Lab 2: Biomolecules
Before coming to the lab:
 Read the General Introduction, along with the Introductions from
Part 1, Part 2, Part 3, and Part 5, to refresh your memory of the
structures and various properties of the macromolecules discussed in
class. Note that the background information provided here is not
complete, and is insufficient for a developing a deep understanding of
the subject. Refer to your textbook to complete this picture.
 Complete the Hypotheses and Predictions for Benedict’s Test,
Lugol’s Test, Emulsion Test, and Biuret Test.
 Complete Part 6. Molecular Visualization of DNA and Hemoglobin,
by going online to view the modules and answering the questions asked
here.
LEARNING OBJECTIVES
By the end of this lab, you should be able to:
Conceptual:
 Describe the structures and properties of the major biomolecules commonly found
in cells, namely carbohydrates, lipids, proteins, and nucleic acids.
 Describe how the presence of the major biomolecules is detected through the use
of the following qualitative assays:
 Benedict’s test for reducing carbohydrates
 Lugol’s test for starch
 Emulsion test for lipids
 Biuret’s test for proteins
 Draw, describe, compare and contrast the 3-D structure of DNA and proteins,
particularly hemoglobin.
Procedural:





Identify reducing sugars using Benedict’s reagent.
Identify polysaccharides using Lugol’s reagent.
Distinguish between lipid and non-lipid substances using the Emulsion test.
Identify proteins using the Biuret test.
Use the knowledge gained from the different biomolecule assays to identify the
biomolecules present in unknown samples.
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GENERAL INTRODUCTION
The properties and functionality of cells are wholly dependent on the chemical properties
of the molecules (primarily organic) of which they are made. The first key to understanding
how cells function is to become familiar with the structure and properties of these
molecules. Most organic molecules found in cells can be classified as carbohydrates,
lipids, proteins, and nucleic acids (Sadava et al., 2008). Each of these classes of
molecules has specific properties that can be identified by simple chemical tests.
In this lab, you will be introduced to these molecules (and their smaller organic molecule
subunits) that make up cells and allow them to function, as well as to five biochemical
qualitative assays (assays that simply test for the presence or absence of a substance):
 Benedict’s test for reducing sugars
 Lugol’s test for starch
 Emulsion test for lipids
 Biuret’s test for proteins
Using computer molecular visualization, you will also study the 3-D structure of DNA,
along with the levels of organization of protein molecules, the latter using the molecular
structure of normal and sickle hemoglobin as an example.
PART 1. TESTING FOR CARBOHYDRATES
Introduction
Carbohydrates (literally, hydrates of carbon) are organic molecules with the general
formula Cn(H2O)n. Carbohydrates, also called saccharides, include monosaccharides
(single sugars), starches, cellulose, chitin and other sugar polymers. They are a very
important and diverse class of biomolecules that serve many functions, including structural
support, intercellular communication, and serving as a source of and storage form for
cellular energy. Individual sugars are called monosaccharides, while chains (polymers)
of sugars are called polysaccharides. Short chains consisting of a couple of dozen or
fewer monosaccharides are often referred to as oligosaccharides (Sadava et al., 2008).
Monosaccharides can have different-sized carbon backbones (e.g., 5-carbon sugars
called pentoses or 6-carbon sugars called hexoses). Glucose, the most important to us
biologically, is a 6-carbon sugar with the formula C6H12O6. Individual monosaccharide
molecules occur naturally as either linear molecules or as rings. The ring forms are more
stable in aqueous solution and hence are more common (Sadava et al., 2008). The linear
and ring forms of glucose and fructose (6-carbon sugar) are illustrated in Figure 2-1.
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Aldehyde
group
Glucose
Ketone group
Fructose
Figure 0-1. Linear and ring forms of glucose and fructose.
Monosaccharides can be covalently linked (polymerized) to form chains of anywhere from
two units (disaccharides) to thousands of monomers. These chains can be linear or
branched, straight or helical.
Disaccharides, oligosaccharides, and polysaccharides are linked by glycosidic bonds
formed by dehydration (also called condensation) reactions— so called because two H
atoms and one O atom are removed to produce a molecule of water (H2O) in the process
(Figure 0-2).
Commonly occurring disaccharides include:
 Sucrose (glucose + fructose), table sugar; found in sugar cane and beets.
 Maltose (glucose + glucose), malt sugar; often the product of enzymatic hydrolysis
of polysaccharides.
 Lactose (glucose + galactose), found in mammalian milk (Sadava et al., 2008).
Figure 0-2. Condensation reaction forming sucrose (a disaccharide).
Depending on the nature of the glycosidic bonds, different polysaccharides will have
different properties and functions within the cell. For example, starch and cellulose are
both polysaccharides composed of individual glucose units, but the links joining these
units together are different in each, resulting in the molecules having different properties
and performing very different functions: starch is an energy storage molecule in plants (a
similar molecule, glycogen, is used for energy storage in animals) that we are able to
digest, while cellulose is a structural component of plant cells that is indigestible by
3
humans (Sadava et al., 2008). As we shall see, these different chemical properties of
carbohydrates are exploited in the laboratory to help characterize their different types.
Benedict’s Test
Benedict’s reagent tests for reducing sugars, which are sugars that contain free carbonyl
groups (aldehyde or ketone functional groups; see Figure 2-1). Monosaccharides and
some disaccharides have these functional groups (though sucrose does not; see Figure
0-2), but polysaccharides do not. Benedict’s reagent contains the blue-coloured, oxidized
form of copper (Cu2+), in an alkaline solution. When added to a sugar solution, the alkaline
solution will linearize any ringed monosaccharides present, making their carbonyl groups
available. The carbonyl groups will then reduce Cu2+ to Cu+, producing a red-coloured
precipitate (Figure 0-3). Thus, a positive Benedict’s test will form a red precipitate,
indicating the presence of reducing sugars. A negative Benedict’s test will remain blue,
indicating that there were no reducing sugars in the test sample (Fylling, 2001; Helms et
al., 1998).
Figure 0-3. The Benedict’s reaction
When Benedict’s reagent is heated with a reactive sugar, such as glucose or maltose, the
colour of the reagent changes from blue, to green, to yellow, and finally to reddish-orange,
depending on the amount of reactive sugar present (Helms et al., 1998). Orange or red
indicates the highest proportion of these sugars (Table 0-1).
Table 0-1. Benedict’s test: colour changes depending on the amount of reactive sugar present.
Colour
Symbol
Description
Amount of sugar present
0
Blue
None
+
Green
Some
++
Yellow
More
+++
Orange
Much
++++
Red
Most
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Hypothesis and Predictions
In Table 0-2, formulate a hypothesis and predict what you might expect to find for each of
the solutions/suspensions to be tested with Benedict’s reagent.
Table 0-2. Hypothesis and predictions for Benedict’s test for reducing sugars.
Solution/Suspension
Hypothesis
Prediction
1. Water
2. 1% starch
3. 1% glucose
4. 1% sucrose
5. Milk
6. Onion juice
7. Potato juice
8. Apple juice
9. Vegetable oil
10. Egg white
11. Egg yolk
What is your null hypothesis for this group of tests?
______________________________________________________________________
______________________________________________________________________
What is the independent variable?
______________________________________________________________________
What is the dependent variable?
______________________________________________________________________
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Benedict’s Test Procedure
1. Set up a row of 11 test tubes and number them 1 through 11.
(Note: This test will be performed on the three unknown samples also;
you may wish to set these up at the same time.)
2. Add 2 mL of each of the solutions listed in Table 0-3 to the test tubes, matching each
number to the number on the tube.
3. Add approximately 2 mL of Benedict’s reagent to each tube.
4. Mix the content of the tubes by agitating the tubes side to side or by using a vortex
mixer. Record the original colour of each tube in Table 0-3.
5. Heat the test tubes in a boiling water bath for 3 minutes. Record any colour change
in Table 0-3.
6. When finished, allow the tubes to cool and pour the waste into the waste located in
the fume hood. Then thoroughly clean the tubes at the sink and place them back in
the test tube rack.
Results and Discussion – Benedict’s Test
Table 0-3. Data table for Benedict’s test.
Tube
Original colour
(before boiling)
Final colour
(after boiling)
Amount of sugar
present
(0 to ++++)
1. Water
2. 1% starch
3. 1% glucose
4. 1% sucrose
5. Milk
6. Onion juice
7. Potato juice
8. Apple juice
9. Vegetable oil
10. Egg white
11. Egg yolk
6
7
What is the purpose of testing water with Benedict’s reagent?
______________________________________________________________________
______________________________________________________________________
Explain your results for each tube.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
Did your results for each tube support your hypothesis/null hypothesis? Did your
results agree with your predictions? Explain.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
8
Lugol’s Test (Iodine Test) for Starch
Starch is a polysaccharide formed entirely of glucose units. Starch does not show a
reaction with Benedict’s reagent. Therefore, you will test for the presence of starch using
Lugol’s reagent (iodine/potassium iodide, I2KI).
Iodine complexes with helically-coiled, linear polysaccharide chains (such as the amylose
form of starch), resulting in a black or dark blue color. Branched polysaccharides (such as
glycogen or the amylopectin form of starch) form a less intense, red-violet color. This
occurs because branched polysaccharides have interrupted, or incomplete, helices which
cannot form a complete complex with the iodine. A negative test will retain the
yellow/brown colour of iodine (Fylling, 2001; Helms et al., 1998).
Hypothesis and Predictions
In Table 0-4, formulate a hypothesis and predict what you might expect to find for each of
the solutions/suspensions to be tested with iodine.
Table 0-4. Hypothesis and predictions for Lugol’s test for starch.
Solution/Suspension
Hypothesis
Prediction
1. Water
2. 1% starch
3. 1% glucose
4. 1% sucrose
5. Milk
6. Onion juice
7. Potato juice
8. Apple juice
9. Vegetable oil
10. Egg white
11. Egg yolk
9
What is your null hypothesis for this group of tests?
______________________________________________________________________
______________________________________________________________________
What is the independent variable?
______________________________________________________________________
What is the dependent variable?
______________________________________________________________________
Lugol’s Test Procedure
1. Set up a row of 11 test tubes as you did for the Benedict’s test.
(Note: This test will be performed on the three unknown samples also;
you may wish to set these up at the same time.)
2. Add 1 mL of each of the solutions listed in Table 0-5 to the test tubes, matching each
number to the number on the tube. Record the original colour of each tube in Table
0-5.
3. Add 3 to 5 drops of Lugol’s reagent to each tube.
4. Mix the content of the tubes and immediately record any colour changes that take
place in Table 0-5. Do not heat the test tubes in the Lugol’s test.
5. When finished, pour the waste into the container located in the fume hood, then
thoroughly clean the tubes at the sink and place them back in the test tube rack.
10
Results and Discussion – Lugol’s Test
Table 0-5. Data table for Lugol’s test.
Tube
Original Colour
(before adding I2KI)
Final Colour
(after adding I2KI)
1. Water
2. 1% starch
3. 1% glucose
4. 1% sucrose
5. Milk
6. Onion juice
7. Potato juice
8. Apple juice
9. Vegetable oil
10. Egg white
11. Egg yolk
What is the purpose of testing starch with Lugol’s reagent?
______________________________________________________________________
______________________________________________________________________
Explain your results for each tube.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
11
Did your results for each tube support your hypothesis/null hypothesis? Did your
results agree with your predictions? Explain.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
From the results of the Benedict’s and Lugol’s tests, what would you conclude
about the type of carbohydrate stored in:
 Onion cells:
____________________________
 Potato cells:
____________________________
 Apple cells:
____________________________
 Chicken egg: ____________________________
12
PART 2. TESTING FOR LIPIDS
Introduction
There are three major types of lipids: fats, phospholipids, and sterols. We will only be
working with fats in the laboratory. Triglycerides, a popular topic in discussions of diet
and nutrition, are the most common form of fat. They are composed of three fatty acids
attached to glycerol (a 3-carbon sugar) by ester linkages (Figure 0-4). At room
temperature, some lipids are solid (generally found in animal tissues) and are referred to
as fats, while others are liquid (generally found in plants) and are referred to as oils
(Sadava et al., 2008).
Figure 0-4. Generalized structure of a triglyceride.
Emulsion Test for Lipids
Since both fats and oils are non-polar compounds, they do not dissolve in water, but do
dissolve in ethanol. This characteristic is used in the emulsion test. Test samples are
mixed with ethanol and are filtered or decanted. The alcohol filtrate is then mixed with
water. If there are lipids dissolved in the ethanol, they will precipitate in the water, forming
a cloudy white (milk-like) emulsion.
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Hypothesis and Predictions
In Table 0-6, formulate a hypothesis and predict what you might expect to find for each of
the solutions/suspensions to be tested with the emulsion test for lipids.
Table 0-6. Hypothesis and predictions for the emulsion test for lipids.
Solution
Hypothesis
Prediction
1. Water
2. Milk
3. Potato juice
4. Vegetable oil
5. Egg white
6. Egg yolk
What is your null hypothesis for this group of tests?
______________________________________________________________________
______________________________________________________________________
What is the independent variable?
______________________________________________________________________
What is the dependent variable?
______________________________________________________________________
Emulsion Test Procedure
1. Label 6 tubes 1 through 6.
(Note: This test will be performed on the three unknown samples also;
you may wish to set these up at the same time.)
2. Add 1 mL of the appropriate sample from Table 0-7 to its labelled tube.
3. Add 1 mL of 95% ethanol to each tube. Vortex to mix.
4. Add 2 mL of water to each tube. DO NOT MIX.
5. Record your colour observations (i.e., emulsion test positive or negative) in Table 0-7.
6. When finished, pour the waste into the container located in the fume hood, then
thoroughly clean the tubes at the sink and place them back in the test tube rack.
14
Results and Discussion – Emulsion Test
Table 0-7. Emulsion test results.
Solution
Observed colour (+/‒)
1. Water
2. Milk
3. Potato juice
4. Vegetable oil
5. Egg white
6. Egg yolk
What is the purpose of testing water with the emulsion test? What about vegetable
oil?
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
Explain your results for each tube.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
Did your results for each tube support your hypothesis/null hypothesis? Did your
results agree with your predictions? Explain.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
15
PART 3. TESTING FOR PROTEINS
Introduction
Proteins are macromolecules that are polymers of amino acids. There are 20 amino acids
common to all life on Earth. An amino acid is characterized by a central carbon atom
attached to an amino group (–NH2), a carboxylic acid (carboxyl group: –COOH), a
hydrogen atom and a side group (called an “R group”; Figure 2-5). Each of the 20 amino
acids has a different R group, giving it a unique set of physical and chemical properties
(e.g., some amino acids are acidic, some are basic, some are non-polar, etc.). In turn, the
combination of individual amino acids that make up a protein determines that protein’s
properties and its function within the cell.
Chains of amino acids are also called polypeptides. Amino acids are polymerized into
polypeptides by dehydration (condensation) reactions to form peptide bonds (Figure 2-5).
Because of the diversity that can be generated by combining 20 amino acids in different
lengths, proteins serve a mind-boggling array of functions in the cell including structural
support, transport, regulation, and enzymatic catalysis (Sadava et al., 2008).
Figure 0-5. Generalized structure of an amino acid and formation of a peptide bond.
Biuret Test for Proteins
Biuret reagent, containing sodium hydroxide and copper sulfate, can be used to test for
the presence of polypeptides. The copper ions in the Biuret reagent react with peptide
bonds, converting the dye from a blue (negative result) to a violet color (positive result).
Free amino acids do not react with the Biuret reagent (Helms et al., 1998; Ninfa and Ballou,
2004).
16
Hypothesis and Predictions
In Table 0-8, formulate a hypothesis and predict what you might expect to find for each of
the solutions/suspensions to be tested with the Biuret reagent.
Table 0-8.Hypothesis and predictions for the Biuret test for proteins.
Solution
Hypothesis
Prediction
1. Water
2. 1% albumin
3. Milk
4. Onion juice
5. Potato juice
…....
6. Egg white
7. Egg yolk
What is your null hypothesis for this group of tests?
______________________________________________________________________
What is the independent variable?
______________________________________________________________________
What is the dependent variable?
______________________________________________________________________
Biuret Test Procedure
1. Label 7 clean test tubes 1 through 7.
(Note: This test will be performed on the three unknown samples also;
you may wish to set these up at the same time.)
2. Add 2 mL of each solution from Table 0-9 to the corresponding tube.
3. Add 2 mL of Biuret reagent to each tube.
4. Mix the content of the tubes by agitating the tubes side to side or by using a vortex
mixer.
5. Incubate the tubes at room temperature for 2 minutes.
6. Record your results/observations in Table 0-9.
17
7. When finished, pour the waste into the container located in the fume hood, then
thoroughly clean the tubes at the sink and place them back in the test tube rack.
Results and Discussion – Biuret Test
Table 0-9. Biuret test results.
Sugar/solution
Colour with Biuret reagent
Protein present (+) or absent ()
1. Water
2. 1% albumin
3. Milk
4. Onion juice
5. Potato juice
6. Egg white
7. Egg yolk
What is the purpose of testing water with the Biuret reagent? What about albumin
solution?
______________________________________________________________________
______________________________________________________________________
Explain your results for each tube.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
Did your results for each tube support your hypothesis/null hypothesis? Did your
results agree with your predictions? Explain.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
18
PART 4. ANALYZING UNKNOWN SAMPLES
Using the reagents and methods from the previous exercises, identify the kinds of
molecules contained in the unknown samples. Your instructor will tell you which
substances are to be tested.
Hypothesis and Predictions
In Table 0-10, formulate a hypothesis and predict what you might expect to find for each
of the unknown samples to be tested.
Table 0-10. Hypotheses and prediction for unknown samples.
Sample
Hypothesis
Prediction
A
B
C
Testing the Unknown Samples for Carbohydrates, Fats, and Proteins
Conduct all tests according to directions in the previous exercises. Record your results in
Table 0-11.
 The success of this lab relies on using clean glassware and avoiding crosscontamination between solutions. Therefore, wash all glassware with warm soapy
water; rinse thoroughly several times with tap water and once with distilled water
before using in the next set of tests.
Results and Discussion - Unknowns
Table 0-11. Results of tests for unknown samples.
Food
Sample
Results: Positive (+) or Negative ()
Benedict’s Test
Lugol’s Test
Emulsion Test
Biuret Test
A
B
C
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Explain your results for each sample.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
Did your results for each tube support your hypothesis/null hypothesis? Did your
results agree with your predictions? Explain.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
Your instructor will provide a list of expected test results for the unknown samples.
Did you predict the contents of each unknown accurately?
______________________________________________________________________
______________________________________________________________________
Challenge Question
Amylase, an enzyme found in your saliva, catalyzes the hydrolysis of starch. The products
of this digestive process are dextrin, maltotriose, maltose, and glucose. Using the
appropriate test(s), design an experiment to visualize the progress of the hydrolysis
reaction.
20
PART 5. DNA EXTRACTION AND DETECTION
Introduction
The DNA molecule is composed of 2 strands of nucleotides hydrogen-bonded together
and twisted to form double-stranded DNA (double helix). The double-stranded DNA helix
is linear and stable because small nucleotide bases called pyrimidines (thymine and
cytosine) always pair specifically with larger nucleotide bases called purines (guanine and
adenine). Adenine (A) always pairs with thymine (T), forming 2 hydrogen bonds, and
cytosine (C) always pairs with guanine (G), forming 3 hydrogen bonds (Figure 0-6).
In a DNA double helix, at one end of each strand is a nucleotide bearing a phosphate
group (OPO3) that is linked to carbon number 5 (5’ carbon) of the sugar deoxyribose. This
is called the 5’ end. At the other end of the chain, an OH group extends from carbon
number 3 (3’ carbon) of deoxyribose. This is called the 3’ end. The DNA double strands
are said to be arranged anti-parallel to each other. This means that one strand runs in a
5’→3’ direction while the other runs in a 3’→5’ direction (Figure 0-6; Sadava et al., 2008).
21
Figure 0-6. Generalized structure of the double stranded DNA helix.
DNA can be isolated from anything that is living. In this lab, you will extract DNA from an
onion. The process of DNA isolation involves 3 steps:
 Homogenization: Cellular structures must be broken down before DNA can be
released from cells. This is done by homogenizing the onion tissue in a blender.
Detergents in the homogenizing medium help to solubilize membranes and denature
proteins.
 Deproteinization: Chromosomal proteins must be stripped from the DNA by
denaturation and precipitation from the homogenate that contains DNA.
 Precipitation of DNA: Ice-cold ethanol is added to the homogenate, causing all
components of the homogenate to stay in solution except DNA, which precipitates
at the interface between the ethanol and the homogenate layers (Helms et al., 1998).
22
DNA Isolation Procedure
The DNA molecule is easily degraded, so it is important to follow all instructions closely.
1. Cut an onion into wedges and place these in a blender.
2. Add 100 mL of chilled buffer/detergent solution. Homogenize the mixture at low
speed for 45 seconds, then at high speed for 30 seconds.
3. Using a funnel, filter the homogenate through cheesecloth into a beaker.
4. Transfer approximately 4 mL of the filtered homogenate to a clean test tube.
5. Add a pinch of meat tenderizer to the test tube and mix gently with a Pasteur
pipette.
6. Tilt your test tube and slowly pour ice-cold 95% ethanol into the tube down the
side so that it forms a layer on top of the onion mixture. Pour until you
have about the same amount of alcohol in the tube as the homogenate.
Ethanol is less dense than water, so it floats on top.
7. Look for clumps of white stringy stuff where the water and
alcohol layers meet (Figure 2-7). Gently swirl the Pasteur
pipette at the interface of the two layers, always rotating
in the same direction. This process is called “spooling”
the DNA. If the DNA has been damaged, it will still
precipitate, but as white flakes that cannot be spooled
Figure 0-7. DNA spooling.
(Genetic Science Learning Centre; Fylling, 2001;
Helms et al., 1998).
Discussion – DNA Isolation
What does the meat tenderizer do?
______________________________________________________________________
______________________________________________________________________
Why does isolated DNA appear stringy?
______________________________________________________________________
______________________________________________________________________
What structural characteristics of DNA allow it to be spooled out on a glass rod?
Why isn’t it possible to spool out precipitated proteins?
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
23
PART 6. MOLECULAR VISUALIZATION OF DNA AND HEMOGLOBIN
Introduction
There are several online molecular visualization programs for displaying, animating, and
analyzing large biomolecular systems using 3-D graphics. In this lab you will use a website
that is enabled with the Jmol (interactive web browser applet) molecular visualization
program. Jmol is a very valuable tool that allows students to learn about molecular
structures. It allows a molecular structure to be displayed in several ways. For example, a
protein can be displayed as a ball-and-stick model, a ribbon diagram, or a space-filled
model. Another powerful capability of Jmol is the ability to rotate molecules in all directions
and to zoom in and out of selected parts of the molecule (Ninfa and Ballou, 2004).
Visualizing DNA
1. On the Lab laptop, open Windows Explorer and go to
http://www.umass.edu/microbio/chime/. Click on “DNA Structure.”
2. The tutorial provides tools for a self-directed exploration. Explore activities A, C,
and D and answer the following questions.
Questions for Activity A
How many H bonds are holding one strand against the other in the double helix?
______________________________________________________________________
How many base pairs are there in the model? How many AT pairs? How many GC
pairs? (Note: Clicking on any base reports its letter and sequence number at the
bottom of the browser window in the status line. Use this feature to obtain the
letters.)
______________________________________________________________________
______________________________________________________________________
How do cells make accurate copies of DNA?
______________________________________________________________________
How can A distinguish T from C?
______________________________________________________________________
______________________________________________________________________
24
Questions for Activity C
If a purine were substituted for a pyrimidine at a single position in one strand of a
DNA double helix, what would happen?
______________________________________________________________________
What information is coded into DNA?
______________________________________________________________________
______________________________________________________________________
Which DNA double helix do you think would be harder to separate into two strands:
DNA composed predominantly of AT base pairs, or of GC base pairs? Why?
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
Questions for Activity D
One base pair is not in position to form normal hydrogen bonds. Can you find it?
(Note: Clicking on any base reports its letter and sequence number at the bottom
of the browser window in the status line. Use this feature to obtain the letters and
sequence numbers of the abnormal base pair, once you find it.)
_The webmaster of this site has changed this question; there is no mutation, anymore_
Visualizing Hemoglobin
1. On the Lab laptop, open Windows Explorer and go to
http://www.umass.edu/microbio/chime/. Click on “Hemoglobin.”
2. The tutorial provides tools for a self-directed exploration. Explore all 7 links and
answer the following questions.
Question for part 2
How many amino acids are there in the peptide chain shown in part 2? List them in
the N→C direction.
______________________________________________________________________
Questions for part 3
How many chains are there in a molecule of hemoglobin? What are they called?
______________________________________________________________________
25
How many levels of structural organization are there in a molecule of hemoglobin?
______________________________________________________________________
How many heme groups are there in a molecule of hemoglobin?
______________________________________________________________________
Which amino acid is bound to Fe in a heme group?
______________________________________________________________________
Questions for part 4
What is the main secondary structure in each hemoglobin peptide chain?
______________________________________________________________________
How many secondary structures are there in each hemoglobin peptide chain?
______________________________________________________________________
Which type of bonds stabilizes these secondary structures?
______________________________________________________________________
Which amino acid is located at the N terminus of the isolated alpha helix? At the C
terminus? (Note: Clicking on any amino acid reports its abbreviation and sequence
number at the bottom of the browser window in the status line. Use this feature to
obtain the 3-letter abbreviation of the amino acid.)
______________________________________________________________________
Question for part 6
The surface of the β chain consists mostly of which kind of amino acids (charged,
polar or non-polar)? What about the centre of the chain?
______________________________________________________________________
Questions for part 7
Compare normal to sickle hemoglobin. How do their primary structures differ?
______________________________________________________________________
______________________________________________________________________
How would sickle hemoglobin behave in the absence of O2? What is the cause of
deoxygenated sickle hemoglobin behaviour?
26
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
Which amino acids and their numbers are responsible for this behaviour?
______________________________________________________________________
What is the effect of sickle hemoglobin on red blood cells?
______________________________________________________________________
______________________________________________________________________
REFERENCES
Fylling M. 2001. Laboratory Manual to Accompany Asking About Life. 2nd edition.
Harcourt; Orlando: FL, p. 16–17, 141–143.
Genetic Science Learning Centre. 2008. How to extract DNA from anything living.
University of Utah. p.1–3.
Helms DR, Helms CW, Kosinski RJ, Cummings JR. 1998. Biology in the Laboratory. 3rd
edition. WH Freeman; New York: NY, p. 5-1–2, 5-4–5, 5-7–8, 16-4–6.
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