Biological Macromolecules Lab Manual Notes

Biological Macromolecules

Additional Personal Protection Equipment (PPE) Required for the Lab

  • Protective Eye Goggles: Essential to shield eyes from chemical splashes.
  • Gloves: Nitrile gloves are recommended to protect skin.
  • Clothing: Attire must cover the torso and legs to prevent exposure.

Learning Objectives

  • Familiarization with Indicator Chemicals: Testing for presence/absence of large macromolecules such as carbohydrates (sugar, starch), proteins, and lipids.
  • Testing Common Food Items: Identify the presence or absence of macromolecules in food items.
  • Understanding Structures and Building Units: Learn the structural components and building units of carbohydrates, proteins, and lipids.

Introduction

  • The chemical composition of all organisms is fundamentally similar, composed primarily of six naturally occurring elements:
    • Carbon (C)
    • Hydrogen (H)
    • Oxygen (O)
    • Nitrogen (N)
    • Phosphorus (P)
    • Sulfur (S)
  • These elements combine to form four major categories of biological macromolecules:
    1. Carbohydrates
    2. Proteins
    3. Lipids
    4. Nucleic Acids
Relationship of Organic Molecules to Cell Function
  • The types of organic molecules found in various body tissues relate to the specific functions of those cells or tissues.
  • Organisms may contain small quantities of additional elements, such as sodium (Na), calcium (Ca), potassium (K), and vitamins.

Four Classes of Macromolecules

  • ### Carbohydrates
    • Main Elements: C, H, O
    • Ratio: Always in a 1:2:1 ratio.
    • Example: Glucose (a monosaccharide) represented as C<em>6H</em>12O6C<em>6H</em>{12}O_6.
  • ### Proteins
    • Main Elements: C, H, O, N, S
    • Composition: Built from 20 different amino acids.
    • Example: Hemoglobin
  • ### Lipids
    • Main Elements: C, H, O, P
    • Structure: Comprised of a glycerol molecule linked to fatty acids.
    • Examples: Cell membranes, fats, hormones
  • ### Nucleic Acids
    • Main Elements: C, H, O, N, P
    • Composition: Built from 5 nucleotides: Adenine, Thymine, Uracil, Cytosine, Guanine.
    • Examples: DNA, RNA
    • Note: Nucleic acids will not be covered in this lab; they are part of a later lab.

Part I: Positive and Negative Indicator Tests for Macromolecules

  • Analyzing food for organic molecules using indicator chemicals can be challenging due to natural colors affecting results. Establishing controls helps validate test results.
Controls
  • Positive Control: Displays an expected positive result, confirming the experiment's effectiveness.
  • Negative Control: Treated identically to other samples but expected to show no change.
Materials Required
  • Equipment and Reagents:
    • 8 test tubes in a rack
    • Marking pencils
    • Ruler
    • Water bottle
    • Hot water bath
    • Benedict’s Solution: For testing reducing sugars (e.g., glucose).
    • Lugol’s Iodine Solution: For testing starches.
    • Sudan IV Solution: For testing lipids.
    • Biuret A and B Solutions: For testing proteins.
    • Starch Solution
    • Glucose Solution
    • Protein Solution
Table 1: Macromolecule Testing Table
  • Fill the following table with results from tests conducted:
    • Macromolecule Testing Solution Used | Positive Test Color | Negative Test Color
    • Starch
    • Glucose
    • Protein
    • Lipid

Testing for Simple Carbohydrate (Glucose)

  • Glucose: A monosaccharide crucial for producing ATP, which powers cellular activities.
  • Molecular Structure: Glucose is a ring structure with six carbon atoms represented as C<em>6H</em>12O6C<em>6H</em>{12}O_6.
Procedure: Testing for Glucose
  1. Safety Precautions: Wear goggles and gloves throughout the experiment.
  2. Prepare Test Tubes: Using a ruler, mark a line at 2 cm from the bottom of each test tube.
  3. Add Deionized Water: Fill to the 2-cm mark in both test tubes.
  4. Add Samples:
    • Test tube one: 10 drops of glucose.
    • Test tube two: 10 drops of deionized water.
  5. Mix Solutions: Invert the tubes with Parafilm over the top to protect fingers.
  6. Add Benedict’s Solution: Add 20 drops to both tubes, mix gently, and note initial color changes.
  7. Heat Samples: Place tubes in a hot water bath for at least 25 minutes.
    • Note: Ensure the tubes are in the bath for the full duration to guarantee accurate results.
Questions (Before Heating)
  1. Positive Control: Color of glucose solution?
  2. Negative Control: Color of the deionized water solution?
Questions (After Heating)
  1. Positive Control Final Color: Result from glucose tube?
  2. Negative Control Final Color: Result from water tube?

Testing for Complex Carbohydrate (Starch)

  • Complex Sugars: Known as polysaccharides, they are composed of long chains of monosaccharides (Figure 2). These function as energy sources in animal cells (glycogen) and structural components in plants (cellulose).
Procedure: Testing for Starch
  1. Safety Precautions: Equip gloves and goggles.
  2. Prepare Test Tubes: Mark at the 2-cm line for each tube.
  3. Add Deionized Water: Fill two tubes to the 2-cm mark.
  4. Add Starch Suspension: 10 drops to one tube and deionized water to the other. Mark accordingly.
  5. Add Lugol’s Iodine: Add 5 drops to each tube, mix, and observe color development.
Questions (Final Color)
  1. Starch Tube (Positive Control): Final color?
  2. Water Tube (Negative Control): Final color?

Testing for Protein

  • Proteins: Macromolecules made of long chains of amino acids. Critical functions include:
    • Catalyzing metabolic reactions
    • DNA replication
    • Response to stimuli
    • Providing structural support
    • Transporting molecules
Procedure: Testing for Protein
  1. Safety Precautions: Wear gloves and goggles.
  2. Prepare Test Tubes: Add deionized water to the 2-cm mark.
  3. Add Protein Solution: 10 drops to one tube, 10 drops of deionized water to the other. Mark accordingly.
  4. Add Biuret Solution: Add 20 drops each of Biuret A and B, mix gently, and observe for color changes.
Questions (Final Color)
  1. Protein Tube (Positive Control): Final color?
  2. Water Tube (Negative Control): Final color?

Testing for Lipids

  • Lipids: Include fats, phospholipids, and others, functioning in energy storage, cellular signaling, and cell membrane structure.
  • Generally hydrophobic, meaning they don’t mix readily with water.
Procedure: Testing for Lipids
  1. Safety Precautions: Wear gloves and goggles.
  2. Prepare Test Tubes: Add deionized water to the 2-cm mark.
  3. Add Vegetable Oil: 10 drops to one tube, 10 drops of water to another. Mark each tube.
  4. Add Sudan IV: 10-15 drops to each tube, shake gently and observe layer separation.
Questions (Final Color)
  1. Lipid Tube (Positive Control): Final color?
  2. Water Tube (Negative Control): Final color?

Part II: Testing Food for Macromolecules

  • Explore the macromolecular content in foods using previously established indicator tests. Note that it will only reveal the presence or absence of macromolecules, not their quantities.
Procedure: Testing Food Samples
  1. Form Predictions: Before testing, predict the presence of macromolecules for foods listed (e.g. Potato, French fries, Milk, Bread, Banana).
  2. Test Setup: Use four clean tubes for each food sample with the appropriate test solution per macromolecule.
  3. Heat Testing Solutions: Place appropriate tubes in hot water bath when testing for glucose.
  4. Compare Results: Analyze against positive/negative controls from Part I.
Table 2: Predictions for Food Experiments
  • Fill the following with predictions:
  • Food | Glucose | Starch | Protein | Lipids
  • Potato
  • French fries
  • Milk
  • Bread
  • Banana
Table 3: Results of Food Experiments
  • Record test results: + for positive, - for negative.
  • Food | Glucose | Starch | Protein | Lipids
  • Potato
  • French fries
  • Milk
  • Bread
  • Banana
Questions Post Testing
  1. List food items tested and macromolecules present.
  2. Explain why potatoes contain starch.
  3. Discuss macromolecules in French fries and their reasoning.
  4. Identify foods testing positive for lipids.
  5. What does a negative Biuret test indicate in orange juice?

Clean Up Procedures

  • Ensure a clean lab space by following these steps:
    1. Dispose of all liquids in the liquid waste flask.
    2. Clean all test tubes with soap and brushes.
    3. Position test tubes upside down in the rack for draining.
    4. Tidy your workstation and wipe down with disinfectant.
    5. Wash hands before exiting the lab.

Part III: Molecular Structure

Molecular Models Procedure
  1. Examine molecular shapes of specified molecules using the model kit.
  2. Assemble glucose and amino acid models; draw them.
  3. Review triglycerides and phospholipids and sketch them as instructed.
Questions for Amino Acids
  1. List three functional groups attached to the central carbon:
    1) .
    2) .
    3) .
  2. Identify the polymer form of amino acids:
    Polymer: _ .
  3. Define the reaction occurring during bond formation:
    Reaction name: ____ .
Carbohydrates: Simple vs. Complex
  1. Construct a glucose model, have it checked, and draw it.
    • Molecular Formula for Glucose: _ .
  2. Identify the bond forming complex carbohydrates:
    • Bond: ___ .
  3. Define the reaction involving water loss during bonding:
    • Reaction name: ___ .
Lipids: Triglyceride vs. Phospholipid

  1. Describe the two components of a triglyceride:

    • 1st Component: _ .
    • 2nd Component: _ .

  2. Assess lipid solubility in water:

    • Mixing: Yes/No. Justification: ___ .

  3. Draw and label a triglyceride.

  4. Describe phospholipids’ structure and function:

    • Amphipathic Molecule: Contains hydrophobic fatty acid tails and a hydrophilic head.