1 GENERAL BIOLOGY LAB 1 (BSC1010L) Lab #4: Biologically Important Molecules ______________________________________________________________________________ OBJECTIVES: • Learn the basic structure of the four types of biological molecules and the importance of each type. • Understand the concepts of hydrolysis and dehydration synthesis. • Become familiar with the tests used to identify macromolecules. o Nucleic acids will be covered in the Molecular Biology lab (Lab #9) • Recognize the difference between a negative and a positive control, and the purpose of each type in experimental design. ______________________________________________________________________________ INTRODUCTION: Biological organisms utilize four major classes of macromolecules: carbohydrates, proteins, lipids and nucleic acids. These organic compounds are important for proper cellular functioning and each plays a different role within the cell. Carbohydrates provide the primary source of energy or “fuel” for cells and are used to support cell walls of bacteria, fungi and plants. Proteins function as “building blocks” or structural elements within the cell and aid in the transport of molecules across membranes (i.e. transmembrane proteins). Proteins also regulate cellular activities (enzymes and hormones) and are important components of the immune system (antibodies). Lipids are not only used for “storage” of excess fuel but are an integral part of cell membranes. Finally, nucleic acids (DNA and RNA), the “information center” of the cell, comprise our genes, regulate cell function, and are involved in energy transfer. In addition, both DNA and RNA participate in cellular replication. Many biological molecules are polymers comprised of smaller subunits (monomers) held together by covalent bonds. Carbohydrates, for example, are composed of varying combinations of monosaccharides (e.g. glucose, ribose, deoxyribose) that can be joined to form disaccharides (e.g. sucrose, maltose) and polysaccharides (e.g. starch, glycogen, cellulose, chitin). Similarly, proteins are made from unique combinations of amino acids. Monomers are linked together by dehydration synthesis (condensation) reactions in which a water molecule is removed, covalently bonding the two subunits (Fig. 1a). Conversely, the bond between monomers can be broken by the addition of a water molecule, a process referred to as hydrolysis (Fig. 1b).2 Figure 1. Dehydration synthesis and hydrolysis reactions Although all macromolecules are characterized by the presence of a carbon backbone, the four classes vary in their elemental structure and thus, their chemical properties (Fig. 2). The disparate functional groups impart different solubilities and polarities to each type of macromolecule. For instance, lipids, which are made of fatty acids and have very little oxygen, are nonpolar and insoluble in water (i.e., hydrophobic). Proteins, on the other hand, are polymers of amino acids covalently linked by peptide bonds. Because amino acids are polar, non-polar, charged or aromatic, the properties of the resulting proteins vary in accordance with the type of amino acids that comprise them. In today’s lab, you will perform a battery of biochemical tests to detect the presence of different organic molecules in known substances. Throughout this process, you will learn about the use of controls as standards for comparison and their role in identifying unknown solutions. Controls are an essential component of every experiment because they help eliminate alternate explanations of experimental results. In general, a control is defined as any variable that is kept constant throughout the entire experiment and is compared to the experimental sample(s) being tested. There are two types of controls: negative and positive. A negative control helps minimize false positives by providing a known negative result for a given experimental treatment. Thus, a negative control provides an example of what the results should look like if the experimental manipulation had no effect on the variable of interest. A positive control, on the other hand, helps minimize false negatives by demonstrating what a positive result should look like if the3 experimental manipulation produces a change. For example, when testing for the presence of salt in a substance, a salt solution would serve as the appropriate positive control and distilled water as the negative control. By applying your knowledge about basic macromolecule structure and using the proper controls you should be able to identify any unknown compound. Figure 2. Basic structures and functional groups of macromolecules4 Task 1: CARBOHYDRATES Carbohydrates are molecules made of simple sugars with Carbon (C), Hydrogen (H) and Oxygen (O) in a ratio of 1:2:1. Monosaccharides (Fig. 2A) are made of single sugar molecules while disaccharides (Fig. 2B) and polysaccharides (Fig. 2C) are composed of two or more sugar molecules, respectively. Figure 3. Carbohydrate Molecules Monosaccharides contain either aldehyde (-CHO) or ketone (-C=O) side groups that reduce oxidizing compounds. A molecule is oxidized if it loses an electron or hydrogen atom and is reduced when it gains an electron or hydrogen atom (Fig. 4). Collectively, the two processes are referred to as a redox reaction because when one molecule is oxidized, another is reduced. Figure 4. Redox reactions C A B5 I. Examine Reducing Sugars Benedict’s reagent can be used to identify the presence of reducing sugars and is therefore a good indicator for the presence of some carbohydrates. At basic/alkaline pHs (8-14) the copper ions (Cu2+) in Benedict’s reagent are reduced by the monosaccharide functional groups (i.e. -CHO or –C=O) to form cuprous oxide. In the Benedict’s test for reducing sugars, the Benedict’s reagent is reduced while the reducing sugar is oxidized. This redox reaction results in a tractable color change going from a light blue solution to a green/reddish orange one. The intensity of the color change is indicative of the amount of reducing sugar present (Fig. 5). Figure 5. Examples of expected test results for positive and negative reactions in Benedict’s test for reducing sugars. Procedure: 1. Obtain seven test tubes and number them 1-7. 2. Add the materials listed in