Proteins
Introduction

Proteins are polymers of amino acids produced
by living cells in all forms of life.

A large number of proteins exist with diverse
functions, sizes, shapes and structures but each
is composed of essential and non-essential
amino acids in varying numbers and sequences.

The number of distinct proteins within one cell is
estimated at 3,000 - 5,000
 The
most abundant organic molecule in cells (50-70%
of cell dry weight)
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Size

A typical protein contains 200-300 amino
acids, but some are much smaller and
some are much larger

Proteins range in molecular weight from
6,000 Daltons (insulin) to millions of
Daltons (structural proteins)
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Peptide bond
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Protein Structure
Primary structure

Sequence of AA

In order to function properly,
proteins must have the correct
sequence of amino acids.

e.g when valine is substituted
for glutamic acid in the  chain
of HbA, HbS is formed, which
results in sickle-cell anemia.
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Protein Structure
Secondary structure

A regularly repeating
structures stabilized by
hydrogen bonds between the
amino acids within the protein

Initial helical folding (α helix)

Beta pleated sheet

Held together by Hydrogen
bonding
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Protein Structure
Tertiary Structure

The overall shape, or
conformation, of the protein
molecule

Chain folds back on itself to
form 3D structure

Interaction of R groups
(hydrogen bonds, and
disulfide bonds)

Responsible for biologic activity
of molecule
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Protein Structure
Quaternary structure

The shape or structure that results
from the interaction of more than one
protein molecule, or protein subunits,
held together by noncovalent forces
such as hydrogen bonds and
electrostatic interactions

2 or more polypeptide chains binding
together

eg. Hemoglobin

Hemoglobin has 4 subunits



Two  chains
Two  chains
Many enzymes have quaternary structures
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Protein Structure

Disruption of bonds holding 2o, 3o or 4o structure
together is called denaturation and can cause
loss of function of the protein

These Bonds that hold the protein together are
weak

It is important in the clinical lab to note that
excessive heat, freeze thaw cycle or vigorous
mixing can break these bonds and denature the
protein
 An
enzyme can loose its activity, Ag can loose its
antigenicity
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Protein Charges

Proteins contain many ionizable groups on the side
chains of their amino acids as well as their N- and Cterminal ends

A protein can bear positive and/or negative charges on
each molecule, due to amino acid composition

pH dependent

Aspartic acid has:



no net charge at pH 2.8,
but a net negative charge at pH 9.47
Lysine has:


no net charge at pH 9.47,
but a net positive charge at acid pH
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Protein Charges
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Protein Charges

Isoelectric point (pI) – The pH at which an
amino acid or protein has no net charge
 The
point at which the number of positively
charged groups equals the number of negatively
charged groups in a protein

When the pH > pI, a protein has a net negative
charge

When the pH < pI, a protein has a net positive
charge
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Solubility

Soluble proteins have a charge on their surfaces.

A protein has its lowest solubility at its isoelectric
point

Without a net charge, protein-protein interactions
and precipitation are more likely

The solubility of proteins in blood requires a pH in
the range of 7.35-7.45.

Differences in solubility can be used to separate
major plasma fractions from each other
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Classification by Protein Structure

Simple Proteins (contain only amino
acids) are classified by shape as –
 Globular
proteins: compact, tightly folded
and coiled chains

Majority of serum proteins are globular
 Fibrous
proteins: elongated, high viscosity
(hair, collagen)
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Classification by Protein Structure

Conjugated proteins contain non-amino acid
groups
 Amino
acid portion is called apoprotein and nonamino acid portion is called the prosthetic group
 The prosthetic group may be lipid, carbohydrate,
porphyrins, metals, and others
 It is the prothetic groups that define the characteristics
of these proteins.
 Name of the conjugated protein is derived from the
prosthetic group
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Conjugated Proteins
Classification
Example
Lipoprotein
Prosthetic
group
Lipid
Glycoprotein
Carbohydrates
Phosphoprotein
Phosphate
Immunoglobulins
Casein of
milk
HDL
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Functions of proteins

Generally speaking, proteins do everything in the living cells

Functional classification of plasma proteins is useful in
understanding the changes that occur in disease:
 Tissue
nutrition
 Proteins of immune defense

Antibodies
 Acute phase proteins
 Proteins associated with inflammation
 Transport proteins( albumin, transferrin)
 Proteins used to bind and transport
 Hemostasis
 Proteins involved in forming clots and acting very closely with
complement
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Functions of proteins
 Regulatory

( receptors, hormones )
 Catalysis,

enzymes
 Osmotic

force
Maintenance of water distribution between cells
and tissue and the vascular system of the body
 Acid-base

balance
Participation as buffers to maintain pH
 Structural,
contractile, fibrous and keratinous
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Catabolism & Nitrogen Balance

Most proteins in the body are constantly being
repetitively synthesized and then degraded

Balance exists between protein anabolism (synthesis)
and catabolism (breakdown)

Turnover totals about 125-220 g of protein each day

Normal, healthy adults are generally in nitrogen balance,


with intake and excretion being equal
Pregnant women, growing children, and adults
recovering from major illness

are often in positive nitrogen balance
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Catabolism & Nitrogen Balance

Conditions in which there is excessive
tissue destruction, such as burns, wasting
diseases, continual high fevers, or
starvation.
 more
nitrogen is excreted than is incorporated
into the body,
 an individual is in negative nitrogen balance
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Plasma Proteins

About 500 proteins have been identified in
plasma

The plasma proteins include the
immunoglobulins, enzymes, and enzyme
inhibitors.

Most plasma proteins, with the notable exception
of immunoglobulins, are synthesized in the liver.

Plasma proteins circulate in the blood and
between the blood and the extracellular tissue
spaces.
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Plasma Proteins

Prealbumin

Albumin

Globulins α1, α2, β, and γ
•
α2 Macroglobulin
•
Transferrin
(Siderophilin) β
•
Hemopexin
•
Acute phase proteins
(CRP)
•
Immunoglobulins
•
α1-Antitrypsin
•
α1-Fetoprotein
•
α1-Acid Glycoprotein

Myoglobin
•
Haptoglobin (α2)

Troponin
•
Ceruloplasmin
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Prealbumin (Transthyretin)

Migrates just ahead of Albumin

When a diet is deficient in
protein, hepatic synthesis of
proteins is reduced
 Indicator
of nutritional status (very
short half life- 2 days)

Transport of thyroid hormones
& retinol (vitamin A)
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Prealbumin (Transthyretin)

Low levels found in:
 Hepatic
damage
 Acute phase responses (-ve acute phase reactant)
 Nutritional deficit – short half-life means decrease
seen early in disease

Increased level found in:
 Steroid
treatment
 Alcoholism
 Chronic renal failure (glomerular filtration rate
decreased)
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Albumin

Synthesized in the liver from 585 amino acids
at the rate of 9–12 grams per day with no
reserve or storage

Highest concentration plasma protein

Albumin also exists in the exrtavascular
(interstitial) space

Two primary functions
 Colloidal
osmotic pressure (80%)
 Bind and transport of numerous substances


Bilirubin, steroids, fatty acids, Ca++, Mg++,
salicylic acid & other medications
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Albumin
Albumin

Decreased Albumin
 Malnutrition & muscle wasting diseases
 Liver diseases – inability to synthesize
 GI loss due to inflammation or mucosal lining
diseases
 Loss in urine due to renal disease
 Genetic analbuminemia (Mutation causes absence of
albumin)
 Bisalbuminemia

results from two copies of different albumin genes,
resulting in different charges.
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Albumin

Increased levels of Albumin
 Seen
in dehydration
 After excessive albumin infusion
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Globulins

The globulin group of
proteins consists of α1, α2,
β, and γ fractions.

Each fraction consists of a
number of different proteins
with different functions.
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Globulins (α1-Antitrypsin)



Major component (90%) of α1 fraction
Acute phase reactant, synthesized in the liver
Most important function the inhibition of the
protease neutrophil elastase


Neutrophil elastase is released from leukocytes to
fight infection, but it can destroy alveoli
Mutations in the SERPINA1 gene cause:
 α1-antitrypsin
deficiency which is associated with
 Severe degenerative emphysema
 Abnormal form of α1-antitrypsin can also accumulate
in the liver and can cause cirrhosis
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α1-Antitrypsin

Increased levels in:
 Inflammatory
reactions
 Pregnancy
 Contraceptive
use

Since major component of α1 band – changes in
levels apparent on protein electrophoresis

Quantitative methods used to confirm
electrophoresis findings are radial immunodiffusion
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α1-Fetoprotein (AFP)

AFP synthesized by fetal yolk sac later by
parenchymal cells of liver

Peaks in fetus at 13 weeks – decreases gradually
by birth

Acts like a fetal “albumin”

Maternal serum testing used to screen fetus for
birth defects


Neural tube defects
Down’s syndrome
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α1-Fetoprotein (AFP)

In adults level is high in:
 80%
of hepatocellular carcinoma
 Gonadal tumors in adults

The methods commonly used for AFP
determinations are radioimmunoassay (RIA) and
enzyme-labeled immunoassay (EIA)
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α1-Acid Glycoprotein (AAG)

Acute phase reactant, synthesized in the liver

Regulates immune responses
 Increased





in:
inflammation,
cancer,
pneumonia,
Rheumatoid arthritis (RA).
The analytic methods used most commonly for
the determination of AAG are radial
immunodiffusion, immunoturbidity, and
nephelometry
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Haptoglobin (α2)

Synthesized in the liver

Considered an acute-phase protein

Binds free hemoglobin

Prevents loss of Hemoglobin & Iron into urine

Used to detect and evaluate hemolytic anemia
and to distinguish it from anemia due to other
causes
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Haptoglobin (α2)

Increased in
 Inflammations,
burns & nephrotic syndrome due to
fluid losses

Decreased in
 with
transfusion reactions, HDN
 Thus, a low plasma haptoglobin concentration can be
indicative of intravascular haemolysis.
 decreased synthesis are seen in chronic liver disease

Radial immunodiffusion has been used for the
quantitative determination of haptoglobin
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Ceruloplasmin (α2)

Synthesized in the liver

acute-phase reactant

Copper carrying protein

90% of serum copper is bound to it

Ordered along with blood and/or urine copper
tests to help diagnose Wilson's disease,
 decreased
levels of ceruloplasmin and excess
storage of copper in the liver, brain, and other organs
resulting in hepatic cirrhosis and neurologic damage.
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Ceruloplasmin

Increased levels
 Pregnancy, inflammatory processes,
 malignancies, oral estrogens & contraceptives

Low levels
 Malabsorption
 Severe Liver Disease
 Nephrotic syndrome
 Menkes’

Syndrome (decreased Cu absorption)
Most assays today use immunochemical
methods, including radial immunodiffusion and
nephelometry
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α2 Macroglobulin

Tetramer of four identical subunits

Synthesized by liver

major component of the α2 band in protein
electrophoresis

Primarily intravascular spaces due to size

Inhibits proteases trypsin, pepsin &
plasmin
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α2 Macroglobulin

Increased levels
 Nephrosis
(Large size prevents loss)
 Oral contraceptives (high estrogens)

Decreased levels
 Pancreatitis

The analytic methods that have been used for
the assay of this protein are radial
immunodiffusion, ELISA, and latex agglutination
immunoassay.
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Transferrin (Siderophilin)







The major component of the -globulin.
Glycoprotein synthesized by liver
Carries 2 ferric iron molecules
Normally 33% saturated
Prevents loss of iron through kidneys
Transports to storage sites where Iron is
transferred to ferritin
Transports to bone marrow for RBC synthesis
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Transferrin (Siderophilin)

Transferrin is abnormally high in iron deficiency
anemia

Decreased in
 general
protein deficiencies
 Liver disease
 Malnutrition
 Inflammations (Negative acute phase protein)
 Hereditary Disorders

The analytic method used for the quantitation of
transferrin is immunodiffusion
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Hemopexin

Beta globulin

acute-phase reactant

Purpose is to remove circulating Heme



Breakdown product of Hemoglobin & myoglobin
Carried to liver – broken down
Increased in


pregnancy,
Some malignancies

Low hemopexin levels are diagnostic of a hemolytic
anemia

Hemopexin can be determined by radial immunodiffusion
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C-Reactive Protein

Acute phase reactant

CRP was so named because
it precipitates with the C
substance, a polysaccharide
of pneumococci.

Antibody-like reactivity for
many bacteria


Opsonization
It is elevated in acute
rheumatic fever, bacterial
infections, myocardial
infarction, rheumatoid arthritis,
etc…
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Immunoglobulins

IgA, IgD, IgE, IgG, IgM

Consist of two identical heavy (H) and two
identical light (L) chains

Decreases seen in general protein deficiencies

Increases
 Chronic
inflammatory processes - polyclonal
 Malignancy (multiple myeloma) - monoclonal
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Myoglobin

Heme protein of skeletal & cardiac muscle

Single polypeptide chain transports
oxygen to muscle tissue

Released into blood when striated muscle
is damaged
 Cardiac
injury (AMI)
 Trauma or crush injuries

Latex agglutination, ELISA
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Troponin

Complex of 3 proteins that bind to filaments of
striated muscle (cardiac & skeletal)
 Troponin
T (TnT)
 Troponin I (TnI)
 Troponin C (TnC)

Regulate muscle contractions
 Calcium
release
attaches
troponin


Increase indicative of myocardial injury

Cardiac troponins can be measured by ELISA
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Hypoproteinemia

Total protein level less than the reference
interval, occurs in any condition where a
negative nitrogen balance exists:
 Malnutrition
and/or malabsorption
 Excessive loss as in renal disease, GI leakage,
 excessive bleeding, severe burns
 Excessive catabolism

Burns, trauma, shock
 Liver

disease
Primary site of protein
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Hyperproteinemia

Dehydration
 Relative due to fluid decrease
 Decreased intake or increased loss
 All

fractions remain normal ratios
Monoclonal increases
 Multiple

Myeloma or related malignancies
Polyclonal increases
 Chronic
inflammatory diseases
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