C4: Identifying Biological Macromolecules

1. Introduction to Biological Macromolecules

Welcome to the lab on biological macromolecules! These are large, organic molecules essential for life, built from smaller repeating units called monomers. They perform a vast array of functions within cells and entire organisms. Understanding their structure and how to identify them is fundamental to biology.

The four major classes of biological macromolecules are:

• Carbohydrates: Primarily serve as an energy source and can also have structural roles.
• Proteins: Incredibly diverse, involved in structure, enzyme activity, transport, signaling, and much more.
• Lipids (Fats): Important for long-term energy storage, insulation, and are major components of cell membranes.
• Nucleic Acids: Store and transmit genetic information (DNA and RNA).

In this lab, you will learn to perform qualitative tests to detect the presence of these different macromolecules in various samples.

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What are the four major classes of biological macromolecules?

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Carbohydrates, Lipids, Proteins, and Nucleic Acids.

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2. Safety First! Important Lab Precautions

Before we dive into the experiments, it’s crucial to review lab safety. We will be working with various chemical reagents, some of which can be hazardous if mishandled.

General Safety Guidelines:

• Always wear appropriate Personal Protective Equipment (PPE): This includes safety goggles, a lab coat, and gloves.
• Know the location of safety equipment: Be aware of the eyewash stations, safety showers, and fire extinguishers in the lab.
• Handle chemicals with care:
  • Read labels carefully before using any reagent.
  • Never taste or intentionally sniff chemicals.
  • Avoid skin and eye contact with all chemicals.
  • Report any spills immediately to your instructor.
• Heating Safety:
  • When heating test tubes in a water bath, always point the opening of the test tube away from yourself and others.
  • Use test tube holders when handling hot test tubes.
• Waste Disposal: Dispose of all chemical waste in the designated containers as instructed by your TA. Do not pour chemicals down the sink unless specifically told to do so.
• General Conduct:
  • No eating, drinking, or applying cosmetics in the lab.
  • Wash your hands thoroughly with soap and water before leaving the lab.
  • Keep your workspace clean and organized.

Specific Reagent Handling Notes (examples, your lab may vary):

• Benedict’s Reagent: Can be an irritant. Avoid skin/eye contact.
• Lugol’s Iodine: Can stain skin and clothing. May be irritating.
• Biuret Reagent: Contains copper sulfate (toxic if ingested) and sodium hydroxide (corrosive). Handle with extreme care to avoid skin and eye burns.
• Sudan IV: Is a dye and can stain. Some Sudan dyes are potential carcinogens, handle with care and avoid inhalation of powder if used in that form.
• Dische Diphenylamine Reagent: Contains strong acids (glacial acetic acid, sulfuric acid). Highly corrosive. Handle with extreme care under a fume hood if possible, or with excellent ventilation.

It is okay to eat a small snack in the lab as long as I clean up afterwards.

True False

3. Testing for Carbohydrates

Carbohydrates range from simple sugars (monosaccharides) to complex polysaccharides like starch. We will perform tests to identify both reducing sugars and starch.

3.1 Benedict’s Test for Reducing Sugars

Principle: Benedict’s reagent tests for the presence of reducing sugars (most monosaccharides like glucose and some disaccharides like lactose and maltose). In alkaline conditions and upon heating, reducing sugars reduce the blue copper(II) sulfate in Benedict’s reagent to red-brown copper(I) oxide, which is an insoluble precipitate. The color change can range from green (small amount) to yellow, orange, or brick-red (large amount of reducing sugars). Sucrose (table sugar) is a non-reducing sugar and will give a negative result (solution remains blue) unless it is first hydrolyzed (broken down) into its monosaccharide components.

Materials (General):

• Test tubes and test tube rack
• Water bath (beaker with boiling water)
• Droppers or pipettes
• Benedict’s reagent
• Sample solutions (e.g., glucose solution, sucrose solution, starch solution, unknown samples)

Procedure:

1. Label your test tubes clearly.
2. Add approximately 1 mL of each sample solution to its respective test tube.
3. Add approximately 2 mL of Benedict’s reagent to each test tube.
4. Mix gently.
5. Place the test tubes in a boiling water bath for 3-5 minutes.
6. Carefully remove the test tubes and observe any color changes against a white background.

Interpreting Results:

• Negative: Solution remains blue.
• Positive (trace amount): Green precipitate.
• Positive (low amount): Yellow precipitate.
• Positive (moderate amount): Orange precipitate.
• Positive (high amount): Brick-red precipitate.

Benedict's Test Results

The color change in Benedict's test indicates the presence and relative amount of reducing sugars. (Image Source: YouTube)

Video Demonstration:

(Video by Amoeba Sisters)

3.2 Lugol’s Iodine Test for Starch

Principle: Lugol’s iodine solution (iodine-potassium iodide, IKI) is used to test for the presence of starch, a complex polysaccharide. Iodine interacts with the coiled structure of amylose (a component of starch), producing a characteristic blue-black color. Other polysaccharides like glycogen may give a reddish-brown color, while simple sugars and cellulose will not react (solution remains yellowish-brown).

Materials (General):

• Test tubes or spotting plate
• Droppers or pipettes
• Lugol’s iodine solution
• Sample solutions (e.g., starch solution, glucose solution, water, unknown samples)

Procedure:

1. Label your test tubes or wells on a spotting plate.
2. Add a few drops (or ~1mL if using test tubes) of each sample to its respective container.
3. Add 2-3 drops of Lugol’s iodine solution to each sample.
4. Observe any color changes.

Interpreting Results:

• Negative: Solution remains yellowish-brown (the color of iodine).
• Positive for Starch: Solution turns blue-black.
• Positive for Glycogen (possibly): Solution may turn reddish-brown.

Iodine Test for Starch Results

The iodine test produces a distinct blue-black color in the presence of starch. (Image Source: Wikimedia Commons)

Video Demonstration:

(Video by The Organic Chemistry Tutor explaining starch tests)

Which of the following tests would you use to specifically detect the presence of starch?

• Benedict's Test
• Lugol's Iodine Test
• Biuret Test
• Sudan IV Test

4. Testing for Proteins (Biuret Test)

Principle: The Biuret test detects the presence of peptide bonds in proteins. In an alkaline solution (provided by sodium hydroxide or potassium hydroxide), copper(II) ions from the Biuret reagent (copper(II) sulfate) react with peptide bonds, forming a purple or violet-colored complex. The intensity of the color is proportional to the number of peptide bonds, so it indicates the presence of proteins (polypeptides). Single amino acids or dipeptides (only one peptide bond) will not give a positive test.

Materials (General):

• Test tubes and test tube rack
• Droppers or pipettes
• Biuret reagent (or separate solutions of ~1% copper sulfate and ~10% sodium hydroxide)
• Sample solutions (e.g., albumin solution, amino acid solution, water, unknown samples)

Procedure:

1. Label your test tubes clearly.
2. Add approximately 1-2 mL of each sample solution to its respective test tube.
3. Add an equal volume (1-2 mL) of Biuret reagent (or if using separate solutions, add 1-2 mL of sodium hydroxide, mix, then add a few drops of copper sulfate solution).
4. Mix gently and let stand at room temperature for about 5 minutes.
5. Observe any color changes. Holding the tube against a white background can help.

Interpreting Results:

• Negative: Solution remains blue (the color of copper sulfate).
• Positive: Solution turns violet or purple.

Biuret Test for Proteins

A purple or violet color indicates a positive Biuret test for proteins. (Image Source: Biology For All)

Video Demonstration:

(Video by Miss Estruch)

A student performs a Biuret test on an unknown solution. The solution turns purple. What does this indicate?

• Presence of reducing sugars
• Presence of starch
• Presence of proteins
• Presence of lipids

5. Testing for Lipids

Lipids are a diverse group of hydrophobic molecules, including fats, oils, and waxes. They are insoluble in water but soluble in organic solvents.

5.1 Sudan IV Test for Lipids

Principle: Sudan IV (or Sudan III) is a dye that is soluble in lipids but not in water. When Sudan IV is added to a mixture containing lipids and water, the dye will selectively dissolve in the lipid layer, staining it red or reddish-orange. If no lipids are present, the dye will not dissolve and may disperse or form small micelles.

Materials (General):

• Test tubes and test tube rack
• Droppers or pipettes
• Sudan IV solution (typically dissolved in ethanol or another organic solvent)
• Sample solutions (e.g., vegetable oil, water, unknown samples)

Procedure (General - can vary):

1. Label your test tubes.
2. Add approximately 2 mL of your sample to a test tube.
3. Add 4-5 drops of Sudan IV solution to each test tube.
4. Mix gently (do not shake vigorously if you want to observe distinct layers, though some protocols call for shaking then allowing to settle).
5. Observe the distribution of the dye. Look for a distinct red-stained layer if lipids are present.

Interpreting Results:

• Negative: The dye does not concentrate in a distinct layer, or the solution remains largely unchanged/dye disperses. The water may appear slightly colored.
• Positive: A distinct red or reddish-orange layer forms, indicating the presence of lipids where the dye has dissolved. Often this is the top layer if the lipid is less dense than water.

Sudan IV Test for Lipids

The Sudan IV test uses a fat-soluble dye to identify lipids. A red-stained layer is a positive result. (Image Source: YouTube - MeitY OLabs)

Video Demonstration:

(Video by MeitY OLabs)

5.2 Emulsion Test for Lipids (Alternative/Additional Test)

Principle: This test relies on the fact that lipids are soluble in ethanol but insoluble in water. When a lipid-containing sample is dissolved in ethanol and then added to water, the lipid will come out of solution and form a cloudy white emulsion.

Procedure:

1. Add about 2 mL of your sample to a test tube.
2. Add about 2 mL of ethanol. Shake vigorously to dissolve any lipids.
3. Pour this mixture into another test tube containing about 2 mL of cold water.
4. Observe.

Interpreting Results:

• Negative: The solution remains clear.
• Positive: A cloudy white emulsion forms. The cloudier the emulsion, the more lipid is present.

Lipids are generally hydrophilic (water-loving).

True False

6. Testing for Nucleic Acids (e.g., DNA - Dische Diphenylamine Test)

Note: This test is often more complex and may not be performed in all introductory UG labs due to reagent handling (strong acids) and heating requirements. Confirm if this will be part of your specific lab.

Principle: The Dische diphenylamine test is used to detect DNA. In acidic conditions and with heating, the deoxyribose sugar of DNA reacts with diphenylamine to form a blue-colored complex. The intensity of the blue color can be proportional to the DNA concentration. RNA, which contains ribose sugar, typically yields a greenish color or no significant color change with this specific test, allowing for differentiation.

Materials (General):

• Test tubes and test tube rack
• Water bath (boiling)
• Droppers or pipettes
• Dische diphenylamine reagent (contains diphenylamine, glacial acetic acid, and concentrated sulfuric acid)
• Sample solutions (e.g., DNA solution, RNA solution, water, unknown samples)

Procedure:

1. Label your test tubes clearly.
2. Add approximately 2 mL of each sample solution to its respective test tube.
3. CAREFULLY add 2 mL of Dische diphenylamine reagent to each tube. This reagent is highly corrosive. Mix thoroughly.
4. Place the test tubes in a boiling water bath for 10-15 minutes.
5. Carefully remove the tubes and allow them to cool.
6. Observe any color changes.

Interpreting Results:

• Negative (No DNA or very little): Solution may remain clear or have a very faint color.
• Positive for DNA: Solution turns blue. The intensity of blue indicates the relative amount of DNA.
• RNA present (may interfere or give different color): Solution might turn greenish.

Dische Diphenylamine Test (Conceptual)

The Dische diphenylamine test yields a blue color in the presence of DNA. (Image is illustrative; actual results depend on sample purity and concentration. Source: YouTube, Science Sauce - general DNA test explanation)

Video on DNA/RNA Chemical Tests (General Overview):

(Video by Bio-Resource covering various nucleic acid tests)

Complete the sentence about the Dische Diphenylamine test:

The Dische Diphenylamine test is used to detect the presence of . A positive result is indicated by a color.

Check Answers

7. Summary Table of Tests

Macromolecule	Test Name	Reagent(s)	Positive Result	Negative Result
Reducing Sugars	Benedict’s Test	Benedict’s Reagent	Green/Yellow/Orange/Brick-Red ppt.	Remains Blue
Starch	Lugol’s Iodine Test	Lugol’s Iodine Solution (IKI)	Blue-Black color	Remains Yellowish-Brown
Proteins	Biuret Test	Biuret Reagent (CuSO₄ + NaOH)	Purple/Violet color	Remains Blue
Lipids	Sudan IV Test	Sudan IV dye	Red/Reddish-Orange stained layer	Dye disperses/No distinct red layer
Lipids	Emulsion Test	Ethanol, Water	Cloudy White Emulsion	Remains Clear
DNA	Dische Diphenylamine	Dische Reagent (Diphenylamine, acids)	Blue color (RNA may give greenish)	Clear/No blue color

8. Pre-Lab Questions

Please answer these questions before coming to the lab.

1. List the four major types of biological macromolecules.
2. Why is it important to wear safety goggles during this lab?
3. What color change indicates a positive Benedict’s test, and what does it signify?
4. Which reagent is used to test for starch, and what is the positive result?
5. The Biuret test detects the presence of what specific chemical structures in proteins?
6. Describe two different methods you could use to test for the presence of lipids.
7. What specific component of DNA does the Dische diphenylamine test react with?
8. If a food sample turns blue-black with Lugol’s iodine but remains blue with Benedict’s reagent, what can you infer about its carbohydrate content?

Make sure you understand the principles behind each test and the expected results. This will help you perform the experiments efficiently and interpret your observations accurately. Good luck!