Download Lab 2: Macromolecules Life on earth is based on the element

Lab 2: Macromolecules
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Lab 2: Macromolecules
THE MOLECULES OF LIFE
Life on earth is based on the element carbon and compounds containing carbon. When carbon is
combined with hydrogen and other atoms this makes organic molecules. Carbon chains form
the skeletons of most organic molecules. The skeleton varies in length and arrangement. This
variation allows for the molecular complexity and diversity seen in living matter. The distinct
properties of these molecules depend not only on this complexity and variation, but also the
chemical groups that are attached to the carbon skeleton. These chemical groups are called
functional groups. Each functional group has a unique structure, and along with it, gives each
molecule unique properties essential for life.
Important Biological Functional Groups
Lab 2: Macromolecules
The large molecules important for all living things fall into four categories: carbohydrates, lipids,
proteins, and nucleic acids. All of these, Proteins, Carbohydrates, Lipids and Nucleic Acids are
very large molecules called macromolecules. In this lab, we will be conducting tests that reveal
properties of carbohydrates, lipids, and proteins. Nucleic acids will be studied in a later lab.
CARBOHYDRATES
Carbohydrates are sugars and polymers of sugars. They occur in a wide variety of monomers
(monosaccharides) and polymers of saccharides (disaccharides or polysaccharides).
Monosaccharides, or simple sugars, are aldehydes or ketones with two or more hydroxyl groups
attached. Examples include glucose, fructose, and ribose. A disaccharide is two joined
monosaccharides and these are the simplest polysaccharides. Examples include sucrose and
lactose. They are composed of two monosaccharide units bound together by a covalent bond
known as a glycosidic linkage formed via a dehydration reaction, resulting in the loss of a
hydrogen atom from one monosaccharide and a hydroxyl group from the other.
A
polysaccharide is a sugar made up of three or more sugar units attached by glycocidic linkages.
Examples include cellulose found in the cell wall of plants and starch used by plants for storage.
Carbohydrates are a major source of fuel for metabolism. Since no single test can be used as a
marker for all of them, you will conduct two tests specific to two of the important classes of
carbohydrates: The Benedict’s test, which tests for reducing sugars (any sugar that has an
aldehyde or can form one) which includes all monosaccharides and some disaccharides and
Lugol’s test which tests for the presence of starch.
1. BENEDICT’S TEST FOR REDUCING SUGARS
Benedict's reagent is used as a test for the presence of reducing sugars. Benedict's test will detect
the presence of aldehydes, and alpha-hydroxy-ketones. A positive test with Benedict's reagent is
shown by a color change from clear blue to a brick-red precipitate. Benedict's reagent contains
blue copper ions (Cu2+) which are reduced to copper (ions (Cu+). These are precipitated as red
copper oxide which is insoluble in water. Benedict's Reagent provides a quantitative test for
reducing sugars along with a qualitative test. The color of the obtained precipitate gives an idea
about the quantity of sugar present in the solution. A greenish precipitate indicates about 0.5%
concentration; yellow precipitate indicates 1% concentration; orange indicates 1.5% and red
indicates 2% or higher concentration.
Demonstration of Benedict’s test:
a. Glucose + Benedict’s solution ______________________________________________
b. Glucose + Benedict’s solution + heat _________________________________________
Lab 2: Macromolecules
Procedure for Benedict’s test
Label five test tubes 1-5 and place 2 ml of the following solutions in the corresponding tubes:
1.
2.
3.
4.
5.
1% glucose
1% sucrose
1% starch
1% ribose
1% distilled water (DI H2O)
Add 2 ml of Benedict’s solution to each tube and heat in a boiling water bath for 5 minutes; note
the color change in the following chart, using the key below to indicate degree of change.
–
++
+++
++++
a.
b.
c.
d.
blue
green
orange
red
Under the conclusion column, note the reason why it did or did not change (ex. It is a
polysaccharide). Use the figures on the next page to assist you with your conclusions. Circle
the aldehyde or ketone on the diagrams (not the whole molecule) on the next page for the
sugars that reacted with Benedict’s solution.
Tube
Contents
1
Glucose
2
Sucrose
3
Starch
4
Ribose
5
DI H2O
Color
(after heating)
Results
(+ or -)
What is the purpose of using the distilled water in this test?
Conclusions
Lab 2: Macromolecules
Carbohydrate Structures
Sucrose
Starch
DI Water
Lab 2: Macromolecules
2. IODINE TEST FOR THE PRESENCE OF STARCH
Starch is a long polymer consisting entirely of glucose (a monosaccharide). Starch is the major nutrient
storage polysaccharide in plants and their seeds. Starch itself is composed of subunits of small
polysaccharides (amylose and amylopectin) which in turn are composed of chains of glucose molecules.
Iodine solution contains iodine and potassium iodide. The coiled structure of the polysaccharide starch
produces a dark, blue-black color in the presence of iodine, which is usually yellow to brownish in color.
Because this reaction does not occur with monosaccharides or disaccharides, the blue-black color is
considered to be positive for starch.
Demonstration of Iodine test:
a. Iodine ___________________________________________________________
b. Iodine + Starch ____________________________________________________
Procedure for Iodine test
Label four test tubes 1-4 and place 2 ml of the following solutions in the corresponding tubes:
1.
2.
3.
4.
1% glucose
1% sucrose
1% starch
1% distilled water (DI H2O)
Add 2 ml of Iodine solution to each tube and mix; note the color change in the following chart.
(Use + or -, note colors and explain conclusions)
Tube
Contents
1
Glucose
2
Sucrose
3
Starch
4
DI H2O
Color
Results
(+ or -)
Conclusions
Lab 2: Macromolecules
LIPIDS
Lipids are a diverse group of large biological molecules that are not made up of true polymers
and are not usually large enough to be considered macromolecules. Members of this class of
molecules have one thing in common, they are hydrophobic. This property means they mix
poorly or not at all. A typical fat molecule is made up of three fatty acids attached to a glycerol
molecule (triglyceride). Lipids can also be in the form of phospholipids and steroids (such
cholesterol). Lipids are used for energy, insulation, membrane structure and as chemical
messengers.
3. VISUAL AND SUDAN TEST FOR THE PRESENCE OF LIPIDS
Lipids are hydrophobic and will separate when itself when dropped in water. Sudan is a special type of
dye called a lysochrome that is typically used by histologists to demonstrate the presence of triglycerides
in frozen tissue sections. It has a distinct orange red color that makes it easy to identify lipids in an
aqueous solution. In our lab, we will use Sudan to detect the presence of lipids in various aqueous
solutions. Exercise caution when using this dye. It is hydrophobic, and will dye your tissues as well as
your clothing. The contents of any test tube containing Sudan must be poured into a discard bottle, not
into the sink! Always tightly cap Sudan dye when you have dispensed the amount you need.
Lab 2: Macromolecules
Procedure for Visual tests
Add three drops of salad oil to 10 ml of water. Mix thoroughly. Immediately observe the mixture. Let it
stand for three minutes and then answer the following questions.
Does the oil separate from the water after standing a few minutes?
Where is the oil located? What can you say about the relative densities of oil and water?
In light of your answer to the previous question, what function (other than energy storage) does fatty
blubber serve in whales?
Procedure for Visual tests (continued)
Fat and oil form a translucent spot when placed on unglazed paper, such as a paper towel or a paper bag.
The translucent spot admits the passage of light, but not sufficiently to distinguish objects by looking
through it. By contrast, water soluble substances will leave a water spot that will dry completely
preventing the passage of light.
Obtain a piece of paper bag.
Use a pencil to draw four circles approximately the size of a silver dollar so that all of the circles are
spatially separated.
Label each circle as Di water, albumin, glucose, salad oil.
Place a drop of each corresponding substance into the appropriate circle.
Allow the paper towel to sit on the desk top undisturbed until the water spot is completely dry
(approximately 30 minutes).
Hold the paper towel up to the light and determine which sample is translucent. Record your data in the
table on the next page, in the column titled “Translucent”.
Lab 2: Macromolecules
Demonstration of Sudan test:
a. Sudan Solution _______________________________________________________
b. Sudan Solution + Lipid ________________________________________________
Procedure for Sudan’s test
Label four test tubes 1-4 and place 10 ml of DI water. Then add 2 ml of the following solutions
in the corresponding tubes:
1.
2.
3.
4.
DI water
1% albumin
1% glucose
salad oil
Add 10 drops of Sudan to each test tube and mix. Record your results in the table below.
Tube
Contents
1
DI H2O
2
Albumin
3
Glucose
4
Salad Oil
Bag
Translucent
(+ or -)
Color
(after Sudan)
Tubes
Results
(+ or -)
Conclusions
PROTEINS
Proteins include a diversity of structures and have a wide range of functions. Proteins are
structural components of cell and organelle membranes as well as muscle and tendons. The can
act as enzymes (biological catalysts), they function as chemical messengers (hormones) and
have a central role in the functioning of the immune system. Proteins are large molecules
composed of amino acids. All amino acids share a common structure. They are a molecule that
has an amino group, a carboxyl group, a hydrogen atom, and an R (side) group which differs
with each of the twenty side chains. The subunits are linked together with the carboxyl group of
one amino acid forming a bond with the amino group of another creating a peptide bond.
Lab 2: Macromolecules
4. THE BUILDING BLOCKS OF PROTEINS
Pepsin is an enzyme that works in the stomach to break down proteins. The silver halide on the
photographic film is held on by an emulsion of protein. If there is protein digestion, the clear
cellulose backing will be exposed. Enzymes work under very specific conditions such as
temperature and pH.
Procedure for Protein Digestion
Label four test tubes 1-4 and place the following solutions in the corresponding tubes:
1.
6 ml of 7.0 buffer
2.
3 ml of 5% pepsin (enzyme) + 3 ml of 0.2% HCL
3.
3 ml of 0.2% HCL + 3 ml of DI H2O
4.
3 ml of 5% pepsin (enzyme) + 3 ml of 7.0 buffer
Add a strip of photographic film to each tube and incubate at 37 degrees Celsius for one hour.
Record the results in the table below.
Tube
1
2
3
4
Contents
Results
(+ or -)
Conclusions
Lab 2: Macromolecules
What conditions do you think are necessary for pepsin to work?
What are the building blocks of proteins?
Practice writing a null and alternative hypothesis for this specific experiment
Ho: _____________________________________________________________________
Ha: __________________________________________________________________________
5. BIURET’S TEST FOR THE PRESENCE OF PROTEINS
Biuret reagent is composed of CuSO4 (copper sulfate) and KOH (potassium hydroxide). It
reacts only with amino acids that are bound together by peptide bonds to form proteins. The
copper sulfate (CuSO4) used in the biuret test is normally light blue colored and turns violet in
the presence of peptide bonds in a strong alkali solution. A negative test, therefore, remains light
blue in color.
Demonstration of Biuret’s test:
a. Biuret’s Solution _________________________________________________________
b. Biuret’s Solution + protein _________________________________________________
Procedure for Biuret’s test
Label four test tubes with the numbers 1-4 and add 3 ml of 10% NaOH to each test tube. Add
the following solutions in the corresponding tubes:
1.
2.
3.
4.
3ml of water
3ml of 1% albumin
3ml of 1% glucose
3 ml of 1% starch
Add 1% CuSO4 to each test tube one drop at a time until a violet or pink color appears. NOTE:
Adding CuSO4 too rapidly can result in a false negative! Start with tube #1 and add one drop to
each tube, then repeat the process until one of the tubes turns pink or violet. Record your results
below.
Lab 2: Macromolecules
Tube
Contents
Color
(Before)
Color
(after)
Results
(+ or -)
Conclusions
1
2
3
4
7. Unknown
Now it is time to apply what you learned. Your instructor will provide you with an unknown.
Each unknown has a unique number. Your unknown may contain: protein, starch, reducing
sugar, fats or distilled water. Remember that your unknown may be a combination of these
things, so be certain you complete all tests before reporting your results. For each test
performed, use 1ml of unknown.
YOU MUST GET YOUR INSTRUCTORS INITIALS WHEN YOU HAVE DETERMINED
THE RESULTS TO GET CREDIT!
Record your unknown number here: _______________________
Test
Benedict’s
Lugol’s
Sudan III
Biuret’s
Tests for
Results
(+ or -)
Conclusions