control we still need to show that any positive reaction is due only to the variable that we are testing. Therefore, we still need the negative control. The negative control does not contain the variable for which you are searching. It contains only the solvent (often distilled water with no solute) and should not react in the test. A negative control shows you what a negative result looks like. In this lab, we are going to use chemical tests to detect macromolecules in known and unknown solutions. We will employ both negative and positive controls to be sure that our tests are working properly and that we can trust our results. You will be working in groups of 4 students (your table) to be “food detectives” and solve the mystery of your unknown solution. Experimental Procedures Carbohydrates: Carbohydrates are made up of monosaccharides. Many monosaccharides such as glucose and fructose are reducing sugars , meaning that they possess chemical groups that can reduce weak oxidizing agents like the copper in Benedict’s reagent. Benedict’s test identifies reducing sugars based on their ability to reduce the cupric ions (blue) to cuprous oxide (green to reddish orange) at high pH. A green solution indicates a small amount of reducing sugars, and reddish orange indicates an abundance of reducing sugars. Non-reducing sugars like disaccharides (sucrose) are not able produce a color change. Starch is a form of carbohydrates produced by plants. It is a highly coiled polymer of individual glucose units. Iodine interacts with only coiled polymer molecules of glucose (like starch) and becomes bluish black. Iodine does not interact with uncoiled sugars such as disaccharides (sucrose) and monosaccharides (glucose) and therefore remains yellowish brown in the presence of these sugars. In summary – what color changes are we looking for with each test? Color Changes Positive Result Negative Result Benedict’s Test Iodine Test For the Carbohydrate tests: 1. Create a water bath using the hot plate and a 250 mL beaker. a. Fill the beaker about half full with water and set the hot plate to 150°C. If you are waiting for the water to boil, you can increase the temperature. However, once the water is boiling, turn it back down between 120°- 150°C. 2

b. Do NOT let the beaker go dry. If the water boils off while you are preparing your tubes, add more water to the beaker. 2. Obtain 6 test tubes. Label one set of 3 (Label “1”, “2”, “3”) for the Benedict’s test and one set of 3 for the iodine test. 3. Add to each tube the materials to be tested (the quantities of these materials are listed in Table 5.1 below). For example, add 1 mL of distilled water to BOTH tubes that are labeled “1”. Benedict’s test for reducing sugars 1. For the first set of 3 tubes, add 2 mL of the blue Benedict’s solution to each tube. 2. Place these test tubes into a gently boiling water bath for 3 minutes and observe color changes at this time. 3. After 3 minutes, use a test tube holder to remove the tubes from the water bath. After giving the tubes ample time to cool to room temperature, record the color of their contents in Table 5.1. 4. Keep the contents of these tubes for comparison later in the experiment. 5. Turn off your hot plate and leave hot plate and beaker to cool . Iodine Test for Starch 1. Use the second set of test tubes prepared at the beginning of the procedure. The tubes should already contain the solutions you are going to test. 2. Add 3-6 drops of iodine into each of the tubes and swirl to mix. 3. Record the color of the tubes’ contents in Table 5.1. 4. Keep the contents of these tubes for comparison later in the experiment. Table 5.1: Solutions and Color Reactions for Carbohydrate Tests Tube Solution Benedict’s Color Reaction Iodine Color Reaction 1 1 mL distilled water 2 1 mL reducing sugar solution 3 1 mL starch solution For the Benedict’s test, what tube # contained the positive control? ________________ For the Benedict’s test, what tube # contained the negative control? _______________ For the Iodine test, what tube # contained the positive control? ____________________ For the Iodine test, what tube # contained the negative control? ___________________ Proteins Proteins are polymers made up of amino acids. A peptide bond forms between the amino acid and the carboxyl group of an adjacent amino acid and is identified by a Biuret test. Specifically, peptide bonds in proteins complex with Cu 2+ in Biuret reagent and produce a violet color. A Cu 2+ must complex with four to six peptide bonds to 3

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produce a color; therefore individual amino acids to not react positively. Proteins have many peptide bonds and produce a positive reaction. Biuret Test for Proteins 1. Obtain three test tubes and number them 1-3. 2. Add the materials listed in Table 5.2. 3. Add 2 mL of 2.5% sodium hydroxide to each tube ****Be careful – NaOH is extremely caustic!!!**** 4. Add three drops of Biuret reagent to each tube and mix. 5. Record the color of the tubes in Table 5.2. 6. Keep the contents of these tubes for comparison later in the experiment. Table 5.2: Solutions and Color Reactions for Biuret Test Tube Solution Biuret Color Reaction 1 2 mL amino acid solution 2 2 mL distilled water 3 2 mL protein solution Which of the solutions is the positive control? ______________________________ Which of the solutions is a negative control? _______________________________ Did the free amino acid solution test postive? ______________________________ Why or why not? _______________________________________________ Lipids Lipids include a variety of molecules that dissolve in non-polar solvents such as ether, acetone, methanol, or ethanol, but not as well in polar solvents such as water. Triglycerides are abundant lipids made of glycerol and three fatty acids. Tests for lipids are based on a lipid’s ability to selectively absorb pigments in fat-soluble dyes such as a red dye, called Sudan IV. If a test is positive for lipids, there will be a red splotch left when testing solutions on a Sudan IV soaked filter paper. We will be conducting two different tests today looking at solubility and the Sudan IV test. Solubility Test for Lipids 1. Obtain two test tubes. To one of the tubes, add 5 mL water. To the other tube add 5 mL acetone. 2. Add a few drops of vegetable oil to each tube and observe the solubility. Which one is vegetable oil soluble in? ____________________ Why? ______________________________________________________________ 4

Sudan IV Test for Lipids **USE PICTURE BELOW** 1. Look at the picture below and answer the questions based on the results of this Sudan IV test: Did the oil leave any residue? _______________________ If so, what color was it? _______________________ Did the water leave any residue? _________________________ If so, what color was it? _______________________ If there was residue – what does this mean? ____________________________ Identifying Different Milks Based on Macromolecule Content You have successfully used different assays to test for carbohydrates, lipids, and proteins in different solutions. Now you get to play the role of a food scientist who is looking for these macromolecules in various milk samples that have been mixed up! You are given samples of each milk type: Whole Milk , Skim Milk , Soy Milk , and Cream , but these samples have been dropped on the floor and are missing their labels! 5 Milk D Milk C Milk A Milk B

Based on what you know about these milk types – the cartons are available for you to peruse ingredient labels, look at nutrition facts, etc. – and the macromolecule assays, you should be able to determine which milk is which. 1. What macromolecules do you expect in each type of milk? a. Whole Milk b. Skim Milk c. Soy Milk d. Cream 2. Think back to the macromolecule assays you tested in the first part of today’s lab. Which assays would be most beneficial for determining the differences between the different milks? Why? 3. Formulate hypotheses that summarize your predictions about how each substance will behave in the assays. 6

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4. Write out which tests you will perform and what order you will perform them. Do you need to perform all of them? Why or why not? What positive and negative controls will you use? Use the image above for the Sudan IV test results. 5. Once you have your experimental design complete, run the assays on your 4 milk samples following the procedures from the first part of the lab. Make sure to include your positive and negative controls! Record your data below . It might be helpful to draw a table or some other visual to record your data. 7

6. Write a paragraph to summarize and interpret your results . Identify which type of milk was in tubes A, B, C, and D and how you determined these identifications. Were your hypotheses supported? Why or why not? Finally – clean up your area. Any solutions in glass tubes will be disposed of in the “Waste” bottles by the sinks. The ethanol/oil solutions can be washed down the drain. Any Sudan IV solution filter papers can be tossed in the trash can. Please wash all of your tubes and return them to your table flipped upside down in your test tube rack. 8

Lab 4 - Macromolecules - updated 2_20_2023 (2)

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