BIOCHEMISTRY HANDOUT

Group 2 | Section 11, BSN 121-122

I. Introduction

A ​Biomolecule ​is any molecule that is produced by a living organism, the four major
classes being proteins, carbohydrates, lipids, and nucleic acids. Biomolecules are large molecules
and are often made up of many repeating units. These repeating units are called monomers
(single unit), dimers (2 units), oligomers (3-10), and polymers (more than 10). Monomers are
connected to other monomers to form polymers by means of dehydration synthesis/condensation
reaction.
Protein ​is naturally occurring, unbranched polymer in which the monomer units are
amino acids held together by peptide bonds. Proteins are responsible for nearly every task of
cellular life, including cell shape and inner organization, product manufacture and waste cleanup,
and routine maintenance. They are the workhorse macromolecules of the cell and are as diverse
as the functions they serve.
Lipids ​on the other hand are an important component of living cells as well as body than
previously believed. It was previously known that lipids played the role of forming cell
membranes alone. In research, it states that lipids have more diverse and widespread biological
role in the body in terms of intracellular signaling or local hormonal regulation etc. The main
role of lipids in our body is for long-term energy storage. The excess energy from the food we
eat is digested and incorporated into adipose tissue, or fatty tissue. These include acting as
chemical messengers, storage and provision of energy and so forth.
In the science of microscopy, staining is a technique wherein it is used to enhance
contrast in the microscopic image. Stains and dyes are often used to highlight structures for
viewing, to define and examine bulk tissues, cell populations, or organelles within individual
cells. In biochemistry In ​biochemistry it involves adding a class-specific (nucleic acids, ​proteins​,
lipids​, ​carbohydrates​) dye to a substrate to qualify or quantify the presence of a specific
compound.
Sudan stains and Sudan dyes are artificial organic compounds that are used as dyes for
various plastics (plastic colorants) and are also used to stain sudanophilic biological samples, like
lipids. Sudan dyes belong to the Solvent dye group. It’s frequently called lysochromes (lyso-
means dissolving and -chrome means color).
The ​Biuret test is a chemical test used for detecting peptide bonds which is commonly
found in amino acids. A Biuret reagent is used as an indicator for the presence of proteins, and it
can be observed by the change of appearance from blue to a violet color.
II. Proteins
STRUCTURE

1. Primary Structure - ​The simplest level of protein structure, primary structure, is simply
the sequence of amino acids in a polypeptide chain.
2. Secondary structure - ​The next level of protein structure, ​secondary structure​, refers to
local folded structures that form within a polypeptide due to interactions between atoms of
the backbone. The most common types of secondary structures are the α helix and the β
pleated sheet. Both structures are held in shape by hydrogen bonds, which form between the
carbonyl O of one amino acid and the amino H of another.
3. Tertiary Structure - ​The overall three-dimensional structure of a polypeptide is called its
tertiary structure. The tertiary structure is primarily due to interactions between the R groups
of the amino acids that make up the protein.
4. Quaternary Structure - ​Many proteins are made up of a single polypeptide chain and
have only three levels of structure. However, some proteins are made up of multiple
polypeptide chains, also known as subunits. When these subunits come together, they give
the protein its quaternary structure.

FUNCTION

Primary Functions of Protein in the Human Body

1.Repair and Maintenance 4. Enzymes

Protein is termed the building block of the Enzymes are proteins that increase the rate of
body. It is called this because protein is vital in the chemical reactions in the body. In fact, most of the
maintenance of body tissue, including development and necessary chemical reactions in the body would not
repair. efficiently proceed without enzymes.

2.Energy 5. Transportation and Storage of Molecules

Protein is a major source of energy. If you Protein is a major element in transportation of
consume more protein than you need for body tissue certain molecules. For example, hemoglobin is a
maintenance and other necessary functions, your body protein that transports oxygen throughout the body.
will use it for energy. If it is not needed due to
sufficient intake of other energy sources such as
carbohydrates, the protein will be used to create fat and
becomes part of fat cells.
3. Hormones 6. Antibodies
Protein is involved in the creation of some Protein forms antibodies that help prevent
hormones. These substances help control body infection, illness and disease. These proteins identify
functions that involve the interaction of several organs. and assist in destroying antigens such as bacteria and
Insulin, a small protein, is an example of a hormone viruses. They often work in conjunction with the other
that regulates blood sugar. immune system cells.

Protein Classification Based on Function

1.Catalytic Protein 7. Transmembrane Protein

Protein is best known for their role as These proteins, which span a cell membrane,
catalysts. Protein with the role of biochemical catalyst help control the movement of small molecules and ions
are called enzymes. Enzyme participate in almost all of through the cell membrane. Many such proteins have
the metabolic reactions that occur in cells. channels through which molecules can enter and exit
cell. Many such proteins are very selective, often
allowing passage of just one type of molecule or ion.

2.Defense Proteins 8.Storage Proteins

These proteins are also called These proteins bind (and store) small
immunoglobulins or antibodies are central to the molecules for future use. During degradation of
functioning of the body’s immune system. They bind to hemoglobin the iron atoms present are released and
foreign substances, such as bacteria and viruses, to help become part of ferritin, an iron storage protein which
combat invasions of the body by foreign particles. saves the iron for use in the biosynthesis of new
hemoglobin molecule.

3.Transport Protein 9.Regulatory Proteins

These proteins bind to particular small These proteins are often found “embedded” in
biomolecules and transport them to other locations in the exterior surface of cells membranes. They act as
the body and then release the small molecules as sites at which messenger molecules, including
needed at the destination location. Hemoglobin is messenger proteins such as insulin, can bind and
example of transport protein because it carries oxygen thereby initiate the effect that the messenger “carries.”
from the lungs to other organ and tissue. Regulatory proteins are often the molecules that bind to
enzyme (catalytic proteins), thereby turning them “on”
and “off” and thus controlling enzymatic action.

4.Messenger Proteins 10.Nutrient Proteins

These proteins transmit signals to coordinate These proteins are particularly important in
biochemical processes between different cells, tissues, the early stages of life, from embryo to infant. Casein,
and organs. A number of hormones that regulate body found in milk, and ovalbumin, found in egg white, are
processes are messenger proteins, including insulin and two examples of such proteins.
glucagon.

5.Contractile Proteins 11. Buffer Proteins

These proteins are necessary for all forms of These proteins are part of the system by which the
movement, Muscles are composed of filament-like acid-base balance within body fluid is maintained.
contractile proteins that, in response to nerve stimuli, Within the blood, the protein hemoglobin has a
undergo conformational changes that involve buffering role in addition to being an oxygen carrier,
contraction and extension. Actin and myosin are Transmembrane proteins regulate the movement of
example of contractile proteins. Sperm can “swim” ions in and out of the cells, ensuring that ion
because of long flagella made up of such proteins. concentrations are those needed for correct acidity or
alkalinity.
 6.Structural Proteins 12.Fluid-balance Proteins
These proteins confer stiffness and rigidity to These proteins help maintain fluid balance
otherwise fluid-like biochemical systems. Collagen is a between blood and surrounding tissue. Two
component of cartilage, and α-keratin gives mechanical well-known fluid-balance proteins, found in the
strength as well protective covering to hair, fingernails, capillary beds of the circulatory system, are albumin
feathers, hooves, and animal shells. and globulin.

III. Lipids

STRUCTURE

1. Triglyceride is an ester derived from glycerol and three fatty acids (from tri- and
glyceride).Triglycerides are the main constituents of body fat in humans and other animals, as
well as vegetable fat.

2. Saturated Fatty Acid do not contain double bonds between the individual carbon atoms.
Most are solid room temperature. ​Unsaturated Fatty Acid on the other hand contains at least
one double bond in the fatty acid chain. Mostly liquid in form.

​4. Phospholipids are a class of lipids that are a

major component of all cell membranes. They can
form lipid bilayers because of their amphiphilic
characteristic. They are composed of one
hydrophilic “head” composed of a phosphate
group and two hydrophobic fatty acid chains.
5. Steroids/ Sterols are lipids that have a structure which includes 4 aromatic rings fused
together.

6. Wax​ is a lipid made up of a chain of alkanes or esters from alcohols and fatty acid.

FUNCTION

Primary Functions of Lipids in the Body

1. Storage of energy 4. Hormonal roles
Carbohydrates provide a readily available All multicellular organisms use chemical
source of energy, lipids function primarily as an messengers to send data between organelles and to
energy reserve. The amount of lipids stored as an different cells. Since lipids are tiny molecules insoluble
energy reserve far exceeds the energy stored as in water, they're best candidates for signaling. The
glycogen since the human body is simply not capable signaling molecules are more attach to the receptors on
of storing as much glycogen compared to lipids. the cell surface and bring about a change that leads to an
Storage lipids are triacylglycerol. These are inert and action. The signaling lipids, in their esterified form can
made up of three fatty acids and a glycerol. infiltrate membranes and to be transported to carry
signals to different cells.

2. Structural Components 5. Protection

Phospholipids are a class of lipids that are a Cholesterol maintains the fluidity of membranes
major component of cell membranes. They form a by interacting with their complex lipid components,
bilayer that consists of two rows stacked together specifically the phospholipids such as
with the hydrophobic fatty acid tails facing each phosphatidylcholine and sphingomyelin. Cholesterol also
other, and the hydrophilic heads on either end. The is the precursor of bile acids, vitamin D and steroidal
ampiphilic characteristic of these phospholipids give hormones. The essential fatty acids, linoleic and linolenic
the cell its semi-permiability, keeping ​ions​, ​proteins acids are precursors of many different types of
 and other molecules where they are needed and eicosanoids, including the hydroxyeicosatetraenes,
prevents them from diffusing into areas where they prostanoids (prostaglandins, thromboxanes and
should not be. prostacyclins), leukotrienes (and lipoxins) and resolvins
etc. these play an important role in pain, fever,
inflammation and blood clotting.

3. Insulation
Maintenance of body temperature is mainly
done by brown fat as opposed to white fat. Babies
have a higher concentration of brown fat. Also,
Layers of subcutaneous fat under the skin also help in
insulation and protection from cold.

IV. Principle of Biuret Test

The Biuret test or Piotrowski's test is a chemical test used for detecting peptide bonds
which is commonly found in amino acids. The sole reagent used for the Biuret test is the Biuret
reagent. A Biuret Reagent is an aqueous solution of potassium sodium tartrate treated with cupric
sulfate and sodium hydroxide. It has the chemical formula C​2​H​5​N​3​O​2​. However, an alternative
reagent such as (1) Sodium Hydroxide and Copper Sulphate Solutions, and (2) Fehling’s
Solutions A and B may be used to yield comparable results
Biuret test is a general test for compounds (proteins and peptides) having two or more
peptide (CO-NH) bonds. It detects peptide bonds, and when they are present in an alkaline
solution, the coordination complexes associated with a copper ion are violet in color. The protein
concentration affects the intensity of the color, and the color will be more intense with more
proteins. ​The normal color of the biuret reagent is blue. With the presence of peptide bonds -the
chemical bonds that hold amino acids together, the reagent would turn violet. The color change
from blue to violet indicates the presence of proteins. The greater the number of peptide bonds in
a protein, the greater the color intensity. Hence, the color change is from blue to pink if the
concentration of peptide bonds is low such as in short-chain peptides. The detected proteins
must have at least three amino acids which implies that the proteins must have at least two
peptide bonds. The reagent’s copper ions, with a charge of +2, are reduced to a charge of +1 in
the presence of peptide bonds, causing the color change. The test may also quantitatively
measure the concentration of total protein using the techniques of absorption spectrometric
methods, which identify the electromagnetic frequencies a sample will absorb.

This experiment requires: Biuret reagent, 3 test tubes (clean and dry, deionized water
(negative control), albumin or egg white (positive control).

Procedure:
1. Take 3 clean and dry test tubes.
2. Add 1-2 ml of the test solution, egg albumin and deionized water in the respective test
tubes.
3. Add 1-2 ml of Biuret reagent to all the test tubes.
4. Shake well and allow the mixtures to stand for 5 minutes.
5. Observe for any color change.
 Observation Interpretation

No color change (solution remains blue ) Proteins are not present

The solution turns from blue to violet (deep Proteins are present
purple)

The solution turns from blue to pink Peptides are present (Peptides or peptones are
short chains of amino acid residues which
contain less number of peptide bonds)

It is also important to take note of the following:

● Histidine is the only amino acid that give biuret test positive.
● Presence of magnesium and ammonium ions interfere in biuret test. This can be
overcome by using excess alkali.

V. Principle of Sudan IV Staining Test

Sudan IV is one of the dyes used for Sudan staining. Sudan II, Sudan III, Oil Red O, and
Sudan Black B are the other similar dyes in this class of compound.
Sudan IV is a lysochrome (fat soluble dye) predominantly used for demonstrating
triglycerides in frozen sections, but which may also stain some protein bound lipids in paraffin
sections. Lipids can be dissolved in non-polar solvents such as chloroform, ether, acetone, or
fat-soluble dyes like Sudan IV. Staining is significant biochemical technique, offering the ability
to visually qualify the presence of the fatty compound of interest without isolating it.

Structure and Components

In structural classification, Sudans are diazo dyes. Azo colourant molecules can be
described as aryl-N=N-R, where R is aryl, heteroaryl or -CH=C(OH)-. Other structures include
heterocyclic rings, metal complex, pyrazolone, xanthene, and ketoamine. Solvent dyes do not
ionise. Sudan IV is soluble in chloroform (1 mg/ml). It is soluble in water (0.7 mg/ml), slightly
soluble in ethanol and very soluble in benzene, methanol, acetone, hydrocarbon solvents, oils,
fats, and waxes.
Purpose
A solvent dye, undergoes molecular rearrangement, which it dissolute in the substrate.
They color and bind to lipids. In industrial fields, they are used to color oils, waxes, greases, fats,
hydrocarbons derivatives, polishes, oily insecticides, and acrylic emulsions. Those containing
azo-structures often produce yellow-red coloration whereas those based on anthraquinones tend
to be green-blue. Sudans are used for demonstrating fats, triglycerides and lipoproteins present in
tissues as biological stains. Used as an oil soluble colorant, fat (lipoprotein) stain.

Lipid Test

● Lipids appear as red after the experiment.

● In a solution with lipid, water and Sudan IV dye, dark red layer on the top will be formed
which indicates that Sudan stained the lipid base substance and separate it from the water.
References:

● Horobin, Richard; Kiernan, John, eds. (2002). ​Conn's Biological Stains: A Handbook of
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