Lab #4: Nutrition & Assays for Detecting Biological Molecules - Introduction

Lab #4: Nutrition & Assays for Detecting Biological Molecules - Introduction Most biological molecules fall into one of four varieties: proteins, carbohydrates, lipids and nucleic acids. These are sometimes called macronutrients - you have probably seen reference to the first three of these on your food labels (lipids are often referred to as fats, and there are several kinds of fats). In today s lab we will use different colorimetric assays to determine the presence or absence of various biomolecules. All four biomolecules are necessary components of our diet, but some are needed more than others. The table below summarizes a health balance of the three primary biomolecules in food. Table 4.1. Recommended Macronutrient Distribution in a Healthy Diet Carbohydrates 45 65% of total calories Lipids (fats and oils) 20 35% of total calories Protein 10 35% of total calories Take a look at the food labels on things you are eating and compare it to the one below. Note that some food labels are small and don t contain the last footnote section. Imagine the food label for something you are eating shows that it contains 3 grams of fiber and that this is 12% of your daily requirement, but it is missing the footnote that shows how much fiber you need each day. You can calculate the total amount of fiber you need per day (X) using the ratio below. Solve for X. 3g = 12%. X g 100 % X = grams of fiber needed per day. hint: remember cross multiply and divide. Is the % daily value recommended for fiber based on a 2000 calorie diet or a 2500 calorie diet? (circle one) Most of us look at food labels because we are interested in the amount of calories in the food we eat. You may think of calories as an indication of how much weight you might gain if you eat the food, but calorie is actually a measure of how much energy is in a chemical. More specifically, a calorie is defined as the amount of energy needed to raise 1 gram of water 1ºC. One dietary Calorie (written as Cal) is equal to 1000 calories (one kilocalorie). Sometimes you may wish to calculate for yourself the amount of dietary Calories in a food given the number of grams of various biomolecules. To do so, you need to remember a few simple facts, shown in the table below. Table 4.2. Dietary Calories per gram of each biomolecule Carbohydrates 1 gram yields 4 Cal Lipids (fats and oils) 1 gram yields 9 Cal Protein 1 gram yields 4 Cal 1

Imagine you get the munchies and eat an entire container of crackers (food label shown below). Use the table 4.2 to calculate the total number of Calories you consumed from each of the three main macronutrients. grams of Total Fat in the entire container. x Calories per gram of Fat = Total Calories from Fat in the entire container. grams of Total Carbohydrates in the entire container. x Calories per gram of Carbohydrates = Total Calories from Carbohydrates in the entire container. grams of Total Protein in the entire container. x Calories per gram of Protein = Total Calories from Protein in the entire container. Add up the Calories from Fat, Carbohydrates and Protein: You may notice the total number of Calories you calculate does not match the label. This is because the Food and Drug Agency (FDA, the federal agency that regulates food labels) requires that food manufacturers round calories to the nearest 5-Calorie (if the food is less than 50 Calories) or the nearest 10-Calorie (if the food has more than 50 Calories). Does this food meets the recommended distribution of calories (Use table 4-1)? yes or no. Examine the bottom of the label. If you consume 2000 Calories per day, how many grams of the following do you require? Carbohydrates: Fat: In addition to Calories, people are very interested in the amount of protein. This is for good reason, as protein is used for building and repairing body tissue, red blood cells, hair, and enzymes (among other things). The amount of protein an individual needs therefore depends on how much tissue they are repairing or building. In order to estimate how much protein you need per day, multiply your body weight by the factor below: Sedentary adult 0.4 x pounds = grams of protein Active adult 0.6 x pounds = grams of protein Growing athlete 0.7 x pounds = grams of protein Adult building muscle mass 0.9 x pounds = grams of protein 2

Qualitative identification of biological molecules Each type of biomolecules has unique chemical properties, and thus can be identified by various chemical tests. The tests involve the use of a chemical called a reagent that reacts with a specific molecule and changes color. If the specific target organic molecule is not present, the reagent will not change color. We will first conduct the various tests using solutions with known contents to familiarize ourselves with how each reagent responds to different compounds. We will also perform each test with distilled water (which serves as a negative control because it contains no organic molecules). When water is mixed with different reagents it turns the color of the reagent, not the color of a positive result. The water control is subjected to the same experimental conditions and treatments that are used on the other substances that are tested. This negative reaction with water is necessary to show that the only color change that occurs is caused by the presence of a specific organic molecule and not by some other variable (e.g. heat). We will then use these tests to determine the constituents in several mystery solutions. Our tests will be qualitative we will determine only if a particular type of biomolecule is present or absent, but not the amount of each biomolecule. If we determined the amount of each compound, then it would be considered a quantitative test. SAFETY NOTE: The chemicals used in this lab should be handled with care because they are hazardous to your health. Be sure to clean your lab area and wash your test tubes between tests and before you leave the lab. Part 1. Practice identifying unique features of the biomolecules shown on page 7 using the textbook (or internet). Part 2A. CARBOHYDRATES - Lugol s Iodine test for starch The Iodine test is used to test for the presence of starch. Iodine solution (I 2KI) known as Lugol s solution contains iodine dissolved in an aqueous solution of potassium iodide. This solution reacts with starch producing a deep blue-black color. Recall that starch is a combination of two polysaccharides, amylose and amylopectin. The amylose forms helices where iodine molecules assemble, forming a dark blue/black color. The reaction is the result of the formation of polyiodide chains. Variation in the intensity of the blue/black color is related to the concentration of starch present in the solution. SAFETY NOTE: Iodine stains clothing and skin; some individuals may have allergic reactions. Procedure for Lugol s Iodine test for starch: 1. Label test tubes as shown in Table 2A. 2. Place 2 ml of the substance indicated in Table 2A. 3. Add 2 drops of concentrated Lugol s iodine solution to each test tube and mix thoroughly. 4. Record the color of the tubes in Table 2A. If starch is present, the iodine will react with the strach to create a dark purple/black colored solution. 3

Part 2B. CARBOHYDRATES - Benedict s test for reducing sugars Many simple sugars (monosaccharides and disaccharides) can be detected using a solution of copper ions because the sugars contain an aldehyde (or they can isomerize to form an aldehyde). We will use this method (called Benedict s test) to determine if a solution contains reducing sugar. Benedict s test is explained below. A carbon atom double bonded to an oxygen atom is called a carbonyl functional group. Carbonyl groups are important because they enable the carbon to react in interesting ways but the reactivity of the carbon depends on what is next to the carbonyl group. If the carbonyl group has a carbon (shown as R ) on one side and a hydrogen on the other, it is called an aldehyde, but if it has a carbon on both sides it is a ketone. Sugars containing an aldehyde are called reducing sugars because they can release electrons to water (forming a carboxylic acid, H 3O + and free electrons). Lobry de Bruyn van Ekenstein transformation R-CHO + 3 H 2O R-COOH + 2 H 3O + + 2e - In a basic solution (ph>7) containing dissolved copper ions (cupric ions, Cu ++, a blue solution known as Benedicts reagent), electrons from reducing sugars can react with OH - and cupric ions to form cuprous ions, Cu +, which then form a copper oxide (CuO) which is a solid precipitate that is either yellow, orange, red, green or brown (the color depends on how strongly the Cu ++ been reduced, not which sugar is present). In the presence of base, some sugars can isomerize (change shape). This is shown to the right, and is called the Lobry de Bruyn van Ekenstein transformation. Which of the sugars shown above contains an aldehyde group? Note: carbon atoms are not always shown, but occur where lines intersect. Which of the sugars above does not contain a reducing sugar, but in the presence of OH - will convert into a sugar that will react with Benedict s reagent? SAFETY NOTE: Benedict s reagents contain copper, which can be toxic in high concentration. Please minimize contact with skin. Be sure to thoroughly wash your hands if any reagent spills on your skin. Dispose of this reagent by pouring it into the hazardous waste container provided in the laboratory. Procedure for Benedict s Test for reducing sugars: 1. Label test tubes as shown in Table 2B. 2. Place 2 ml of the substances indicated in Table 2B. 3. Add an equal amount of Benedict s solution (2 ml) to each test tube and mix thoroughly. 4. Place the test tubes in a hot-water bath for 10 minutes. 5. Allow the tubes to cool and then examine the tubes for changes in color record your results in Table 2B. A reducing sugar will convert the Benedict solution from blue to green, orange or red (depending on the strength of the reaction). 4

Part 2C. LIPIDS (FATS AND OILS) brown paper bag test for hydrophobic molecules Lipids are a class of molecules whose only distinguishing feature is that they are mostly non-polar. They include fats (triglycerides), steroids, and phospholipids but this exercise focuses on fats, also called oils, which contain one glycerol molecule attached to three fatty acids (see figure in Part 1; glycerol is green and fatty acids are red). Unsaturated fats are primarily found in plants and are liquid at room temperature, and are often called oils. Saturated fats are solid at room temperature and are found in animals as well as in some plant fats such as coconut and palm oil. In this exercise you will test for the presence of fats using a simple test that involves applying a test substance to brown paper. If the substance is nonpolar it will penetrate the fibers of the paper and produce a translucent or greasy spot. Procedure for the Paper Test for Lipids: 1. Draw dime-sized circles on pieces of unglazed brown paper and number them as shown in Table 2C. 2. Apply a small drop of each of the substances listed in Table 2C to the paper. After a few seconds, shake the paper to remove the droplet. 3. Record your results in the Table 2C. If a lipid is present, there will be a greasy, translucent spot. Part 2D. PROTEINS - Biuret s Test for Proteins (peptide bonds) Proteins contain a linear sequence of amino acids linked together by peptide bonds (sometimes a protein is called a polypeptide). The name amino acid comes from the fact that each of monomer contains a basic amino group and an acidic carboxyl group. The Biuret test identifies the presence of a peptide bond, and hence indicates the presence of a protein. The peptide bond between amino acids can be detected by using a Biuret test. Biuret s reagent is prepared from urea and contains peptide bonds, as well as copper sulfate in an alkaline solution. When mixed with a polypeptide, a purple color develops. The reaction is based on the formation of a purple-colored complex between copper ions and two or more peptide bonds. Proteins give a strong biuret reaction because they contain large numbers of peptide bonds. The intensity of the color can be used to estimate the quantity of protein present. SAFETY NOTE: Use caution when handling biuret reagent, which is strongly alkaline. Dispose of this reagent by pouring it into the hazardous waste container. Procedure for Biuret s test for peptide bonds (in proteins): 1. Label test tubes as shown in Table 2D. 2. Place 2 ml of the substance indicated in Table 2D. 3. Add 1 ml (~10 drops) of Biuret s solution to each test tube and mix thoroughly. 4. Allow 5 minutes for the color to develop. 5. Record the color of the tubes in Table 2D. If a protein is present, the solution will appear purple. Part 3. FINALE - Analysis of Unknown solutions. Procedure: 1. Obtain one unknown solution per student from your instructor. 2. Determine what each unknown solution contains by individually repeating each of the tests for biomolecules: a. Lugol s Iodine Test for starch b. Benedicts Test for reducing sugars c. Paper Test for lipids d. Biuret s Test for proteins. 3. Record the results of each test in Table 3. 5

What is the FDA and how is it related to nutrition labels? Review Questions True or False: True or False: Today s lab exercise uses two reagents (Benedict s and Biuret s) that contain copper. Solutions that contains copper are toxic at high concentrations and should not be discarded down the drain unless you spill it on your skin. Provide an example (ex) of a suitable positive control and negative control for each of the following tests. Benedict s test Lugol s iodine Biuret s test Negative control Ex: Water Positive control Ex: starch Complete the following table: Biomolecule tested for Negative result (result if biomolecule is absent) Positive result (result if biomolecule is present) Benedicts test Ex: Reducing sugar Ex: orange Lugol s iodine paper test Ex: did not absorb, no greasy spot Ex: absorbed, greasy spot Biuret s test 6

Lab #4: Nutrition & Assays for Detecting Biological Molecules team member names Group score (part 1 and part 2) Individual score (unknown: part 3) Total score Part 1 Use the word bank to the right to label the structures below: Glucose Fructose Sucrose Maltose Starch (amylose) Polypeptide Oil (triglyceride) Proteins are made of what kind of subunit? What is the name of the covalent bond connecting these two subunits? (it is unique to proteins) Examine the structure of starch: Does it have any polar (O H) bonds? Yes or no Will it hydrogen bond with water (H O H)? Yes or no Is it hydrophilic? Yes or no Examine the structure of the triglyceride (oil): Does it have any polar (O H) bonds? Yes or no Will it hydrogen bond with water (H O H)? Yes or no Is it hydrophilic? Yes or no 7

Tube # 2A. Lugol s Iodine Test for Starch substance Result (color) 1 Glucose solution 2 Fructose solution 3 Banana juice 4 Salad oil 5 cornstarch 6 Egg albumin 7 Distilled water Tube # 2B. Benedicts test for Reducing Sugars substance Result (color) 8 Glucose solution 9 Fructose solution 10 Banana juice 11 Salad oil 12 cornstarch 13 Egg albumin 14 Distilled water Part 2 paper # 2C. Spot Test for Lipids substance Glucose solution bag #1 Fructose solution bag #2 Banana juice bag #3 Salad oil bag #4 bag #5 cornstarch Egg albumin bag #6 Distilled water bag #7 Tube # Result (absorb or not absorb?) 2D. Biuret Test for Proteins substance Result (color) 15 Glucose solution 16 Fructose solution 17 Banana juice 18 Salad oil 19 cornstarch 20 Egg albumin 21 Distilled water Unknown #. Test Unknown Tube A Lugol s Iodine Test for Starch Unknown Tube B Benedict s Test for Reducing Sugars Unknown Bag C Spot Test for Lipids Part 3 Result (what is the color? or did it absorb into the brown paper?) Unknown Tube D Biuret Test for Proteins Your unknown solution could contain which of the following (circle all that apply): Glucose salad oil egg albumin cornstarch water What should you do with your test tubes before leaving lab? (Hint you will lose half your points if your test tubes have orange coloring on the bottom) 8