Bio 3A Lab: Biologically Important Molecules Procedures Page 1 of 7 Figure 1. Dehydration synthesis and hydrolysis of a polymer. BIO 3A LABORATORY Biologically Important Molecules Carbohydrates, Lipids, Proteins and Nucleic Acids Objectives • To perform tests that detect the presence of carbohydrates, lipids, proteins, and nucleic acids in known and unknown samples • To recognize the importance of a control in a biochemical test • To use biochemical tests to identify an unknown compound Introduction Organic molecules are those primarily made up of carbon, hydrogen and oxygen. The common organic compounds of living organisms are carbohydrates, proteins, lipids, and nucleic acids. Each of these macromolecules (polymers) are made of smaller subunits (monomers). The bonds between these subunits are formed by dehydration synthesis. This process requires energy; a molecule of water is removed (dehydration) and a covalent bond is formed between the subunits (Fig.1). Breaking this bond is called hydrolysis; it requires the addition of a water molecule and releases energy. Each class of these macromolecules has different structures and properties. For example, lipids (composed of fatty acids) have many C-H bonds and relatively little oxygen, while proteins (composed of amino acids) have amino groups (-NH3+) and carboxyl (-COOH) groups. These characteristic subunits and chemical groups impart different properties to the macromolecules. For example, monosaccharides such as glucose are polar and soluble in water, whereas lipids are non-polar and insoluble in water. Methods for Identifying Organic Compounds There are several chemical tests available for the identification of the major types of organic compounds in living organisms. Typically these tests are used to determine the makeup of an unknown material. For instance, a forensic detective may be interested identifying a fluid found at a crime scene. The collected fluid is the unknown. As the tests are carried out, the detective will also use known compounds, or controls, for comparison. During the experiment the detective compares the unknown's response to the experimental procedure with the control's response to that same procedure. Bio 3A Lab: Biologically Important Molecules Procedures Page 2 of 7 Controls are important because they reveal the specificity of a particular test. For example, if water and a glucose solution react similarly in a particular test, the test cannot distinguish water from glucose. But if the glucose solution reacts differently from distilled water, the test can distinguish water from glucose. In this instance, the distilled water is a negative control for the test, and a known glucose solution is a positive control. A positive control contains the variable for which you are testing. It produces a positive reaction and demonstrates the test's ability to detect what you expect. A positive reaction to a positive control demonstrates that your test reacts correctly. A negative control does not contain the variable for which you are searching. It contains only the solvent (often distilled water with no solute) and does not react in the test. A negative control demonstrates what a negative result looks like. There are literally hundreds of tests available for biological molecules. In today’s lab we will be using some of the more common methods to look for the presence of simple (or reducing sugars), starch, protein, lipid, and DNA. Testing For Carbohydrates: Simple Sugars Carbohydrates are molecules made up of carbon (C), hydrogen (H), and oxygen (O) in a ratio of 1:2:1 (for example, the chemical formula for glucose is C6H12O6). Carbohydrates may be monosaccharides, or simple sugars, such as glucose or fructose; they may be disaccharides, paired monosaccharides, such as sucrose (a disaccharide of glucose linked to fructose, Fig. 2); or they may be polysaccharides, where three or more monosaccharides form chains, such as starch, glycogen, or cellulose (Fig. 3). As already mentioned, the linkage of subunits in carbohydrates, as well as other macromolecules, involves the removal of a water molecule (dehydration). Figure 4 depicts how dehydration synthesis is used to make maltose and sucrose, two common disaccharides. Many monosaccharides such as glucose and fructose are reducing sugars, meaning that they possess free aldehyde (-CHO) or ketone (-C=0) groups that reduce weak oxidizing agents such as the copper in Benedict's reagent. Benedict's reagent contains cupric (copper) ion complexes with citrate in alkaline solution. Benedict's test identifies reducing sugars based on their ability to reduce the cupric (Cu2+) ions to cuprous oxide at basic (high) pH. Cuprous oxide is green to reddish orange. In this test, a green solution indicates a small amount of reducing sugars, and reddish orange Figure 2. Glucose, a monosaccharide and Sucrose, a disaccharide. Figure 3. Polymers of glucose: starch and celluloseBio 3A Lab: Biologically Important Molecules Procedures Page 3 of 7 indicates an abundance of reducing sugars. Non-reducing sugars, such as sucrose produce no change in color (the solution remains blue). Procedure 1. Benedict's test for reducing sugars 1. Obtain seven test tubes and number them 1-7. 2. Add to each tube the materials to be tested (see Table 1 ). Your instructor may ask you to test some additional materials. If so, include additional numbered test tubes. Add 2 ml of Benedict's solution to each tube and mix. 3. Place all of the tubes in a boiling water-bath for three minutes and observe color changes during this time. 4. After 3 minutes, remove the tubes from the water-bath and let them cool to room temperature. Record the color of each test tube in Table 1. Starches Staining by iodine (iodine-potassium iodide, I2KI) distinguishes starch from monosaccharides, disaccharides, and other