PROTEINS

• Proteins are extremely complicated molecules and are the nitrogenous compounds
made up of a variable number of amino acid residues joined to each other by a
specific type of covalent bond called peptide bond or peptide linkage
• They are the most abundant molecules in the cell interior where they constitute
50% of dry cell mass
• The name protein is derived from the Greek word protos meaning supreme or
first.
• Proteins are one of the macronutrients which can yield energy in the body.

CHEMISTRY

• Proteins are large molecules; their basic units are alpha amino acids.
• The amino acids are joined to each other through -CONH- linkages
• There are 20 different amino acids present in proteins, and a protein may have
some or all of them.
Peptide bond
• In a peptide molecule the amino acids are attached to their
neighboring amino acids by –COOH on one side and by –NH2
groups on the other; one molecule of water is eliminated in this
process. In this way an acid –amide bond is formed which is called
a peptide bond.

CLASSIFICATION OF PROTEINS

Simple proteins Compound proteins Derived proteins

Albumins Nucleoproteins Primary derived proteins
Globulins Phosphoproteins Secondary derived
Globins Lipoproteins proteins
Prolamins Glycoproteins • Proteoses
Histones Chromo proteins • Peptones
Protamines Metalloproteins • Polypeptides
Albuminoids • Oligopeptides
Energy value
1gram of protein can yield 4kcal of energy.

Food Sources
Protein-containing foods are grouped as either complete or incomplete proteins.
Complete proteins contain all nine essential amino acids. Complete proteins are
found in animal foods such as meat, fish, poultry, eggs, milk, and milk products such as
yogurt and cheese. Soybeans are the only plant protein considered to be a complete
protein.
Incomplete proteins lack one or more of the essential amino acids. Sources of
incomplete protein include beans, peas, nuts, seeds, and grain. A small amount of
incomplete protein is also found in vegetables.
Plant proteins can be combined to provide all of the essential amino acids and
form a complete protein. Examples of combined, complete plant proteins are rice and
beans, wheat cereal, and corn and beans.

Recommended Daily Allowance

Age, Gender, Life group/stage Protiens (g/day)

Infants
0 – 6 mo 9.1
7 – 12 mo 13.5
Children
1 – 3 yrs 13
4 – 8 yrs 19
Male Adults
9 – 13 yrs 34
> 14 yrs 56
Female Adults
9 – 13 yrs 34
>14 yrs 46
Pregnancy
14 – 50 yrs 71
Lactation
14 – 50 yrs 71
 Plasma Proteins
The proteins in the plasma are
• Serum albumin
• Serum globulins(IgG,IgM,IgA,IgD,IgE)
• Fibrinogen

Normal values

• Total proteins 7.3gm%

• Serum albumin 4.7gm%
• Serum globulin 2.3gm%
• Fibrinogen 0.3gm%

Origin of plasma proteins

A. In embryo
In embryonic stage .plasma proteins are synthesized by mesenchyme cells.
First the albumin is produced in the embryo and then the other proteins
B. In adults
In adults the plasma proteins are synthesized by reticuloendothelial cells of liver.
• They are also synthesized from spleen bone marrow and disintegrating blood cells
and general tissue cells
• Gamma globulins are synthesized by B lymphocytes

PROPERTIES

1. Molecular weight
The molecular weight of albumin 69000
The molecular weight of globulin 156000
The molecular weight of fibrinogen 400000

2. Osmotic pressure

Plasma proteins are responsible for osmotic pressure of blood. The

osmotic pressure exerted by proteins in the plasma is called colloidal
osmotic pressure. Normally it is about 25mmHg.albumin plays an
important role in that.

3. Specific gravity

The specific gravity of plasma proteins is 1.026

4. Buffer action
Plasma proteins have 1/6 of total buffering action in the blood
 SEPARATION OF PLASMA PROTEINS

The plasma proteins and their fractions can be separated by following methods;
• Precipitation method
• Salting out method
• Electrophoretic method
• COHN’s fractional precipitation method
• Ultracentrifugation
• Immunoelectrophoretic method

FUNCTIONS OF PLASMA PROTEINS

Coagulation of blood:
Fibrinogen is essential for coagulation of blood. it is converted into fibrin

Defense mechanisms:
Antibodies are specialized proteins involved in defending the body
from antigens (foreign invaders).

Transport:
Transport Proteins are carrier proteins which move molecules from one
place to another around the body. Examples include hemoglobin and cytochromes.
Hemoglobin transports oxygen through the blood. Cytochromes operate in
the electron transport chain as electron carrier proteins.
They are important for transport of hormones, enzymes, respiratory gasses and metals
across the cell membranes.

Contractile Proteins :
Contractile Proteins are responsible for movement. Examples include actin and
myosin. These proteins are involved in muscle contraction and movement.

Enzymes :
Enzymes are proteins that facilitate biochemical reactions. They are often referred to
as catalysts because they speed up chemical reactions. Examples include the enzymes
lactase and pepsin. Lactase breaks down the sugar lactose found in milk. Pepsin is a
digestive enzyme that works in the stomach to break down proteins in food.

Hormonal Proteins
Hormonal Proteins are messenger proteins which help to coordinate certain bodily
activities. Examples include insulin, oxytocin, and somatotropin. Insulin regulates
glucose metabolism by controlling the blood-sugar concentration. Oxytocin
stimulates contractions in females during childbirth. Somatotropin is a growth
hormone that stimulates protein production in muscle cells.

Structural Proteins
Structural Proteins are fibrous and stringy and provide support. Examples include
keratin, collagen, and elastin. Keratins strengthen protective coverings such as hair,
quills, feathers, horns, and beaks. Collagens and elastin provide support
for connective tissues such as tendons and ligaments.

Storage Proteins
Storage Proteins store amino acids. Examples include ovalbumin and casein.
Ovalbumin is found in egg whites and casein is a milk-based protein.

Maintenance of osmotic pressure:

They are also important for maintenance of osmotic pressure. The osmotic
pressure exerted by plasma proteins is about 25 mm Hg

Maintenance of pH:
They play an important role in maintenance of pH

Role in viscosity of blood:

They play an important role in providing viscosity to blood. Albumin provides
more viscosity than other plasma proteins

Reserve proteins:
During periods of fasting they are utilized by body hence called reserve proteins

Suspension stability of RBC:

They are important as they increase the suspension stability of RBC
 ESTIMATION OF TOTAL PROTEINS IN SERUM

PRINCIPLE:
Protein peptidic bond react with cupric ion in alkaline medium, rendering a violet
complex with a maximum absorption at 540nm, being its intensity proportional to the
total proteins concentration of the sample.

PROVIDED REAGENT:
13mmol/l EDTA/Cu complex in 875 mmol/l NaOH and alkyl aryl polyether (AAP)

NON-PROVIDED REAGENTS:
Suero Patron: separately provided by Wiener Lab

SAMPLE:
Serum

a. Collection: obtain non-hemolyzed serum

b. Additives: not required
c. Known interference substances: No interference from bilirubin upto 100mg/l or
from mild hemolysis is observed. Turbidity caused by chylomicrons has never
been observed.
d. Stability and storage instructions: if the serum is not immediately tested, it can
be stored up to 3 days in refrigerator (2-10 0C) or a week in freezer.

REQUIRED MATERIAL:

 Spectrophotometer or photocolorimeter
 Micropipettes and pipettes to measure the stated volumes.
 Spectrophotometer cuvettes or photocolorimeter tubes
 Water bath at 37 0C.
 Stop watch

ASSAY CONDITIONS:

 Wavelength: 540 nm (520-560nm)

 Reaction temperature: 37 0C
 Reaction time: 15 min
 Sample volume: 20 µl
 Reagent volume: 2.0 ml
 Final reaction volume: 2.02 ml
PROCEDURE:
In three photocolorimeter test tubes labeled B (Blank), S (Standard) and U
(Unknown), place:

B S U
Suero patron - 20 µl -
Sample - - 20 µl
Reagent 2.0 ml 2.0 ml 2.0 ml

Mix with rod. Incubate 15 minutes at 37 0C. Read in photocolorimeter at 540nm

setting the instrument to zero O.D. with Reagent Blank.

STABILITY OF FINAL REACTION:

The reaction is stable for 12 hours, thus readings should be performed within this
period.

CALCULATIONS:
Using the Suero patron as indicated in Procedure, the calculations are performed
as follows:

Concentration of Standard = 4.8 g/dl

Absorbance of Standard = ---------------------------------------

Absorbance of Sample = ---------------------------------------

Total Proteins (g/dl)= Absorbance of sample

---------------------------- X Conc. of standard
Absorbance of standard