Download Molecules of Life

Biomolecules
Lab #3
1
Today
Quiz #2 – 15 minutes
Objectives and Introduction to Biomolecules –
20 minutes
Safety
Today’s Lab Instructions – 10 mins
Experiments - ~ 70 minutes
Clean Up
Summary
Objectives
Describe the distinct features of three of four classes of
organic molecules
Differentiate between monomers and polymers
Identify reducing sugars, polysaccharides, lipids, amino
acids and proteins using diagnostic reagents
Conduct nutritional assessments from case studies
OBJECTIVES
Describe the functions of the major classes of biomolecules
Explain how dehydration synthesis causes molecular bonds
Compare the different bonds that help define the major
classes of biomolecules
Describe the reactive groups of the major biomolecules &
explain how they relate to the basic chemical structure of
each class of biomolecules
Use diagnostic reagents to identify biomolecules & explain
the chemical basis for the tests
Design & present an experiment to test for the presence of
biomolecules in food materials
Explain how the chemistry of biomolecules relates to FDA
food labels, & how you can use this knowledge to make wise
food choices
Biomolecules: The Basics
Organic molecules are necessary for biological
processes.
Organic molecules contain carbon.
4 major types of organic molecules:
–
–
–
–
Carbohydrates
Lipids
Proteins
Nucleic Acids (We’ll look at these in a future lab.)
Similarities Between Biomolecules:
– All Contain Carbon
– Most Contain H, O, N, S and/or P
– ALL Life on Earth is made up of Carbon
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Building Blocks of Life
Each Macromolecule is comprised of building block
molecules called monomers:
– Monosaccharides  carbohydrates
– Fatty Acids and Glycerol  Lipids
– Amino Acids  Protein
Macromolecule: A giant molecule formed by the joining of
smaller molecules, usually by a condensation reaction.
Monomer- The subunit that serves as the building block of a
polymer.
Polymer- A long molecule consisting of many similar or identical
monomers linked together.
Functional groups
The components of organic
molecules that are most
commonly involved in
chemical reactions are known
as functional groups
Each functional group behaves
consistently from one organic
molecule to another
Provide a way to differentiate
between different
biomolecules
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/Groups
_5.gif
Hydroxyl Group
– Found in alcohols (ex. ethanol) and
sugars
– Polar - increases the solubility of
molecules
– OH (not OH- hydroxide ion)
Where are the Hydroxyl groups in the
glucose and in the ethanol?
ethanol
Carbonyl Group
Found in
Carbohydrates:
Two types
– Aldehyde
terminal
– Ketone
internal
– One or the other on
every monosaccharide
Carboxyl Group
In fatty acids AND amino
acids
Acidic properties
Double Bond between
Carbon and Oxygen
Single Bonds: Carbon to
Hydroxyl
Written as COOH or
C02H
Amino Group
Amino Group
– In every amino acid
– Nitrogen atom attached to two hydrogen atoms
Monomer – a single molecule that can
combine to form polymers
Polymer – long chains of monomers
Dehydration Synthesis and Hydrolysis
Dehydration Synthesis
– Monomers are connected by an enzymatic removal of water
– One monomer provides a hydroxyl group and the other provides
a hydrogen
Result is the release of a water molecule
Hydrolysis
– Covalent Bonds connecting monomers into polymers
– A Hydrogen atom and hydroxyl group from a split water molecule
attaches where the bond used to be
1 H(+) Atom
1 Hydroxyl Group (split from a water molecule)
Attach where the bond used to be
Examples of
Dehydration and
Hydrolysis
Which one of these is
also called a
condensation
reaction?
Carbohydrates (Saccharides)
Four major functions:
–
–
–
–
Supply carbon for synthesis of other biomolecules
Structural components in cells and tissues
Fuel (glucose)
Store glucose (polysaccharides)
Three types:
– Monosaccharides
– Disaccharides
– Polysaccharides
Defined by carbonyl group
– Aldehyde
– Ketone
Monosaccharides (Basic Unit of Carbohydrates)



Simple sugars
Classified by the number of carbons they contain

Trioses

Pentoses

Hexoses
May Exist as Rings (Stable) or Linearly (Unstable)
glucose
Disaccharides

Consist of 2
monosaccharides

Linked via a condensation
reaction (Dehydration
Synthesis)

Linked by a Glycosidic
Bond
Polysaccharides
Chains of linked
monomers
Amylose
– Linear chain of
several hundred
glucose molecules
Amylopectin
– Branched chains of
several thousand
glucose molecules
Starch in plants
Glycogen in animals
Reducing Sugars:
Contain a free aldehyde or ketone
group (carbonyl)
Reducing sugars reduce something
else
– Includes:
All monosaccharides
Disaccharides where one aldehyde or
ketone group is free
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Testing for Saccharides
– Benedicts Test
Tests for the presence or absence
of reducing sugar
– A reducing sugar is a sugar with
free aldehyde or ketone group
Free- not used in the bond
between monosaccharides. All
monosaccharides are reducing
sugars.
Positive test results red-orangeyellow color
Testing for Saccharides
Lugol’s Test
– Indicates the presence
or absence of starch
– Positive test results
black/purple color
All our tests today will be
qualitative not
quantitative (specific
numerical measurement)
Lipids
Nonpolar molecules
– Composed of long chains of carbon and
hydrogen
Lipid molecules include:
– Fats, Oils, Steroids, Phospholipids
Monomers (for triglycerides)
– glycerol and fatty acids
Major functions include:
– Energy storage
– Material for membranes
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Lipids
Saturated vs. Unsaturated
– Saturated lipidsAll bonds between carbon & hydrogen are single bonds
No carbon-carbon double bonds
Straight Chains
Solid at Room temperature
– Unsaturated lipidsSome carbon-carbon bonds are double bonds
– Kinked Chains where double bonds are present
Kinks prevent molecules from packing tightly together
Plants and Fish Fats (oils) are liquid at room temperature
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Saturated Fats
have fatty acids with no carboncarbon double bonds
There is a hydrogen at every
possible location
The molecule has straight chains
Saturated fats are solid at room
temperature
Most animal fats are saturated
Contributes to cardiovascular
disease
Unsaturated Fats
have fatty acids with one or more
carbon-carbon double bonds
The molecule has kinked chains
where there is a carbon-carbon
double bonds
Plants and fish fats, known as oils,
are liquid at room temperature
The kinks provided by the double
bonds prevent the molecules from
packing tightly together
Lipids
Triglycerides are solid lipids (fats) that are stored in human tissue
OR liquid lipids (oils) that are stored in plant tissue.
– Store more energy than any other molecule
Triglycerides are formed by attaching 3 fatty acid molecules to a
glycerol molecule through a condensation reaction.
– Functional group for glycerol (an alcohol) = hydroxyl group (OH)
– Functional group for fatty acid (a carboxylic acid) = carboxyl group
(COOH)
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Phospholipids
Make up the cell
membrane
Consist of:
– a polar head (hydrophilic)
– a nonpolar tail (hydrophobic)
Testing for Lipids
Sudan IV
– A nonpolar dye
– Bonds to other nonpolar molecules
– Positive test results
Cloudy emulsion of dispersed lipid droplets
which are red color due to the Sudan IV
Proteins
Structural support, storage, transport of other substances, and intercellular
signaling
– ALL Enzymes are Proteins
All Proteins have a complex 3D Shape
Monomers = amino acids (there are 20 amino acids used by biological organisms)
Amino acids contain:
– An amino (NH3 ) group
– A carboxyl (COOH) group.
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Proteins and Amino Acids
When amino acids are linked together via a condensation reaction, they
form a protein.
The chemical bond between each pair of amino acids is called a peptide
bond.
– Carboxyl Group of one Amino Acid joins the amino group of another amino
acid
Polypeptides: polymers of proteins
– Proteins consist of one or more
Polypeptides folded and coiled
into a specific formation
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Proteins and Amino Acids
When long chains of amino acids form
proteins, the chemical properties of the
amino acids cause the protein to take
on more complex shapes:
Proteins are used for:
–
–
–
–
Structural support
Transport of substances
Cell signaling
Enzymes
All enzymes are made of protein.
Enzymes regulare metabolism by
selectively accelerating chemical reactions
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Testing for Proteins
Biuret Test
– React with peptide bonds
– Positive test results purple
Ninhydrin
– Tests for single amino acids
– Reacts with free amino
groups
– Positive test results
Purple- straight chain
amino acid
Yellow- ring chain amino
acid (proline)
Lab Safety – Biology 111 Lab 3 Biomolecules
Goggles and closed-toed shoes are required!
Gloves need to be worn when handling all reagents. Refer to the lab manual for
safety notices. Some materials such as Ninydrin and Ninhydrin-collidine are corrosive
and toxic. Other chemicals are flammable.
Closely monitor all hot plates. Immediately turn off when not in use and use hand
protectors when handling any beakers placed on hot plates. Keep all solvents away
from heat sources.
Chromatography solvent is highly flammable with toxic, volatile fumes. Keep tightly
closed when not in use and keep away from all heat sources.
Dispose of any solutions containing the following reagents in the appropriately
labeled containers on the side counter:
– Dispose of chromatography solvent, ethanol, and Sudan IV in the solvent waste
container.
– Dispose of Lugol’s solution, Benedicts reagent, and Biuret’s reagent in the base waste
container.
– Dispose of ninhydrin filter papers in the ninhydrin waste bucket.
Keep waste containers closed when not disposing of waste, and do not fill past the
indicated line on waste containers. Inform your TA of any full waste containers.
Lab Activity: Testing for the
presence of organic molecules
Each table will work as a single group.
– Within each group, students will perform different tests.
– When all tests are complete, group members will teach each
other how to conduct all of the tests.
Procedure
How does the test work?
What is a positive / negative result?
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Lab Activity: Testing for the
presence of organic molecules
Organization for each table (8 students):
–
–
–
–
–
Benedict’s Test for Reducing Sugars- 2 students
Lugol’s Test for Starches- 2 students
Sudan IV Test for Lipids- 1 student
Biuret Test for Proteins- 2 students
Ninhydrin Test for Amino Acids- 1 student
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Benedict’s Test
Tests for the presence of reducing sugars (reacts with the free aldehyde or
ketone groups)
You will fill in the data for Table 3-1.
– You will need 8 test tubes to test the 8 solutions in Table 3-1.
Make sure you gently shake the solution bottles before you use
them.
10 drops = 1 mL
Use parafilm to mix your solutions with the Benedict’s reagent in the
test tubes.
– WARNING! BENEDICT’S REAGENT IS A CORROSIVE BASE.
DO NOT COME IN CONTACT WITH THIS CHEMICAL AND
DISPOSE IN BASE WASTE CONTAINER.
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Benedict’s Test
A positive test result is any color
change.
Color changes range from:
– Blue > Green > Yellow > Reddish
Orange
Orange and Red indicate the strongest
reducing sugars.
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Lugol’s Test
Tests for the presence of starches
You will fill in the data for table 3-1.
– Procedure begins on pg. 30
– You will need 8 test tubes to test the 8 solutions in Table 3-1 (these are
the same solutions used in the Benedict’s test).
– Make sure you gently mix the solution bottles before you use them.
– 10 drops = 1 mL
– Use parafilm to mix your solutions with the Lugol’s reagent.
– WARNING! LUGOL’S IS A TOXIN (ESPECIALLY
FOR PEOPLE WITH IODINE ALLERGIES OR
WILSON’S DISEASE). DO NOT COME IN CONTACT
WITH THIS CHEMICAL AND DISPOSE IN BASE
WASTE CONTAINER.
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Lugol’s Test
A positive test result is a blue-black color, which indicates
the presence of starch.
Monosaccharides and Disaccharides will test negative for
Lugol’s.
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Emulsification Test for lipids
– Use Ethanol + Sudan IV
• A non-polar dye
• Bonds to other non-polar molecules
• Positive test results red color
Positiv
e
Negativ
e
Sudan IV Test
A positive test is a red
stain left on the filter
paper after rinsing.
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Thin Layer Chromatography
•Mixed lipids can be separated
•Yields a chromatogram
•Mixed molecules interact with
the solvent (mobile phase) and
the silica gel (stationary phase)
to separate as solvent moves
up
•Relationship between distance
moved by molecule to distance
moved by solvent is specific for
the matrix conditions & solvent
•Relationship is given by Rf
Solvent
front
Triglyceride
Fatty acids
Phospholipi
ds
Origi
n
s
Biuret Test
Tests for the presence of proteins by reacting with peptide bonds
You will fill in the data for Table
–
–
–
–
–
Procedure begins on pg.
You will need test tubes to test the solutions
Make sure you gently mix the solution bottles before you use them.
10 drops = 1 mL
Use parafilm to mix your solutions with the Biuret reagent.
WARNING! BIURET IS A STRONG BASE. AVOID
CONTACT AND DISPOSE OF IN BASE WASTE
CONTAINER.
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Biuret Test
A positive test is a violet
color.
– Any other color change is a
negative result.
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Ninhydrin Test
Tests for the presence of free amino acids by reacting
with their amino groups.
You will fill in the data for table.
Make sure you gently shake the solution bottles before
you use them.
The test will be done on a piece of filter paper folded
into eights.
– Use pencil to label each test area.
– Dry each solution on the filter paper before you test it
with the ninhydrin solution.
– Dry the filter paper again after the ninhydrin is added.
WARNING! NINHYDRIN IS POISONOUS. AVOID
CONTACT AND DO NOT INHALE.
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Ninhydrin Test
A positive test is either a
purple/violet color OR a yellow
color.
– Yellow indicates the presence of a
specific amino acid, proline.
– Purple indicates the presence of any
other free amino acid.
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Ninhydrin
Ninhydrin - Collidine
Nutrition Activity
When the students at your table have shared all of their
data and taught each other these different tests:
– Read the section on Nutritional Analysis
You may do this individually or with others.
– You need to know:
How to convert pounds (lbs) to kilograms (kg)
How to calculate BMR (know the equation)
Have a basic understanding of the Prudent Diet
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Clean up
•
Dispose of any solutions containing the following reagents in the
appropriately labeled containers on the side counter:
– Dispose of chromatography solvent, ethanol, and Sudan IV in the
solvent waste container.
– Dispose of Lugol’s solution, Benedicts reagent, and Biuret’s reagent in
the base waste container.
– Dispose of ninhydrin filter papers in the ninhydrin waste bucket.
•
Keep waste containers closed when not disposing of waste, and do not fill
past the indicated line on waste containers. Inform your TA of any full waste
containers.
• Rinse all used test tubes (after dumping contents in proper waste container)
and mortar and pestles and return to the middle of the bench.
• Replace all bottles and other equipment to the middle of the bench in an
orderly manner.
• Make sure all hot plates are off
• Clean all pipettes and return them to the “clean pipette” container
Learning Objectives
Be able to identify the three classes of organic molecules based on their
functional groups.
Know the difference between a monomer and a polymer.
– Know the monomers for the organic molecules discussed.
Know each of the diagnostic tests performed in class
– Know the general procedure
– Know how each test works
– Know what positive and negative results look like.
Nutritional Analysis- You need to know:
How to convert pounds (lbs) to kilograms (kg)
How to calculate BMR (know the equation)
Have a basic understanding of the Prudent Diet
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Chemical Tests:
Test
Benedicts
Lugols
Sudan IV
Biuret
Ninhydrin
Test for
Color
before
Color
after
Detects
Biomolecules Summary
Biomolecule
Building
Blocks
Type of
Bond
Carbohydrates
Simple
Sugars
Glycosidic
Lipids
Gylcerol
Fatty Acids
Proteins
Amino Acids
Binding
Mechanism
Disaccarides
Polysaccharides
Dehydration
Peptide
Products
Triglycerides
Phospholipids
Steroids
Polypeptides
Proteins
Test
Test for
Color before
Color after
Positive control,
negative control
Benedicts
Reducing
sugars
Pale blue
Pale blue – none, Glucose
green – low,
solution,
brown –
distilled water
intermediate,
orange red - high
Lugols
Starch
Yellow
Blue - black
Potato, distilled
water
Sudan IV
Lipids
Light red
Red stained oil
droplets
Any oil,
distilled water
Biuret
Peptide
bonds
Very pale
blue
Violet
Milk, distilled
water
Ninhydrin
Free amino
acids
No Color
On drying, may
be either purple
or yellow
Amino acid
methionine,
distilled water