Medical Biochemistry and Molecular Biology Department
MEDICAL BIOCHEMISTRY
AND
MOLECULAR BIOLOGY DEPARTMENT
PRACTICAL GUIDE NOTES
ON
BIOLOGICAL MOLECULES
LABORATORY INVESTIGATIONS
Medical Biochemistry and Molecular Biology Department
BIOLOGICAL MOLECULES
LABORATORY INVESTIGATIONS
ILO of the current topic:By the end of this topic, the student will be
able to: Identify bio-molecules and enzymes.
IDENTIFICATION OF BIOLOGICAL MOLECULES
A cell may be compared to a living chemistry laboratory. Most functions
within the cell take the form of interactions between organic (carboncontaining) molecules. Organic molecules found in living systems can be
classified as carbohydrates, fats, proteins, or nucleic acids. Each of
these classes of molecules is made of smaller units and both the smaller
and larger units have specific properties that can be identified by simple
chemical tests. In this laboratory investigation, you will learn to identify biomolecules as carbohydrates, fats, and proteins.
The tests for the different types of organic molecules will be done:
1. On water or a similar colored solution which does not contain the
molecules being tested (to demonstrate negative results).
2. On the solution which contain the molecules being tested (to demonstrate
positive results).
TESTING FOR CARBOHYDRATES
Monosaccharides are characterized by the presence of a carbon-oxygen
bond. If found at the end of the molecule it is called a terminal aldehyde
group or if found in the interior it would be called a ketone group. Both of
these groups contain double-bonded oxygen that reacts with Benedict's
solution to form a colored precipitate.
When two monosaccharides are bonded together, they form a disaccharide.
If the reactive aldehyde or ketone groups are involved in the bond between
the monosaccharide units (as in sucrose), the disaccharide will NOT react
with Benedict's solution (needs Acid hydrolysis ketose test). If only one
group is involved in the bond (as in maltose), the other is free to react with
the Benedict's reagent. Sugars with free aldehyde or ketone groups, whether
monosaccharides or disaccharides, are called reducing sugars. These
sugars are oxidized (lose electrons) to the copper ions in the Benedict's
reagent which becomes reduced (gains electrons), hence the name
reducing sugar. The color of the precipitate (material that settles to the
bottom of the tube) varies dependent on the strength of the reducing sugar
present.
Monosaccharides may join together to form long chains (polysaccharides)
that may be either straight or branched. Starch is an example of a
polysaccharide formed entirely of glucose units. Starch does not show a
reaction with Benedict's reagent because the number of free aldehyde
groups (found only at the end of each chain) is small in proportion to the rest
of the molecule. Therefore, we will test for the presence of starch with
Lugol's reagent (iodine/potassium iodide, I2KI).
ACTIVITY 1A. BENEDICT'S TEST FOR REDUCING SUGARS
Medical Biochemistry and Molecular Biology Department
Objectives:
Identify reducing sugars using Benedict's reagent
When Benedict's reagent is heated with a reactive sugar, such as glucose,
the color of the reagent changes from blue to yellow to reddish-orange,
depending on the amount of reactive sugar present. Orange and red indicate
the highest proportion of these sugars.
Left to right:
Benedict`s with no reducing
sugar (blue), Different grades
of reducing sugars (greenorange –brown to red
Procedure:
In a test tube place 5 ml solution,
add 3 ml Benedict's reagent to the solution in the test tube and place the
tube in the boiling water bath for five minutes.
Activity A2: LUGOL'S TEST FOR STARCH
Objectives:
Identify Starch molecules as Lugol's reagent changes from a brownish or
yellowish color to blue-blue-black when starch is present, but there is no
color change in the presence of monosaccharides or disaccharides.
Procedure: To 2ml of starch add two drops of IKI
TESTING FOR LIPIDS
Left to right:
IKI only (yellow), Starch solution
(white), Starch with IKI (deep
blue)
ACTIVITY B: TESTING FOR LIPIDS
Objectives: Identify lipid molecules
The word lipid refers to any of the members of a rather diverse group of
organic molecules. Lipids are soluble in non-polar solvents such as ethyl
alcohol, ether, chloroform, but are insoluble in water. Although lipids include
fats, steroids, and phospholipids, this exercise will focus primarily on fats.
At room temperature, some fats are solid (generally those found in animals)
and are referred to as fats, while others are liquid (generally those found in
plants) and are referred to as oils. Vegetable oil, a liquid fat, is a mixture of
triglycerides.
Basis of test:
Medical Biochemistry and Molecular Biology Department
Lipids are immiscible with water. Adding water to a
solution of the lipid in alcohol results in emulsion of
tiny droplets in the water which reflect light and give
an opalescent appearance.
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Procedure:
Add 2ml of colored water to the following:
2ml of oil.
2ml of oil in absolute alcohol (oil dissolved in
alcohol by shaking vigorously).
2ml of water (-ve control)
Right to left:
Oil+ Colored Water = immiscible
Colored Water alone= soluble
Oil in Alcohol+ Colored water opaque cloudy suspension (emulsion)=
miscible
TESTING FOR PROTEINS
Proteins are made up of one or more polypeptides which are linear polymers
of smaller molecules called amino acids. Polypeptides are formed when
amino acids are joined together by peptide bonds between the amino group
of one amino acid and the carboxyl group of a second amino acid.
The Biuret reagent reacts with peptide bonds and will, therefore, react with
proteins, like egg albumin. The reagent will not react with free amino acids
like glycine or alanine.
ACTIVITYC: TESTING FOR PROTEIN WITH THE BIURET REAGENT
Objectives:
proteins (polypeptides) react with Biuret reagent .
The Biuret reagent is light blue. In the presence of proteins it turn violet.
Other types of molecules may cause other color changes, but only the violet
color indicates the presence of polypeptides.
Left to right:
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Biuret's reagent (BrR),
water + BrR,
Protein(egg albumin
solution),
Protein(egg albumin
solution)+ BrR.
Procedure: Add equal amounts of the sample and buiret reagent.
RESULTS: Denim-blue = negative. Violet color = positive.
TESTING FOR CATALASE ENZYME
Medical Biochemistry and Molecular Biology Department
Catalase is a common enzyme found in nearly all living organisms that are
exposed to oxygen, where it catalyzes the decomposition of hydrogen
peroxide to water and oxygen.
2H2O2------CATALASE-------- O2+2H2O
ACTIVITYD: TESTING FOR CATALASE ENZYME
Catalase -Positive =copious bubbles;
Catalase-Negative = No bubbles.
Objective: Identify catalase enzyme
Principle of the activity
The enzyme catalase mediates the breakdown of hydrogen peroxide into
oxygen and water. The presence of the enzyme in yeast is evident when a
small amount is introduced into hydrogen peroxide (3% solution), and a
rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident
by a lack of or weak bubble production.
Procedure:
• Place few drops of 3% hydrogen peroxide (H2O2) into the tube containing
the enzyme.
• Observe for the evolution of oxygen bubbles.
Notes to be observed
1- Don’t use media containing blood, because the red blood cells
contain Catalase and it will give us a false positive test. Always use a
fresh H2O2 because it’s unstable and check it.
Questions:
1. What is the purpose of a positive control? Give an example of a positive
control in the Benedict's Test.
2. Why do fat droplets float, rather than mix with the water?
3. Why was water used as a test solution in each of the tests? What was its
function or role in the exercises?