Chapter III:

Proteins
 Definition
 Large biological molecules, or macromolecules, consisting
of one or more long chains of amino acid residues
 The most abundant and functionally diverse molecules in
living systems
 Biological Importance of Amino Acids
1- Amino acids serve as building blocks of proteins.

1- Building of new cells

2- Maintenance of existing cells

3- Source of energy (1 g protein gives 4 Kcal)

4- They are involved in:

- Hormones - regulation of metabolic process
- Enzymes - catalysis of biochemicalreactions
- Hemoglobin - transport of oxygen
- Antibodies - body's defense against infection
- Nerves - transmission of impulses
- Contraction - muscle activity
- Components of skin, hair, nails, connecting and supporting tissues.
 Chemical Nature of Amino Acids
Among the different amino acids (> 300), only 20 are
found as constituents of mammalian proteins
common to all a.a. of proteins

H O

H2N C C OH
 carboxyl group

R
carbon is between the carboxyl and
amino group the amino group
side chain:
distinctive for each a.a

+
or H3N C COOH

R
 Structure of amino acids
At physiologic pH (~ 7.4)

peptide bond
H O H O H O H O

+ - + -
-H2O
H3N C C O + H3N C C O +
H3N C C N C C O-

R1 R2 R1 H R2

side chains determine the

Thus, more amino and properties of proteins
carboxyl groups add on into
forming a peptide chain
(that is not available for
reactions) Their nature dictates role of
an a.a plays in a protein
 Classification of a.a.
Classified according to the properties
of their side chains

non-polar
side chains
polar side
chains
acidic side
chains
basic side
chains
 Classification of a.a.
Amino acids with non-polar side chains
O O

H2N CH C OH H2N CH C OH

CH2 CH CH3

CH CH3 CH2

CH3 CH3 -oily (lipid-like), thus

promoting hydrophobic
Leucine = Leu Isoleucine = Ile interactions

-don’t bind
-don’t give proton
-don’t participate in
hydrogen or ionic bonds
 Classification of a.a.
Amino acids with non-polar side chains
H H O

N C C Ile

HC CH3

CH2

CH3
Cluster together, acting
much like droplets of oil
polypeptide backbone
Hydrophobic interactions
coalescing in aqueous
environment
H 3C CH3
CH

CH2
Leu
N C C

H H O

Stabilize the protein

structure
 Classification of a.a.
Amino acids with non-polar side chains
O

C OH
amino group
O Alanine = Ala
Proline = Pro
H2N CH C OH
HN
CH3

imino group

its unique geometry

contributes to the formation
of the fibrous structure of
collagen
 Classification of a.a.
Amino acids with uncharged polar side chains
O

H2N CH C OH

CH2
O O

H2N CH C OH H2N CH C OH

CH2 CH OH

OH CH3
OH

Serine = Ser Threonine = Thr Tyrosine = Tyr

Polar hydroxyl group (OH) can participate in

hydrogen bond formation
 Classification of a.a.
Amino acids with uncharged polar side chains
O

O H2N CH C OH

H2N CH C OH CH2

CH2 CH2

C O C O

NH2 NH2

Asparagine = Asn Glutamine = Gln

carbonyl group (C=O), and amide group (NH2) can

also participate in hydrogen bond formation
 Classification of a.a.
Amino acids with uncharged polar side chains
O

H2 N CH C OH

CH2

Tyrosine = Tyr

hydrogen bond

C carbonyl group
 Classification of a.a.
Amino acids with uncharged polar side chains
O O

N CH C
N CH C

H CH2 H CH2

SH S
oxidant: O2 disulfide bond

SH
H CH2

H CH2
N CH C

N CH C
O

2 cysteines residues cystine residue

Active site of many enzymes

 Classification of a.a.
Amino acids with acidic side chains
O O

H2N CH C OH H2N CH C OH

CH2
At physiologic pH CH2
fully ionized,
C O CH2
containing negatively
OH charged carboxylate C O

group (COO-) OH

Aspartic acid = Asp Glutamic acid = Glu

- proton donors
- called aspartate and glutamate (a.a.
negatively charged at physiological pH)
 Classification of a.a.
Amino acids with basic side chains O
O
H2N CH C OH
H2N CH C OH
CH2
CH2
CH2
CH2 At physiologic pH
CH2
CH2
fully ionized,
NH
containing positively
CH2
groups C NH

NH2
NH2

Lysine = Lys Arginine = Arg

proton acceptors
 Classification of a.a.
Amino acids with basic side chains
O

H2N CH C OH

CH2 can be positively charged or neutral

(depends on ionic environment
N
provided by polypeptide chains of
the protein)
NH

Histidine = His

Important property that helps in the

role histidine plays in functioning of
proteins (ex: hemoglobin)
 Optical properties of a.a.

chiral
optically active H O

H2N C C OH
 carboxyl group

R
carbon is between the carboxyl and
amino group the amino group
side chain:
distinctive for each a.a

Exception

optically
Glycine
inactive

2H
 Optical properties of a.a.

COOH COOH

+
H3N C H H C NH3+

CH3 CH3
Left side Right side
L-alanine D-alanine
mirror

- All a.a found in proteins are of the L configuration

- D-amino acids are found in some antibiotics and in
bacterial cell walls
 Protein modification and a.a. derivatives
Decarboxylation
CO2

glutamate -aminobutyrate (GABA): neurotransmitter in brain

CO2

Histidine Histamine

controls the constriction of certain

blood vessels and the secretion of HCl
by the stomach.
 Protein modification and a.a. derivatives
Oxidation

oxidation
Proline hydroxyproline

controls the synthesis and stabilization

of collagen fiber.

its insufficient hydroxylation

caused by deficiency in
vitamin C leads to scurvy
 Protein modification and a.a. derivatives
Carboxylation

CO2

glutamate -carboxyglutamate

its insufficient carboxylation in

prothrombin (a clotting protein)
caused by vitamin K deficiency
can lead to hemorrhage.
 Structure of Proteins
Primary structure of proteins

In other terms, the primary structure is

the sequence of the a.a.

Genetic diseases in proteins can cause

abnormal sequences of a.a.

Improper folding and loss of

impairment of normal function
 Structure of Proteins
Primary structure of proteins
H O H O

+H3N C C O- + +H3N C C O-

HC CH3 CH3
alanine
CH3

valine - H2O

Free carboxyl
H O H O
end of peptide
+H3N C C N C C O-

H3C CH H - not broken by conditions that denature

CH3

CH3 peptide bond

proteins (ex: heating, high [urea]…
valylalanine (amide linkages) - Can be hydrolyzed nonenzymically by
long exposure to a strong acid or base at
elevated T°
 Structure of Proteins
Primary structure of proteins

H O H O
N-terminal C-terminal
+H3N C C N C C O-
(free a.a.) (free carboxyl)
LEFT H3C CH H CH3 RIGHT
CH3

All a.a. are read from left to right, that

is from N-terminal to C-terminal

its a.a. if with suffixes (-ine, - its a.a. if with suffixes (-ine, -
an, -ic) change to –yl after an, -ic) stay the same
bonding
 Structure of Proteins
Secondary structure of proteins
In other terms, the secondary structure is
the regular recurring of a.a. chain (α-helix)
 Structure of Proteins
Secondary structure of proteins
Many proteins contain α-helices. Ex.
keratin (in tissues such as hair and skin)

its rigidity is determined by the # of

disulfide bonds between constituent
polypeptide chains

Note: each turn of an α-helix contain

3.6 a.a. residues, so a.a. in primary
structure that are 3 or 4 spaced apart
become close when folded
 Structure of Proteins
Tertiary structure of proteins
results from folding of a linear protein
chain with secondary structure

a.a. residues that are far apart

in 1 structure are brought
together (interactions occur
among their side chains)

This structure is stabilized by non-covalent interactions

(mostly hydrophobic effect) between the side chains of a.a.
 Structure of Proteins
Quaternary structure of proteins
results from combination of several protein units (each
with its 1, 2, and 3 structure) to form a complex unit

Can function together, or

independently from each other
Ex. hemoglobin: binding of O
to one subunit of the tetramer
increases the affinity of the
other subunits of O
 Denaturation of Proteins

Protein denaturation results in the unfolding and

disorganization of the protein’s II and III structures
(without hydrolysis of peptide bonds).

It can be reversible under ideal conditions

(i.e. refolds to its original structure when
the denaturing agent is removed)

Heat, organic solvents, mechanical

mixing, strong acids or bases, ions Under physiological
of heavy metals (Pb, Hg) conditions, most proteins
are stable at temperatures
up to 50° to 60°C.
Selected plasma proteins (body fluids proteins

30 000 to 50 000 structural genes code for synthesis of

human proteins among which more than 300 are in the
plasma

Most plasma proteins are are synthesized in the liver

with exception of immunoglobulins and protein
hormones

Albumin
α1-Globulins
α2-Globulins
β-Globulins
γ-Immunoglobulins
 Selected plasma proteins (body fluids proteins
Albumin

 Present in high [] in the serum

 Represents 50-60% of total proteins
 synthesized in the liver
 normal value: 35-55 g/l

2 well-known functions:
-Because of its high concentration, albumin is responsible
for nearly 80% of the colloid osmotic pressure of the
intravascular fluid, which maintains the appropriate fluid
in the tissue.
- Its propensity to bind various substances in the blood.
Ex. albumin binds bilirubin, salicylic acid
 Selected plasma proteins (body fluids proteins)
Variation in normal value of albumin
Hypoalbuminaemia (decreased [albumin] in serum)
Can be caused by:
- Malnutrition (inadequate source of amino acids)
- Burns
- Hemorrhage
- Fever
- Liver disease (inability of hepatocytes to synthesize albumin)
- Nephrotic syndrome (loss of albumin in the urine).

Hyperalbuminaemia (increased [albumin] in serum)

 Selected plasma proteins (body fluids proteins)
Albumin

 [albumin] in plasma used as a test of liver function

 [albumin] is normal in acute hepatitis because of its long

half-life in plasma

 low [albumin] indicate a chronic liver disease (can be

due to both decreased synthesis and an increase in the
volume of distribution as a result of fluid retention)

 Many drugs bind to albumin in blood stream, thus

decreasing [albumin] which can have pharmacokinetic
consequences. Ex. increasing [free drug] leading to toxicity
 Selected plasma proteins (body fluids proteins)
α1-Globulins
α1-Fetoprotein

 present in tissues and plasma of the fetus

 Plasma concentrations falls rapidly after birth ( trace
amounts ~15 μg/l are present in plasma from adults)
 Their function are unclear, but may play an
immunoregulatory role during pregnancy
 Increased amniotic fluid and plasma AFP are present in
women carrying a fetus with open neural tube defects
 Dncreased plasma AFP are present in women carrying a
fetus with Down’s syndrome
 Selected plasma proteins (body fluids proteins)
α2-Globulins
Ceruloplasmin
 A deficiency of this copper-carrying α2 -globulin is
characteristic of Wilson's disease (disease related to bad
transport of copper and precipitation of this in the liver).

 90% of total serum copper is found in ceruloplasmin.

 [ceruloplasmin] is increased in pregnancy and by

oestrogen-containing oral contraceptives.

 It is also an acute phase protein.

 Selected plasma proteins (body fluids proteins)
β-Globulins
Transferrin

 Major iron-transporting protein in the plasma and is

synthesized by the liver.

 Transports iron to its storage sites, where it is incorporated

into apoferritin, another protein, to form ferritin.

 Carries iron to cells, such as bone marrow, that synthesize

hemoglobin and other iron-containing compounds.

 Normally about 30% is saturated with iron

 Selected plasma proteins (body fluids proteins)
β-Globulins
Transferrin

In hemochromatosis (transferrin is characteristically 100%

saturated with iron) in which excess iron is deposited in the
tissue, especially the liver and the pancreas.
This disorder is associated with bronze skin, cirrhosis, diabetes
mellitus, and low plasma transferrin levels.

Measurement of ferritin plasma concentration is the best single

test now available for determining body iron stores.
 Selected plasma proteins (body fluids proteins)
β-Globulins
C-Reactive Protein (CRP)

 Synthesized in the liver

 Appear in the blood of patients with diverse
inflammatory diseases.
 CRP was so named because it precipitates with the C
substance, a polysaccharide of pneumococci.
 CRP rises sharply whenever there is tissue necrosis,
whether the damage originates from a pneumococcal
infection or some other source.
 elevated in acute rheumatic fever, bacterial infections,
myocardial infarcts, and viral infections.
 Selected plasma proteins (body fluids proteins)
β-Globulins
C-Reactive Protein (CRP)
Reduced CRP levels and reduced vascular risk can result
from interventions such as weight loss, diet, exercise, and
smoking cessation and administration of pharmacologic
agents such as statins.

CRP is very useful because it can differentiate between the

viral infection (low CRP) from bacterial infection (CRP very
high; > 6 mg/dl) especially in newborns and children.
 Selected plasma proteins (body fluids proteins)
γ-Immunoglobulins (Igs)
5 groups in the serum: IgA, IgC, IgM, IgD, IgE
Function as antibodies (recognize and bind foreign antigens)
Every immunoglobulin is sepcific for one antigenic detremination

IgG is increased in liver disease, infections and collagen disease.

IgA is present in the respiratory and gastrointestinal mucosa. The
increase in the serum IgA is found in liver disease, infections,
and autoimmune diseases.

IgM is the first antibody that appears in response to antigenic

stimulation. Increased IgM concentration is found in
toxoplasmosis, rubella, herpes, syphilis, and various bacterial
and fungal diseases.
 Selected plasma proteins (body fluids proteins)
Variation in normal values of γ-Immunoglobulins (Igs)

Physiological hypogammaglobulinaemia is one of the

reasons for the susceptibility of infants (especially the
premature) to infection.

Hypergammaglobulinaemia: increased levels of

immunoglobulins are seen in both acute and chronic
infections. For example, rheumatoid disease and in chronic
liver diseases, some of which have an autoimmune basis.
 Determination of serum total protein
Biuret method

- Cupric ions (Cu2+) complex with the groups invloved in

the peptide bond
-With the presence of at least two peptide bonds in an
alkline medium a violet colored chelate is formed
- Reference range: 6-8 g/dl or 60-80 g/l

Intensity of colore is
propotional to # of
peptide bonds
 Determination of serum total protein
Kjeldahl method

- Nitrogen is determined

- average of 16% nitrogen mass in protein calculates

[protein]

- not used in laboratories because it is time consuming

and too tedious for routine use
 Determination of serum total protein
Dye-Binding methods

- Based on ability of most proteins in serum to bind dyes

- The affinity with which proteins bind may vary

- Coomassie Brilliant Blue or Amido Black or Panceau S

stain protein bands after electrophoresis or determination
of total protein by spectrophotometry
 Serum Protein Electrophoresis

Electrophoresis: migration of charged solutes or particles in

a liquid medium under the influence of an electric Field. The
migration distance varies directly with the charge carried by
the proteins.

albumin
α1-globulins
α2-globulins
β-globulins
- arranged into five bands γ-globulins
- stained after separation
- appear as bands on support medium after dye is added
Anode Cathode
 Serum Protein Electrophoresis

The width of the band of proteins in a fraction depends on

the number of proteins with slightly different molecular
characteristics that are present in that fraction.

Area of the peak is

integrated by
densitometer
indicating [] of each
fraction of the. And is
[total protein] is
known then [protein
fraction] is calculated
 Serum Protein Electrophoresis

Reference serum control should be run with each electrophoretic

Reference values for each fraction:

Albumin: 53-65 % of total protein (3.5-5 g/dl)

α1-globulins: 2.5-5 % of total protein (0.1-0.3 g/dl)
α2-globulins: 7-13 % of total protein (0.6-1 g/dl)
β-globulins: 8-14 % of total protein (0.7-1.1 g/dl)
γ-globulins: 12-22 % of total protein (0.8-1.6 g/dl)
 Serum Protein Electrophoresis

In nephrotic syndrome: a dramatic decrease in the relative

amount of albumin and a significant increase in the relative
amounts of α2-globulin and β2-globulin fractions.
 Serum Protein Electrophoresis

An inflammatory pattern indicating an inflammatory

condition is seen where there is a decrease in albumin and an
increase in the α1-globulins, α2-globulins, and β-globulin band
(CRP). This type of pattern, also called an acute-phase
reactant pattern, is seen in trauma, burns, infarction, and liver
disease.
 Serum Protein Electrophoresis

The electrophoretic pattern of serum proteins in liver disease

shows the decrease in serum albumin concentration and the
increase in γ-globulin. In addition, there are some fast moving
γ-globulins that prevent resolution of the β- and γ-globulin
bands. This is known as the β-γ bridge of cirrhosis.

CIRRHOSIS
 Proteins in urine: Proteinuria

- Reference range: < 0.15 g/l or 0.12 g/24hr

- Normal subjects excrete up to 0.08 g of protein per day in the

urine (undetectable by usual screening tests)

- Proteinunria > 0.15g per day indicates disease

- Significant proteinuria may be due to renal disease or, more

rarely, may occur because large amounts of low-molecular-
weight proteins are circulating and therefore being filtered.

- Blood and pus in the urine and also urinary infection give
positive tests for protein.
 Microalbumin

- microalbuminuria is abnormal excretion of small amount of

albumin in the urine

- Routine tests give only positive reading at levels > 250 mg/day

- Reference range: < 20 mg/l or > 30 mg/24hr

- Patients with diabetes mellitus who excrete more than 0.05g,

but whose total urinary protein excretion is “normal”, are said to
have microalbuminuria and to be at greater risk of developing
progressive renal disease than those whose albumin excretion is
normal.