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North Star Academy College Preparatory High School

Name: ______________________________ Team_____________________

EDUCATION IS FREEDOM! Date__________/Mr. McIntire

Honors Biology Organic Macromolecules Lab

Macromolecular Architecture

Lab modified from Biology in the Laboratory, 3rd Ed. By DR Helms, CW Helms, RJ Kosinski and JR Cummings, W.H. Freeman and Co: New York, 1998.


Introduction

Biological macromolecules are defined as large molecules made up of smaller organic molecules. There are four classes of macromolecules that are essential to living things: carbohydrates, lipids, proteins and nucleic acids. These macromolecules are said to be organic because they are principally composed of carbon, a tetravalent atom that forms four single nonpolar covalent bonds with other atoms. The base elements of carbohydrates and lipids are Carbon (C), Hydrogen (H) and Oxygen (O). Protein is also made up of these base elements but it also contains Nitrogen (N). When viewing the chemical structures of carbohydrates, lipids and proteins you can distinguish proteins from the other two by the presence of N in its chemical structure. Alone, these elements comprise more than 96% of the human body.

Moreover, each macromolecule is made up of smaller organic molecules. For carbohydrates and proteins these smaller molecules are known as monomers. These similar or identical monomers are covalently bonded together to create a large polymer molecule. The monomer unit for carbohydrates is a monosaccharide or a simple sugar. When two of these monosaccharides are linked by covalent bonds a disaccharide is created. When several monosaccharides are bonded together a polysaccharide, or complex sugar, is created. Polysaccharides are the polymers of carbohydrates. Proteins are made up of monomers called amino acids. There are twenty amino acids and they can be strung together in unique combinations known as polypeptide chains, the polymer unit for proteins. A protein is only complete and functional when the polypeptide chain is folded into a unique 3-D shape, a concept discussed in your textbook.

In this lab, you will learn about the first three, nucleic acids will be covered in a later lab (DNA Structure). Whereas some foods, such as table sugar, only contain one of these nutrients, most foods, contain mixtures of carbohydrates, lipids, and proteins. In this investigation, you will use chemical substances called indicators to identify the presence of specific nutrients. You will first work with known carbohydrates, lipids, and proteins to determine how each indicator reacts to each type of molecule. Next week, you will then analyze several unknown substances to determine which macromolecules are present and or absent, as the basis of a lab practical exercise.

Objectives

1. Explain how indicators are used to test for the presence of organic compounds.2. Identify the presence of reducing sugars (both monosaccharides and disaccharides) using

Benedict’s reagent. 3. Identify the presence of polysaccharides using Lugol’s reagent. 4. Distinguish between lipid and non-lipid substances using the Sudan IV test.5. Distinguish between free amino acids and proteins (polypeptides) on the basis of their ability to

react with either biuret reagent or ninhydrin

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Materials

Benedict’s reagent Lugol’s solution Sudan IV reagent Biuret reagent Ninhydrin Starch solution Glucose solution

Sucrose solution Milk Potato slices Vegetable oil Orange juice Bacon grease Egg albumin

Test tubes Test tube racks Beakers Hot plate Brown paper bag

Safety Note: Your safety goggles MUST be word at all times during this lab. Use caution when handling iodine potassium iodide solution. It can stain skin and clothing. Do not touch the sides of test tubes or beakers as they might be hot. Use test tube clamps for this purpose.

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Activity 1: Testing for Carbohydrates

Introduction

The basic structural unit for carbohydrates is the monosaccharide (or simple sugar). Monosaccharides are classified by the number of carbons they contain: for example, trioses have three carbons, pentoses have five carbons, and hexoses have six carbons. They may contain as few as three or as many as 10 carbons.

Monosaccharides are also characterized by the presence of a terminal aldehyde group or an internal ketone group. Both of these groups contain a double-bonded oxygen atom that reacts with Benedict’s reagent to form a colored precipitate

When two monosaccharides are joined together, they for a disaccharide. If the reactive aldehyde or ketone groups are involved in the bond between the monosaccharide units (as in sucrose), then the disaccharide will not react with Benedict’s reagent. If only one group is involved in the bond (as in maltose), the other is free to react with the reagent. Sugars with free aldehyde or ketone groups, whether monosaccharides or disaccharides, are called reducing sugars. In this exercise, you will use Benedict’s reagent to test for the presence of reducing sugars.

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Monosaccharides may also join together to form long chains (polysaccharides) that may be either straight or branched. Starch is an example of a polysaccharide formed entirely of glucose units. Starch does not show a reaction with Benedict’s reagent because the number of free aldehyde groups (found only at the end of each chain) is small in proportion to the rest of the molecule. Therefore, you will test for the presence of starch with Lugol’s reagent (iodine/potassium iodide, I2KI).

Before carrying out the Benedict’s and Lugol’s tests for reducing sugars and starches, respectively, predict whether each of the sugars to be tested, below, will result in a positive result for the Benedict’s or Lugol’s test.

1. Water: _____________________________________________________________________

2. Starch: ____________________________________________________________________

3. Glucose: ____________________________________________________________________

4. Sucrose: ____________________________________________________________________

5. Potato Slice: ________________________________________________________________

6. Milk: _______________________________________________________________________

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Procedure:

Benedict’s Test for Reducing Sugars

1. Set up a row of 6 test tubes. Use a marker to number them 1-6.

2. To each test tube, add 2mL of the solutions listed in Table 1, below, matching each number to the number on the test tube.

3. Add one dropperful (approximately 2mL) of Benedict’s reagent to each tube.

4. Mix the reagent and the sample by agitating the solution in each tube from side to side. Record the original color of each tube’s contents in Table 1, under “Benedict’s Test.”

5. Heat the test tubes in a boiling water bath for 3 minutes. Record any color changes Table 1, under “Benedict’s Test”

Lugol’s Test for Starch

1. Prepare another 6 test tubes as indicated in step 1, above.

2. To test the tubes, add 1mL of the solutions listed in Table 1, matching each number to the number on the tube.

3. Record the original color of each tube’s contents in Table 1.

4. Add several drops of Lugol’s reagent (I2KI) to each tube, mix, and immediately record your observations in Table 1 for any color changes that take place. Do NOT heat the test tubes in the Lugol’s test.

Table 1: Observations for Benedict’s and Lugol’s Tests.

Benedict’s Test Lugol’s Test

TubeOriginal Color Before Boiling

Final Color After Boiling

Original Color Before Adding I2KI

Final Color After Adding I2KI

1. Water

2. Starch

3. Glucose

4. Sucrose

5. Potato Slice

6. Milk

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Benedict’s and Lugol’s Test Analysis Questions

1. Why did you test water with Benedict’s reagent?__________________________________________________________________________________

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2. Which sugars reacted with Benedict’s reagent?__________________________________________________________________________________

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3. Which sugars did not react with Benedict’s reagent?__________________________________________________________________________________

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4. Did the results for each test agree with your predictions? Explain any discrepancies.__________________________________________________________________________________

__________________________________________________________________________________

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5. Which sugars reacted with Lugol’s reagent? Why?

__________________________________________________________________________________

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6. Which sugars did not react with Lugol’s reagent? Why not?__________________________________________________________________________________

__________________________________________________________________________________

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7. Explain your results for the following:

a. Starch: _____________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

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b. Potato slice: _________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

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c. Milk: _______________________________________________________________________

___________________________________________________________________________

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8. Did the results for each test agree with your predictions? Explain any discrepancies.

__________________________________________________________________________________

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9. From the results of the Benedict’s and Lugol’s tests, would you conclude that a potato stores its carbohydrates as sugars or as starch? How do you know?__________________________________________________________________________________

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Activity 2: Testing for Lipids

Introduction

The word lipid refers to any of the members of a rather heterogeneous group of organic molecules that are soluble in nonpolar solvents such as chloroform (CHCl3) but insoluble in water. Although lipids include fats, steroids, and phospholipids, this exercise will focus primarily on fats.

Triglycerides , a popular topic in discussions of diet and nutrition, are the most common form of fat. They consist of three fatty acids attached to a glycerol molecule. Triglycerides are found predominantly in adipose tissue and store more energy per gram than any other types of compounds.

At room temperature, some lipids are solid (generally those found in animals) and are referred to as fats, while others are liquid (generally those found in plants) and are referred to as oils. Vegetable oil, a liquid fat, is a mixture of triglycerides.

Since both solid and liquid fats are nonpolar, you will test for their presence using Sudan IV, a nonpolar dye that dissovles in nonpolar substances such as fats and oils but not in polar substances such as water.

Before carrying out the Sudan IV test for lipids, predict whether each of the substances to be tested, below, will result in a positive result for the Sudan IV test.

1. Water: ________________________________________________________________________

2. Vegetable Oil: __________________________________________________________________

3. Orange Juice: ___________________________________________________________________

4. Bacon Juice: ____________________________________________________________________

5. Milk: __________________________________________________________________________

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Procedure:

Brown Paper Bag Test for Lipids

1. Divide a piece of brown paper bag into 8 equal sections. In each section, write the name of one test substance.

2. Place 2 drops of each substance onto the brown paper bag in the appropriate section.

3. Wait until all spots have dried.

4. Hold the paper up to a bright light. Some foods leave a transluscent spot on the brown paper. This transluscent spot indicates the presence of lipids. In the data table below, place a check mark next to any substances that test positive for lipids.

Table 2: Observations for Brown Paper Bag Test.

Substance Observations After Spotting on Brown Paper Bag

1. Water

2. Vegetable Oil

3. Orange Juice

4. Bacon Juice

5. Milk

Sudan IV Test for Lipids

1. Label 5 test tubes in sequence, 1 through 5.

2. Add 1 dropperful (1mL) of each substance you have listed in Table 2 to the appropriate tube.

3. Add 3 drops of Sudan IV to each tube. Mix.

4. Add 2mL of water to each tube.

5. If fats or oils are present, these will appear as floating red droplets or as a floating red layer colored by Sudan IV. In Table 2, record the reactions that occur in each of the test tubes

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Table 3: Observations for Sudan IV Solubility Test.

Substance Sudan IV Solubility Reaction

6. Water

7. Vegetable Oil

8. Orange Juice

9. Bacon Juice

10. Milk

Sudan IV Test Analysis Questions

1. Why do the droplets float rather than mix with water?

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2. Which substances reacted with the brown paper bag?

__________________________________________________________________________________

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3. Which substances reacted with Sudan IV?

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4. Did your results agree when comparing the two tests?

__________________________________________________________________________________

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5. Which substances did not react with Sudan IV? Why not?

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Activity 3: Testing for Proteins

Introduction

Proteins are made up of one or more polypeptides, which are linear polymers of smaller molecules called amino acids. Amino acids derive their names form the amino group and the carboxyl group (acidic) that each possesses. Polypeptides are formed when amino acids are joined together be peptide bonds between the amino group of one amino acid and the carboxyl group of a second amino acid.

Above, review the structure of an amino acid. Note the presence of the amino (-NH2) group and a carboxyl (-COOH) group. Two amino acids are joined by a peptide bnod when a molecule of water is split out from their amino and carboxyl groups. A polypeptide is made up of many amino acids joined in this way. “R” represents a side group that is characteristic for each amino acid. Here, the amino acids are shown in their ionized form.

The Buiret reagent reacts with peptide bonds and therefore reacts with proteins, such as egg albumin, but not with free amino acids, such as glycine and alanine. On the other hand, the reagent ninhydrin reacts with the amino group of free amino acids, but not with polypeptides.

The Biuret reagent is light blue, but in the presence of proteins it turns violet. Other types of molecules may cause other color changes, but only the violet color indicates the presence of polypeptides.

Before carrying out the Biuret test for proteins, predict whether each of the substances to be tested, below, will result in a positive result for the Biuret test.

1. Water: ________________________________________________________________________

2. Egg Albumin: ___________________________________________________________________

3. Potato Starch: __________________________________________________________________

4. Glucose: _______________________________________________________________________

5. Amino Acid: ____________________________________________________________________

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Procedure:

Biuret Test for Proteins

1. Obtain five clean test tubes and use an overhead marker to number them 1-5.

2. To the test tubes, add 2ml of the solutions listed in the table below, matching each number to the number on the tube.

3. Add one dropperful (approximately 2ml) of Biuret reagent to each tube.

4. After an incubation period of 2 minutes, record your results in the data table below and determine whether the solution treated contains protein. Base your conclusions only on the presence or absence of the violet color.

Table 4: Observations for Biuret Test.

Substance Color with Biuret Reagent After 2 min Protein Present (+) or Absent (─)

1. Water

2. Egg Albumin

3. Potato Starch

4. Glucose

5. Amino Acid

Biuret Test Analysis Questions

1. What does this test tell you about the biochemical composition of starch or glucose?

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2. Why has water been included as one of the test substances?

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3. Which substances reacted with Biuret reagent? Why?

__________________________________________________________________________________

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4. Which substances did not react with Biuret reagent? Why?

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