Biologically Important Molecules

Biologically Important Molecules

Carbohydrates, Proteins, Lipids, and Nucleic Acids

PRE-LAB ASSIGNMENT:

Students are expected to read pages 1 to 3 before coming to the lab to complete the experiments.

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Print this entire lab packet and bring it to the laboratory. You must submit the completed lab worksheet for credit. Please provide a FULL lab report for this experiment.

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.

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.

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

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LAB DATASHEET

Part 1: Carbohydrates

Benedict’s test

What color(s) represents a positive result? Any other color than blue is positive (Onion, Glucose & starch solution)

What color(s) represents a negative result? Blue (Sucrose, distilled water)

Exercise 1: Perform the Benedict’s test for reducing sugars

1. Obtain eight test tubes and number them 1-8.

2. Add the solutions to be tested from the table to the appropriately labeled test tube.

3. Add 2 mL of Benedict’s solution to each tube.

4. Place all tubes in a gently boiling water bath for 3 min; observe the color changes during this time.

5. After 3 min, remove the tubes from the water bath and give the tubes ample time to cool to room temperature.

6. Record the color of the contents in each tube in the data table.

TubeSolutionBenedict’s Color Reaction
110 drops onion juiceBrown/Orange
210 drops potato juiceOrange/Yellow
310 drops sucrose solutionBlue
410 drops glucose solutionRed/Orange
510 drops distilled waterBlue
610 drops starch solutionRust Red
710 drops unknown solution #Blue
Analysis Questions

1. Which functional groups of a glucose molecule are involved in forming a polysaccharide? Two hydroxyl groups

2. Which of the solutions is the positive control? Which is the negative control?

The positive control is tube #6 the reducing sugar, While the Negative control is tube # 5 distilled water, and tube # 7 unknown solution.

3. Which is a reducing sugar, sucrose or glucose? How do you know?

The reducing sugar is Glucose, because it reduces the Benedict’s reagent to a green to reddish orange color.

4. Which contains more reducing sugar, potato juice or onion juice? How do you know? Onion juice contains more reducing sugar, because its’s reaction with Benedict’s reagent is more intense.

5. What does this tell you about how sugars are stored in onions and potatoes? Onions store reducing sugars while potatoes non-reducing sugars.

Iodine Test

What color(s) represents a positive result? A blue-black

What color(s) represents a negative result? Yellow / Brown

Exercise 2: Perform the iodine test for starch

1. Obtain eight test tubes and number them 1-8.

2. Add the solutions to be tested from the table to the appropriately labeled test tube.

3. Add three drops of iodine to each tube.

4. Swirl lightly to mix.

5. Record the color of the contents in each tube in the data table.

Tub eSolutionIodine Color Reaction
110 drops onion juiceYellow
210 drops potato juiceBlue-Black
310 drops sucrose solutionYellow
410 drops glucose solutionYellow
510 drops distilled waterYellow
610 drops reducing sugar solutionYellow
710 drops starch solutionBrown
810 drops unknown solution #Yellow
Analysis Questions

6. Which of the solutions is the positive control? Which is the negative control?

Starch is the positive control in the Iodine test and distilled water is the negative control.

7. Which has a more intense color, onion juice or potato juice? Why?

Potato juice has more starch, since, the iodine reacts with starch, potato juice produces and dark blue color.

8. In what part(s) of a plant is the most starch typically stored?

Starch is stored in a plant organelle called amyloplast.

Part 2: Proteins

Biuret Test

What color(s) represents a positive result? Purple

What color(s) represents a negative result? Light Blue

Exercise 3: Perform the Biuret test for protein

1. Obtain six test tubes and number them 1-6.

2. Add the solutions to be tested from the table to the appropriately labeled test tube.

3. Make sure to wear proper protection (eye goggles, apron, gloves) as the reagents you will be handling next can be dangerous

4. Add ten drops of potassium hydroxide (KOH) to each tube.

IMPORTANT: Use caution when handling KOH. It is EXTREMLY caustic. Please DO NOT spill it and if it comes in contact with your skin, rinse immediately and let the instructor know.

5. Add five drops of Biuret reagent to each tube and swirl lightly to mix.

6. Record the color of the contents in each tube in the data table.

TubeSolutionBiuret Color Reaction
12 mL egg albumenPurple
22 mL honeyClear
32 mL amino acid solutionLight blue
42 mL distilled waterLight blue
52 mL protein solutionLight blue
62 mL unknown solution #Purple
Analysis Questions

9. Which of the solutions is the positive control? Which is the negative control?

Tube #5 the protein solution is the positive control and tube # 4 distilled water is the negative control.

10. Which contains more protein (C-N bonds), egg albumen or honey? How do you know?

Egg albumen contains more protein (C-N bonds) because biuret reagent use to test the presence of protein becomes purple upon reacting with egg albumin.

11. Do free amino acids have peptide bonds? In a protein, each amino acid is connected to another amino acid via covalent bond also known as peptide bonds, so, to form a peptide bond, there should be at least two amino acids. Free amino acids don’t have peptide bonds.

Part 3: Lipids

Solubility Test

What represents a positive result? Soluble

What represents a negative result? Not soluble

Exercise 4: Perform the solubility test for lipids

1. Obtain four test tubes. Label them 1-4.

2. Add 5 mL of water to test tube 1 and 3.

3. Add 5 mL of acetone to test tube 2 and 4.

IMPORTANT: Use caution when handling the acetone. It is EXTREMLY toxic. Please DO NOT spill it and if it comes in contact with your skin, rinse immediately and let the instructor know.

4. Add a few drops of vegetable oil to tubes 1 and 2 and unknown to tubes 3 and 4.

5. Swirl lightly to mix.

6. Record your observations in the data table.

TubeSolutionSolubility Observations
1Water and OilNot soluble
2Acetone and OilSoluble
3Water and Unknown #Not soluble
4Oil and Unknown #Soluble
Analysis Questions

12. What can you conclude about the solubility of lipids in polar solvents such as water?

Lipids are insoluble in polar solvents, and soluble in non-polar solvents

13. What can you conclude about the solubility of lipids in non-polar solvents such as acetone? Lipids are NOT able to dissolve in water because of their hydrophobic nature but able to dissolve in non-polar solvents such as acetone.

Grease Spot Test

What represents a positive result? Translucent / Transparency

What represents a negative result? No Transparency

Exercise 5: Perform the grease spot test for lipids

1. Obtain a piece of brown paper from the lab instructor.

2. Use a dropper to add a drop of oil near a corner of the brown paper.

3. Use a dropper to a drop of water near the opposite corner of the brown paper.

4. Let the fluids evaporate.

5. Hold the brown paper up to the light to view both corners.

6. Record your observations in the data table.

7. Repeat the procedure for food products 3,4 and your unknown

Food ProductGrease Spot Observations
1OilPositive (Translucent)
2WaterNegative (Opaque / No transparency)
3HoneyNegative (Opaque / No transparency)
4Potato JuiceNegative (Opaque / No transparency)
5Unknown Solution #Positive (Translucent)
Analysis Questions

14. Which of the food products that you tested contained large amounts of lipids? How do you know? The vegetable oil,

Exercise 6: Determine the macromolecule composition of your unknown solution

Using the results that you generated, determine if the unknown solution tested positively or negatively for each test. In your conclusion, report which macromolecules are in your

Unknown and how you know.

ObservationsUnknown Results
Biochemical TestNegative Positive(+/-)
Benedict’s test (reducing sugars)Orange/YellowOrange/Red(+) Positive
Iodine (starch)Dark GreenNavy Blue(-) Negative
Biuret test (protein)Blue/BrownPurple(-) Negative
Acetone solubility (lipids)InsolubleSoluble(+) Positive
Grease spot test (lipids)No TransparencyTranslucence(+) Positive

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