Biologically Important Molecules

Objectives:

After completing this laboratory assignment, students will be able to:

· Understand which assay is used to detect the presence of carbohydrates, proteins, lipids, and nucleic acids.

· Perform assays to detect the presence of carbohydrates, proteins and lipids.

· Explain the importance of controls in biochemical tests.

· Use biochemical tests to identify an unknown compound.

Every biological material you find is composed of macromolecules. One part of being a scientist is identifying the presence of macromolecules, so that we can better describe the composition of products. To do this, we use test (or assays) to measure for the presences of substances.

Part 1: Macromolecules
Macromolecules are large molecules that are essential to the structure and function of the cell. The four groups necessary for life are carbohydrates, proteins, lipids, or nucleic acids. Each of these macromolecules is made up of smaller subunits called monomers. Monomers are linked together by dehydration synthesis to form the macromolecules which are polymers. Dehydration synthesis also called condensation reaction is an energy-requiring process in which a molecule of water is removed as the two subunits are bonded covalently. When polymers are broken apart to form the individual monomers, a water molecule must be added

in an energy-releasing process is called hydrolysis.

Each macromolecule has different structures and chemical properties. For example, lipids (made up of fatty acids) have many C-H bonds and relatively little oxygen molecules and are insoluble in water, while proteins (made up of amino acids) have amino groups (-NH2) and carboxyl (-COOH) groups that makes them dipolar ions and able to serve as biological buffers. Carbohydrates such as glucose are polar and soluble in water, whereas nucleic acids are acidic.

Identifying Macromolecules
Most foods that we consume often consist of substances derived from plants or animals; therefore, these foods are combinations of macromolecules. Some of these macromolecules can be detected by taste, while others cannot. Therefore, scientists have devised biochemical tests to identify the presence of these unknown macromolecules in food samples. During the experiment, one must compare the unknown solution’s response to that of a known solution or control using the same procedure. Often these tests utilize colorimetry (color changes) to indicate positive results.

Only a carefully conducted experiment will reveal the content of the food in question. Therefore, each of these tests utilizes controls to provide standards for comparison. The controls are known solutions and are used to validate that the procedure is only detecting what it is expected it to detect.

A positive control contains the variable for which you are testing: it reacts positively and demonstrates the test’s ability to detect what you expect. For example, if you are testing for the presence of protein in an unknown solution, then an appropriate positive control is a solution known to contain proteins. A positive reaction shows that your test reacted correctly: it also shows you what to expect for a positive result.

A negative control does not contain the variable for which you are searching. It contains only the solvent that the molecules may be dissolved in which is often distilled water with no solute. A negative control does not react in the test and shows you what to expect for a negative result.

Part 2: Carbohydrates
Carbohydrates are molecules made up of carbon, hydrogen, and oxygen in a ratio of 1:2:1 (e.g., the chemical formula for glucose is C6H12O6). Carbohydrates are composed of monosaccharides, or simple sugars. Two monosaccharides bonded together form a disaccharide—for example, sucrose (table sugar) is a disaccharide of glucose linked to fructose. Similarly, three or more monosaccharides linked together form a polysaccharide such as starch, glycogen, or cellulose. As mentioned above, the bonding of monomers in carbohydrates, as well as other macromolecules, involves the removal of a water molecule (dehydration synthesis).

Many monosaccharides such as glucose and fructose are reducing sugars, meaning that they possess free aldehyde (-CHO) or ketone (-C=O) groups that reduce weak oxidizing agents such as a copper. Benedict’s reagent contains cupric (copper) ion complexed 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 pH.

Cuprous oxide is green to reddish orange. A green solution indicates a small amount of reduction sugars, and the reddish orange indicates an abundance of reducing sugars. Nonreducing sugars such as sucrose produce no change in color (i.e., the solution remains blue).

Since all carbohydrates cannot be detected using Benedict’s test, there are multiple tests to detect different types of carbohydrates. The iodine test is used to test for the presence of starch, a polysaccharide, in a solution. The brown iodine solution consists of iodine dissolved in potassium iodide. When ions from the iodine solution bond with starch, it produces a dark blue/purple color.

Part 3: Proteins
Proteins are remarkably versatile molecules found in all life forms. They are made up of amino acids, each of which has an amino group (-NH2), a carboxyl (acid) group (-COOH), and a variable side chain (R). Adjacent amino acids bind together through a peptide bond. This bond forms between the amino group of one amino acid and the carboxyl group of an adjacent amino acids.

Biuret’s test Identifies C-N bonds in proteins and causes the nitrogen molecules to form a complex with the Cu2+ in Biuret reagent, a 1% solution of CuSO4

(copper sulfate). The reaction produces a violet color. A Cu2+ must complex with four to six peptides bonds to

produce a color; therefore, long-chain polypeptides produce a positive reaction and individual amino acids do not react positively. The intensity of the color is related to the number of peptide bonds that react.

Part 4: Lipids
Lipids include a variety of molecules that are characterized by their ability to dissolve in nonpolar solvents such as ether, acetone, methanol, or ethanol, but not as well in polar solvents such as water. Triacylglycerol, also called triglycerides, are commonly referred to as fats. They are the most abundant lipids and are composed of one glycerol and three fatty acids. Not all lipids contain fatty acids.

Similar to carbohydrates, there are several tests that can detect the presence of lipids. One test for lipids is based on a lipid’s ability to selectively absorb pigments in fat-soluble dyes such as Sudan IV. Another test is the solubility of lipids in polar and non-polar solvents. A simpler test for lipids is based on their ability to produce translucent grease-marks on unglazed brown paper.

Part 5: Nucleic Acids
Nucleic acids are made up of nucleotide subunits which consist of a five carbon sugar, a phosphate group and a nitrogenous base. DNA and RNA are nucleic acids. One major difference between DNA and RNA is the type of five carbon sugar: DNA contains deoxyribose, whereas RNA contains ribose. Because of this difference in composition, DNA can be identified chemically using the Dische diphenylamine test. If deoxyribose is present, the solution will be a blue color and the intensity of the color correlates to the concentration of DNA.

Instructions:

Each group will perform biochemical tests to identify known and unknown solutions. For each experiment, there is only ONE positive control and ONE negative control. The positive control is the material that is known to have the substance for which you are testing. It is known that it will test positive even BEFORE you perform the test. The negative control is the material that is known to lack the substance for which you are testing even BEFORE you perform the test.

Each group MUST obtain and use your unknown solution for ALL of the tests in order to determine the polymer composition of your unknown solution. You MUST record the number of your unknown (#1, #2, or #3, ) and include this information in your lab report.

Follow the specific directions in each experiment to successfully identify each solution and then determine what macromolecules are present in your unknown solution