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LAB 4 – Macromolecules
Overview
In addition to water and minerals, living things contain a variety of organic molecules. Most of the organic molecules in living organisms are of 4 basic types: carbohydrate , protein , lipid and nucleic acid. Many of these molecules are long polymers , and thus collectively referred to as macromolecules. In this laboratory you will learn how organic molecules are put together, with an emphasis on proteins and carbohydrates. In addition, you will learn several methods of detecting carbohydrates and proteins in complex samples such as foods.
Part 1: BUILDING MACROMOLECULES
The Structure of Organic Molecules
All living things consist of both organic and inorganic molecules. Organic molecules contain the elements carbon (C) and hydrogen (H), and more specifically, carbon–hydrogen bonds. Molecules lacking C–H bonds are considered to be inorganic. For example, oxygen gas (O 2 ), water (H 2 O) and carbon dioxide (CO 2 ), despite their obvious importance for life, are inorganic molecules. Methane (CH 4 ), ethanol (C 2 H 6 O) and glucose (C 6 H 12 O 6 ) on the other hand, are all organic. In general, organic molecules are derived from living organisms, hence the association of the word organic with natural, living things.
It is helpful to think of organic molecules as skeletal carbon structures ( carbon skeletons ) to which various chemical groups are attached. To illustrate this, let’s take a look at the structure of a simple organic molecule that we are all familiar with, ethanol :
Notice that a molecule of ethanol contains a core carbon structure or skeleton consisting of 2 carbon atoms connected to each other by a single covalent bond. The remaining unpaired electrons in the carbon atoms are involved in covalent bonds with individual hydrogen atoms or a hydrogen-oxygen combination known as a hydroxyl group. The single hydrogen atoms and the hydroxyl group are examples of common functional groups (though a hydrogen atom technically is not a “group”) that are attached via covalent bonds to carbon skeletons. Let’s look at another slightly larger organic molecule, the simple sugar glucose :
ethanol
If you look carefully you’ll notice that the glucose molecule above (shown in both its linear and ring forms) is simply a 6-carbon skeleton to which numerous hydrogens and hydroxyl groups are attached (as well as a double-bonded oxygen).
Hydrogen atoms and hydroxyl groups are by no means the only functional groups found in organic molecules, so let’s get acquainted some other common functional groups in addition to these two and take note of their chemical properties when bound to a carbon framework:
functional group molecular formula property (at pH 7)
hydrogen – H non-polar
hydroxyl – OH polar
methyl – CH 3 non-polar
amino – NH 2 basic (binds H+)
carboxyl – COOH acidic (releases H+)
(or carboxylic acid)
Exercise 1A – Constructing functional groups
Much like you did in a previous lab, diagram the structural formulas for the functional groups shown above on your worksheet and then build them with your molecular model kit. In your structural formulas, represent the bond that will connect to a carbon skeleton as a line sticking out from your function group. In your models, each functional group should have a covalent bond connector that is not connected to anything on one side.
Here is a key to the components of your kit:
WHITE = hydrogen atom RED = oxygen atom BLACK = carbon atom BLUE = nitrogen atom short connectors (use for single covalent bonds) long connectors (use in double & triple covalent bonds)
glucose
(a monosaccharide)
All amino acids have in common the first 3 functional groups: the hydrogen, amino and carboxyl groups. Proteins are constructed from up to 20 different amino acids, and the “R” group is different for each giving each amino acid its unique properties. Let’s examine the “R” groups (highlighted in green) of six different amino acids, after which you and a partner will assemble one amino acid using a molecular model kit:
Exercise 1B – Building an amino acid
1. On your worksheet, diagram the structural formula for the amino acid you are assigned to build.
Note: the diagrams shown are partial structural formulas to help guide you in determining the complete structural formula (with all covalent bonds shown)
2. Working in pairs, build the amino acid with your molecular model kit as follows:
a) to a central carbon atom, attach the following functional groups you’ve already made: a hydrogen atom an amino group a carboxyl group
b) construct the “R” group for your amino acid separately
c) attach your “R” group to the remaining bond in your central carbon atom
Assembling and Breaking Down Polymers
Living organisms such as yourself are continuously building polymers and breaking them down into monomers. For example, when you eat a meal you ingest large amounts of polymers (proteins, starch, triglycerides) which are subsequently broken down into monomers (amino acids, glucose, fatty acids) within your digestive system. Within your cells there is a continuous cycle of building new protein, carbohydrate, lipid and nucleic acid polymers, and breaking down “old” polymers into their respective monomers (amino acids, sugars, fatty acids, nucleotides).
There is a common theme to the building and breaking down of biological polymers. Whenever a monomer is added to a growing polymer, a molecule of water (H 2 O) is released in a process called condensation or dehydration. For example, when two amino acids are joined in a growing polypeptide, the – OH of the carboxyl group of the first amino acid will combine with an H from the amino group of the second amino acid. This results in the simultaneous formation of a covalent bond between the two amino acids, a peptide bond , and release of a water molecule:
Exercise 1D – Breaking down a polypeptide
Break down the polypeptide your group has just assembled as follows:
1. Break the peptide bond between the last 2 AAs in your polypeptide. 2. Use your water molecule to restore the H on the amino group of AA just removed and the – OH on carboxyl group of the other AA. 3. Repeat steps 1 and 2 for each successive peptide bond until the polypeptide is completely broken down into its original amino acid monomers.
Part 2: DETECTING MACROMOLECULES
In the exercises to follow, you will test various food items for the presence of simple sugars, starch and protein using chemical reagents specific for each. When doing such tests it is always important to include control reactions. As you learned in the first lab, a control experiment is one in which the independent variable (e.g., the source of sugar, starch or protein in test samples) is “zero” or some background level. For example, if you are testing for starch you want to be sure to include a sample that you know does NOT contain starch. The perfect negative control for this and other such experiments is plain water since it does not contain starch or anything else. This sort of control is referred to as a negative control since it is negative for what you are trying to detect. The importance of performing a negative control is two-fold:
To verify that a negative sample actually gives a negative result with the reagents you are using.
To allow you to see what a negative result looks like for the sake of comparing with your other test samples.
You will also want to include a positive control for each of your experiments, i.e., a sample that DOES contain the substance you are testing. For example, when you test various foods for the presence of starch you will want to include a sample that you know contains starch. The ideal positive control in this case would be simply a starch solution (water with starch and nothing else). The importance of performing a positive control is also two-fold:
To verify that a positive sample actually gives a positive result with the reagents you are using.
To allow you to see what a positive result looks like for the sake of comparing with your other test samples. If either control fails to give the predicted outcome in a given experiment, then the results for all of your test samples are suspect. If your controls give the expected outcomes, then you can be confident that the results for your test samples are reliable.
Now that you understand the importance of performing positive and negative controls, you are ready to test the following foods for the presence of simple sugars , starch and protein :
milk banana extract coconut extract peanut extract potato extract butter*
*place in wide test tube and melt in hot water before testing
Exercise 2A – Detection of simple sugars
Benedict’s reagent is a chemical reagent that will reveal the presence of any monosaccharide as well as the disaccharides lactose, maltose or mannose (not sucrose). The reagent itself is blue, however when it reacts with monosaccharides (or the disaccharides indicated) it will change to a green, yellow, orange or reddish brown color depending on how much sugar is present (green to yellow if low levels, orange to reddish brown if high levels). Materials you will need include:
Benedict’s reagent 8 test tubes and a rack 6 food samples to test deionized water (negative control) glucose solution (positive control) boiling water
NOTE: Before you start, remove the hot plate from your drawer and plug it in. Half fill a large beaker with water, place it on the hotplate and turn on the heat dial ~halfway.
Test each of your 8 samples as follows:
1. Label each of your 8 test tubes accordingly (e.g., A1, A2, A3…). label the upper part of each tube so it won’t come off when you boil! 2. Add 1 ml of Benedict’s reagent (1 squeeze of dropper) to each tube. 3. Add 0.5 ml of the appropriate test sample to each tube, mix. 4. Boil all samples for 5 minutes. be sure beaker is no more than half full with boiling water to avoid overflowing! 5. Record the colors for each tube and determine whether or not sugars are present.
LABORATORY 4 WORKSHEET Name ________________________
Section_______________________
Exercise 1A – Constructing functional groups
Draw the structural formulas for the following functional groups:
**- OH – CH 3
Match each functional group below with the correct chemical property on the right ( choices may be used more than once ).
____ amino group A. acidic
____ carboxyl group B. basic
____ hydrogen C. polar
____ hydroxyl group D. non-polar
____ methyl group
Exercise 1B – Building an amino acid
Draw the structural formula for the amino acid you built with your molecular model kit. Circle and label the amino , carboxyl and R groups , and mark the central carbon with an asterisk (*).
amino acid ______________
Exercises 1C & 1D – Assembling and hydrolyzing a polypeptide
Draw the complete structural formula for the dipeptide your group assembled, and circle the peptide bond. Be sure to show your instructor your dipeptide and demonstrate its hydrolysis.
