Download Biochemical Testing of Macromolecules

Pre-Lab 6: Biochemical Testing of Macromolecules (10 pts)
Name: _______________________________ Lab Section: _______________ Grade: _______
1. Which
a.
b.
c.
of the following is a mismatch between the subunit and the polymer?
Amino acids ------- protein
Deoxyribonucleotides ------ RNA
Glucose ------- starch or glycogen
2. Which
a.
b.
c.
d.
test will indicate the presence of starch?
Biuret test
Benedict’s test
Iodine test
None of the above
3. To test for the presence of proteins, you use _______________________.
4. Benedict’s solution is commonly used to test for the presence of _________________.
5. In Biuret’s test, what indicates a positive result?
6. Which
a.
b.
c.
d.
of the following is NOT a reducing sugar?
Fructose
Glucose
Lactose
Sucrose
7. Sudan
a.
b.
c.
d.
IV stains the lipid layer what color?
Blue
Red
Purple
Black
8. Can the diphenylamine test differentiate DNA and RNA? Why or why not?
9. Name the two tests in this experiment that require boiling.
10. Explain the difference between a negative control and a positive control.
BIOL 2281, Spring 2016
E6: Testing of Biomolecules Procedure/Report
Experiment 6: Biochemical Testing of Macromolecules
Objectives: At the end of this exercise, you should be able to
1. Describe the structures and functions of the four main categories of biologically important
macromolecules.
2. Perform chemical tests to identify the presence of lipids, proteins, two forms of
carbohydrates, and DNA.
3. Understand and explain the importance of control experiments.
Introduction
Cells are composed of five broad categories of chemical molecules: inorganic molecules,
carbohydrates, lipids, proteins and nucleic acids. The last 4 are organic molecules. For the most
part, each of these four types of macromolecules is composed of smaller subunits held together by
covalent bonds resulting in very large molecules (macromolecules). These macromolecules then
provide much of the structure and function of living cells.
Carbohydrates:
Carbohydrates are composed primarily of the elements carbon (C), hydrogen (H) and oxygen (O) at a
ratio of 1:2:1, respectively. An important characteristic of carbohydrates is that they are polyhydroxyl
aldehydes and ketones, meaning that they have a double-bonded oxygen (=O) attached to one of
their carbon atoms and hydroxyl (-OH) groups attached to the others. “Simple sugars” is just another
name for mono- and disaccharides: sweet substances with which we are familiar, such as glucose,
fructose, and sucrose (table sugar). Polymers of simple sugars bound by covalent bonds are called
polysaccharides. Starch, glycogen, and cellulose are all examples of polysaccharides. Carbohydrates
function primarily as energy storage and structural molecules in cells.
Benedict’s reagent can be used for a certain class of sugars, the reducing sugars. Reducing
sugars possess free aldehyde (-CHO) or ketone (-C=O) groups that reduce weak oxidizing agents
such as Benedict’s reagent. Most monosaccharides, but only some disaccharides, are reducing
sugars. Benedict’s reagent contains a cupric (copper) ion complexed with citrate in an alkaline
solution. It will react with reducing sugars, turning the solution from blue to green in the presence of
small amounts of these molecules, and turning the solution yellow-orange or even red in the presence
of larger amounts.
The iodine test is used to determine the presence of starch. The basis for this test is that starch is
a coiled polymer of glucose; iodine (iodine-potassium iodide, I2KI) interacts with these coiled
molecules and becomes bluish-black. A yellowish-brown color (i.e. no color change) is a negative test
for starch.
Proteins:
Proteins are polymers of amino acids joined together by peptide bonds. Each amino acid
contains a carbon to which an amino group (-NH2), a hydrogen (H), a carboxyl group (-COOH) and
another variable group (generically designated as –R), that varies in each of the 20 naturally-occurring
amino acids, are attached (See Figure below). Peptide bonds (See Figure below) form between the
carboxyl group in one amino acid and the amino group of another to form a chain designated as
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BIOL 2281, Spring 2016
E6: Testing of Biomolecules Procedure/Report
primary protein structure. Secondary protein structure forms when the chain coils in an alpha () helix
or when two or more sections of the chain lie next to each other and fold into beta () pleaded sheets.
Tertiary structure forms when the chain with its alpha helices and beta pleaded sheets fold back on
itself. This structure is then held together by a variety of hydrogen bonds, ionic bonds, disulfide
bonds, van der Waals interactions and hydrophobic interactions. If two or more proteins join, they
form quaternary protein structure.
The Biuret test detects proteins by reacting specifically with the peptide bonds (C-N bonds) and
produces a violet color. The Biuret reagent (1% solution of copper sulfate) must complex with four to
six peptide bonds to produce the purple color. Free amino acids, or very short chain peptides, are not
able to turn Biuret reagent purple, but they may produce a pink color instead.
Lipids
Lipids are actually a heterogeneous group of substances but can generally be characterized by the
fact that these molecules are hydrophobic (do not dissolve in polar solvents like water). One of the
most common types of lipids is the triglyceride. This molecule consists of a glycerol backbone that is
connected to three fatty acids by ester linkages. The fatty acids consist of long chains of carbons
bound to hydrogen only. Lipids usually function as energy storage molecules in cells or as structural
components of the membranes (phospholipids and sterols). The presence of lipids can be detected
by lipid’s ability to selectively absorb pigments in fat-soluble dye, such as Sudan IV (which
stains the lipid layer red).
Nucleic Acids
Nucleic Acids are polymers of individual nucleotide subunits joined together by phosphodiester
linkages. Each nucleotide (see Figure below) consists of three parts: a pentose sugar (deoxyribose in
DNA and ribose in RNA), a nitrogenous base (thymine, adenine, guanine or cytosine in DNA or uracil,
adenine, guanine or cytosine in RNA) and a phosphate group. RNA consists of a single chain of
nucleotides and DNA consists of two chains that coil into a double helix. The nucleic acids DNA and
RNA function in information storage and transfer based on the sequence of nitrogenous bases in the
molecules.
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BIOL 2281, Spring 2016
E6: Testing of Biomolecules Procedure/Report
DNA can be chemically identified by the Dische diphenylamine test. Deoxyribose reacts with
diphenylamine to form a blue complex under acidic conditions. The intensity of the blue color is
proportional to the concentration of DNA.
Controlled Experiments
A control is an experiment that is carried out to provide a basis of comparison to other
experiments. In our case, controls are known solutions which we can use to make sure our
experimental procedure is working. A positive control contains the variable you are attempting to
detect. If your positive control reacts in the way you expect, it indicates that your experimental
procedure and reagents are working correctly. A negative control does not contain the variable you
are attempting to detect and in our case includes only the solvent (water). It shows what a negative
result looks like so that they can be differentiated from positive results.
Procedures: (work in a group of 4 students)
Material on the supply bench for each group:

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

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
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4 Orange racks for small test tubes
30 clean, dry small test tubes
1 small beaker (250 ml) for boiling some of the reactions
1 hot plate
2 Metal clamps for holding hot tubes in water bath.
Masking tape
Marker pen
PARAFILM: 3 small pieces.
Testing materials (onion juice, potato juice, 1% sucrose, 1% glucose, 1% starch, milk,
eggwhite, BSA protein solution, 1% glycine, vegetable oil)
Testing reagent (Benedict’s, Iodine, Biuret, Sudan IV, 2.5% NaOH)
Demo material (total yeast RNA, calf thymus DNA, diphenylamine)
2 clean slides in blue slide box (to be reused between sections)
Unknown sample for each student
The Benedict’s Test For Reducing Sugars:
1. Using masking tape label a set of test tubes with A to G.
2. Add about 10 drops of each test solution to the appropriate test tube according to Table 1,
then add 0.5 ml of the Benedict’s solution to each test tube. Mix sample thoroughly before
transfer it to your tubes.
3. Shake the tubes gently to mix and then place them in a boiling water bath for 5 minutes.
4. Note any color changes. Any color that is not blue indicates the presence of reducing sugars.
Record your results in Table 1.
The Iodine Test for Starch (a polysaccharide):
1. Using masking tape label a set of test tubes with A to G (Table 1).
5. Add about 10 drops of each solution to the appropriate test tube according to Table 1. Mix
sample thoroughly before transfer it to your tubes.
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BIOL 2281, Spring 2016
E6: Testing of Biomolecules Procedure/Report
2. Add 5 drops of the Lugol’s Iodine (I2KI) solution and note any color changes. A blue to blueblack color usually indicates the presence of starch.
3. Record your results in Table 1.
Table 1: Solutions and Color Reactions for Benedict’s Test and Iodine Test
Benedict’s Color Reaction Iodine Color Reaction
Tube Solution
10 drops of onion juice
A
B
10 drops of potato juice
C
10 drops of 1% sucrose solution
D
10 drops of 1% glucose solution
(Control for benedict’s)
10 drops of 1% starch solution
(control for Iodine)
10 drops of milk
E
F
10 drops of distilled water
(Control)
G
The Biuret Test for the Presence of Proteins:
1. Using masking tape label a set of test tubes with H to L (Table 2).
2. Add 1 ml of each test solution to the appropriate test tube according to Table 2.
3. Add 1 ml of 2.5% sodium hydroxide (NaOH) solution to each test tube and gently mix.
CAUTION: SODIUM HYDROXIDE CAN CAUSE BURNS.
4. Add 5-10 drops of the Biuret reagent (1% solution of copper sulfate (CuSO4)).
5. Observe for a color change. Violet color usually indicates the presence of proteins.
6. Record your observations in Table 2.
Table 2: Solutions and Color Reactions for the Biuret Test
Tube
H
Solution
1 ml egg white
I
1 ml milk
J
1 ml amino acid solution (1% glycine)
K
1 ml distilled water (control)
L
1 ml Protein solution (1% Bovine Serum Albumin)
(Control)
Color
The Sudan IV Test for Lipids:
1. Label a set of test tubes with the following labels: M, N, O.
2. Add solutions according to Table 3 and shake well with the stretched Parafilm on top of the
tube. When a lipid is added to water, you will observe two layers and Sudan IV should be
present in the top lipid layer.
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BIOL 2281, Spring 2016
E6: Testing of Biomolecules Procedure/Report
3. Record your results in Table 3.
Table 3: Solutions and Color Reactions for the Sudan IV Test
Tube
M
Solution
1ml water l + 5 drops of Sudan IV
N
1ml water + 10 drops of vegetable
oil + 5 drops of Sudan IV
1ml water + 10 drops of vegetable
oil + 5 drops of detergent (Triton
X-100 ) + 5 drops of Sudan IV
O
Description of Reaction
The Dische diphenylamine Test for DNA (demonstration in chemical hood)
1. (TA) Add the material listed in Table 4 to each tube and then add 1 ml of the Dische
diphenylamine. Mix thoroughly.
2. (TA) Place the tubes in a gently boiling water bath to speed up the reaction for 10 minutes.
3. (TA) Gently mix and observe the colors. (Student) Record results in Table 4.
Table 4: Dische Diphenylamine Test for DNA (DEMO – observe in fume hood)
Tube
Solution
1
1ml calf-thymus DNA solution
3
1ml yeast total RNA solution
4
1ml water
Color
Unknown Identification:
1. Each student obtains a numbered tube of an unknown solution. The possibilities are: sucrose,
glucose, starch, and BSA protein. Mix the sample thoroughly before you transfer it to
your experiment test tubes.
2. Run the necessary tests to figure out the identity of the solution. Be sure to include positive
and negative controls for your tests. Return the sample tube and dropper to TA.
3. Show your results to your TA before you clean the tubes.
4. Present your procedure (listed steps) and result (tables) clearly in the lab report.
Cleaning Up:
Each group must:
a) Dispose of the test tubes containing vegetable oil in the broken glass box.
b) Dispose of slides and slide covers in the broken glass box.
c) Rinse all of the used test tubes with tap water a couple of times, SCRUB with the tube
brush, rinse again, hold up to the light to make sure tube is clean, and then place the
clean tubes upside down in the metal basket.
Lab Report: (20 pts total): Must have a title page.
Part I: Answer the following questions. Include questions 1-10 in your report.
1) Present a typed version of Table 1, complete with your observations. Which of the solutions is
a negative control? What does this result tell you? (2 pts)
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BIOL 2281, Spring 2016
E6: Testing of Biomolecules Procedure/Report
2) Glucose in the urine, glycosuria, can be an indicator of diabetes. Which test could you use to
determine if a patient’s urine contained glucose? (1 pt)
3) How can a substance taste sweet, yet give a negative reaction with the Benedict’s test? (1pt)
4) What have you learned about how carbohydrates are stored in onions and potatoes from the
experiments performed in Table 1? (1 pt)
5) Present a typed version of Table 2, complete with your observations. Which of the solutions in
Table 2 is a positive control? What is the purpose of this control? (list 2 purposes) (2 pts)
6) Suppose you have tested an unknown sample with Biuret and Benedict’s reagents. The
solution mixed with Biuret reagent is blue. The solution boiled with Benedict’s reagent is also
blue. Can you conclude the identity of the sample? Why or why not? (1 pt)
7) What can you conclude about the molecular content of milk based on all of the tests you
performed on milk? (1 pt)
8) Present a typed version of Table 3, complete with your observations. What can you conclude
about the function of the detergent comparing test “N” and “O” in table 3? (In other words,
what did the detergent do?) (2 pts)
9) Present a typed version of Table 4, complete with your observations. Could the Dische
diphenylamine test tell you if a DNA sample is contaminated with RNA? Why or why not?
(2 pts)
10) Hand-draw (2 pt)
a) The ring form of a glucose molecule
b) A generic amino acid structure
c) A generic nucleotide structure
d) A triglyceride molecule
Part II: Unknown identification
1) Present the results of your tests in typed table format (with proper titles). Use the tables in
this handout as examples. You should describe the color of each reaction in your tables
AND indicate (+) or (-) for the test result. If you did not perform a test, indicate that in the
table. (2 pts)
2) Write a simple summary or conclusions paragraph (4-5 sentences) stating the identity of
your unknown and how you ruled out other possibilities. TO RECIEVE CREDIT YOU
MUST INDICATE THE NUMBER OF YOUR UNKNOWN IN THIS PARAGRAPH. (3 pts)
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E6: Biochemical Testing of
Macromolecules
The “Suspects” and Their Tests
• Carbohydrates Structure and Tests
– Benedict’s Test
– Iodine Test
• Protein Structure and Test
– Biuret Test
• Lipid Structure and Test
– Sudan IV Test
• Nucleic Acid Structure and Test
– Diphenylamine Test
Polymer Principle
• Macromolecules (except lipids) are polymers
consisting of many small repeating molecules
called the monomers
•
•
•
•
Carbohydrates: Polymer of monosaccharides
Proteins: Polymer of amino acids
Nucleic Acids: Polymer of nucleotides
Lipids: Based on hydrocarbons
Monosaccharides
• Chemically,
monosaccharides
are either
polyhydroxyaldehydes (aldose)
or polyhydroxyketones (ketose)
• Can be also
classified by the
number of carbons.
Cyclization of aldoses and ketoses
•
•
The carbonyl carbon of an aldose (having at least 5-C) or of a ketose (having at least
6-C) can react with an intramolecular hydroxyl group to form a cyclic form.
In solution, cyclic aldoses and ketoses exist in equilibrium with noncyclic versions
Dr. Pickett & Dr. Lin – Spring 2016
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Polysaccharides--Cellulose
Polysaccharides: Starch and Glycogen
Benedict’s Test for Reducing Sugars
Benedict’s Test Results
• Benedict’s test is used for detecting reducing sugars:
Boil Mixture for 3 min
• Sugars that contain aldehyde groups that are oxidized to
carboxylic acids are classified as reducing sugars.
• Ketoses can also be reducing sugars if they contain alphahydroxy-ketones or if they can isomerise to aldoses
during the reaction.
• Reducing sugars include: glucose, fructose,
glyceraldehyde, arabinose, lactose, and maltose
• Sucrose is not a reducing sugar (the glycosidic bond
between it’s components, fructose and glucose, prevents
their isomerization)
Iodine Testing for Starch
• Iodine interacts with coiled polymer of glucose
and becomes black
•
Blue: -
• Green: +
• Yellow: ++
• Orange: +++
•
Red/Brown: ++++
• Why is water blue??????
Iodine Testing for Starch
• Procedure and Results
– add drops of Iodine Solution (iodinepotassium iodide: I2KI) to the substance
being tested
– At room temperature
• Bluish Black: ++
• Yellow Brown/orange: -
Dr. Pickett & Dr. Lin – Spring 2016
2
Proteins: Polymers of amino acids
Peptide Bonds
in Proteins
Biuret Test for Proteins
• Cu++ react with at least 4-6 peptide bonds to
produce color change to the Biuret reagent
• Procedure and Result
Four Levels
of Protein
Structure
Lipids: Fats, Steroids, and
Phospholipids
– Mix drops of Biuret Reagent to the material and
NaOH
– Results at Room Temperature
• Light blue: • Slight purple: +
• Deep violet: ++
Dr. Pickett & Dr. Lin – Spring 2016
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Sudan IV Test for Lipids
• Sudan IV is a fat-soluble dye; will stain the lipid
layer only
• Emulsifier: a type of surfactant with hydrophilic
and hydrophobic groups. (ex: Triton X-100)
Diphenylamine Test For DNA
• Acidic conditions convert deoxyribose to a
molecule that binds with diphenylamine to
form a blue complex
Unknown Identification
• Possible candidate solutions
–
–
–
–
Glucose
Sucrose
Starch
Protein
• Available reagents:
– Benedict’s
– IKI (Lugol’s Iodine)
– Biuret’s
The Importance of Control
Reactions
• Positive Control
– Shows that your test or experiment runs
correctly (procedures, reagents, equipment)
• Negative Control
– Does not contain the variable for which you are
testing; it shows you what a negative result
looks like.
Dr. Pickett & Dr. Lin – Spring 2016
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