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Chemistry of Proteins

Chemistry of Proteins
Definition:
Proteins are organic compounds with a
high molecular weight formed of
carbon, oxygen, hydrogen and nitrogen
and may also contain sulfur,
phosphorus coloring non-protein
organic groups and metal ions.
They are polymers formed of subunits
called amino acids linked together by
peptide linkage.

Biological importance of proteins


1. Nutritional role: Provide the body with
essential amino acids, nitrogen and sulfur.
2. Catalytic role: All enzymes are proteins in
nature.
3. Hormonal role: Most of hormones and all
receptors are protein in nature.
4. Defensive role: The antibodies
(immunoglobulins) that play an important
role in the bodys defensive mechanisms
are proteins in nature.

5-Plasma proteins are responsible for most


effective osmotic pressure of the blood.
This osmotic pressure plays a central role in
many processes, e.g., urine formation.
Blood clotting factors are proteins.
6-Transport role: Proteins carry lipids in the
blood forming lipoprotein complexes.
Proteins also carry, hormones, e.g., thyroid
hormones and minerals, e.g., calcium, iron
and copper. Hemoglobin (a chromo-protein)
carries O2 from the lung to tissues is a
protein.
7-Structural role: Proteins are the main
structural component in bone, muscles,
cyto-skeleton and cell membrane.
8-Control of gene expression: Most factors
required for DNA replication, transcription
and mRNA translation are protein in nature.

Amino Acids
Amino acids are organic acids that contain NH2
group.
They are the structural units of proteins and are
obtained from them by hydrolysis.
The general formula of any amino acid is as
follows:
O

H2N

CH

OH

All these amino acids are alpha-amino acids and


the metabolizible form of them are L-amino acids.
Alpha-amino acid means that the amino group is
attached to the -carbon atom,

Each amino acid (except proline and


hydroxyproline) has a carboxyl group
(COOH), an amino group (NH2) and a
characteristic side chain (R).
All amino acids (except glycine) are
optically active, i.e., can rotate plane
polarized light.
This is because the 4 groups attached
to -carbon are different. In glycine,
the -carbon is attached to 2 hydrogen
atoms, therefore, is optically inactive.

Classification of Amino Acids


Amino acids can be classified by one of
three methods:
I-Chemical classification: Based upon the
number of amino groups or carboxyl
groups in the amino acid:
1. Neutral amino acids (mono-amino, monocarboxylic).
2. - Acidic amino acids (mono-amino,
dicarboxylic).
3. - Basic amino acids (diamino, monocarboxylic).

II-Biological classification: Based upon


whether the amino acids can be
synthesized in human body or not:
1. Essential amino acids: Not
synthesized in the body and must be
supplied in the diet.
2. Non-essential amino acids: Can be
synthesized in the body and is not
essential to be present in diet.

III-Metabolic Classification: Based upon


the fate of amino acid inside the body :
1. Glucogenic amino acids, that can be
converted to glucose.
2. Ketogenic amino acids, that can be
converted to ketone bodies.
3. Mixed function amino acids, i.e., can
be converted to both glucose and
ketone bodies

I-Chemical classification
(According to number of carboxyl and
amino groups)
Amino acids can be classified into:
a) Neutral amino acids.
b) Acidic amino acids.
c) Basic amino acids.

A) Neutral amino acids


They contain one amino group and
one carboxyl group. They have 5
types:
1-Aliphatic
O : e.g.,
O amino acids
O
H2N CH

OH

H2N CH

OH

CH3

Glycine

H2N CH

CH CH3

Alanine

CH3
O

O
H2N

CH

OH

H2N

CH

CH2

CH CH3

CH CH3

CH2

CH3

CH3

Leucine

OH

Isoleucine

OH

Valine

2. Hydroxy amino acids: e.g.,


serine, threonine,.

O
H2N CH

C OH

H2N CH

C OH

CH2

CH OH

OH

CH3

Serine

Threonine

3. Aromatic amino acids: e.g.,


phenylalanine and tyrosine .
Tyrosine is synthesized from phenyl alanine
and both give triiodothyronine and thyroxin,
adrenaline and noradrenaline.
Melanin pigment and cresol ,phenol in the
body, e.g.,
O

O
H2N

CH

OH

CH2

Phenyl alanine

H2N

CH

CH2

OH

Tyrosine

OH

4-Sulfur-containing amino acids: e.g.,


Cysteine gives cystine and its SH
group is very essential in activity of
many proteins particularly the active
sites of enzymes.
O
O
H2N CH C OH H2N CH C OH
CH2
CH2
S
SH

Cysteine

S
CH2
H2N CH C OH
CystineO

O
H2N CH C OH
CH2
CH2
S
CH3

Methionine

5-Heterocyclic amino acids: e.g.,

Histidine gives histamine a very important


inflammatory mediator.
Proline gives hydroxyproline that is essential for
collagen cross-linking.
Tryptophan gives nicotinic acid, melatonin,
serotonin and indican in the body.
H2N CH

O
C OH

CH2
N
NH

imidazole group
Histidine

H2N CH

O
C OH

CH2
HN

indole group
Tryptophan

O
C OH
CH
HN
CH2

CH2
CH2

Proline

O
C OH
CH
HN
CH2

CH2
CHOH

Hydroxyproline

B) Acidic amino acids


They contain 2 carboxyl groups and one amino
group, e.g., glutamic acid and asparatic acid.
These acidic amino acids can occur in the tissue
in the form of amides, e.g., glutamic acid
glutamine and asparatic acid asparagine.
O
H2N CH C OH
CH2
CH2

C O
OH

Glutamic acid

O
H2N CH C OH
CH2
CH2

C O
NH2

O
H2N CH C OH
CH2

C O

amide
OH
group Asparatic acid
Glutamine

O
H2N CH C OH
CH2

amide
NH2 group
Asparagine
C O

C) Basic amino acids


They contain 2 amino groups and one carboxyl
group, e.g., Ornithine and Arginine. Ornithine does
not enter in the synthesis of proteins and is usually
present in the free form. It is synthesized from
arginine. Citrulline is formed from ornithine during
urea synthesis
O
O
H2N

CH

CH2

OH

H2N

CH

O
OH

CH2

H2N

CH

CH2

CH2

CH2

CH2

NH

NH

NH2

CH2

Ornithine

CH2
NH2

urido group
Citrulline

OH

CH2
NH2

NH

guanido group
Arginine

Lysine and Hydroxy lysine: They


participate in protein cross-linking.
O
H2N

CH

CH2
CH2
CH2
CH2
NH2

Lysine

OH

H2N

CH

O
C

CH2
CH2
CHOH
CH2

OH

NH2

Hydroxy lysine

II-Metabolic classification:
classification
Amino acids may be classified into
A-glucogenic amino acids, i.e., those
which can be converted into glucose,
B-ketogenic amino acids, i.e., those
which can be converted into ketone
bodies
C-mixed amino acids, i.e., those which
can be converted into both glucose and
ketone bodies.

Ketogenic

Leucine

Ketogenic & glucogenic

Glucogenic

Lysine

Rest of amino acids

Isoleucine
Tyrosine
Tryptophan
Phenyl alanine

III-Biological or Nutritional Classification:


Some amino acids can not be synthesized
inside the body.
If these amino acids are not taken in diet they
will affect the growth and the health. Thus,
amino acids may be classified into:
A- Essential amino acids: These are amino
acids that can not be synthesized in the
human body and should be taken in the diet,
otherwise their deficiency will lead to a
nutrition deficiency disease that affect both
growth and health.

Valine
Leucine

Isoleucine Threonine
Lysine

Tryptophan

Methionine Phenylalanine

Arginine
Histidine

B- Non essential amino acids:


acids - The rest
of amino acids can be synthesized inside
the human body and their deficiency in
diet does not affect the growth or the
health.

I. Physical properties of amino acids:


1. Solubility:
All amino acids are soluble in water, diluted
acids and alkalis.
2. Optical activity:
All amino acids, except glycine, are optically
active, i.e., they contain asymmetric carbon
atom ( -carbon), thus they can deviate the
plane polarized light either to the right or to
the left.
3. Absorption of ultraviolet light:
Aromatic amino acids (tryptophan, tyrosine
and phenylalanine) can absorb ultraviolet
light.

Structure of Proteins
There are 4 levels or orders of
organization of the structure protein
molecule: primary, secondary, tertiary
and quaternary structures.
This complication gives the molecule
its functional domain to explain its
structure-function requirements that if
changes due to mutation will give nonfunctional protein and, therefore, a
disease.

1. Primary structure:
Primary structure is the linear form of
the polypeptide illustrating the total
number, chemical nature, and linear
order of all of the amino acid residues
in the polypeptide chain or chains of a
protein and position of disulfide bonds
if present.
The peptide bonds (primary bond) are
responsible for the primary structure.

2. Secondary structure:
It is the fine folding of polypeptide chain into
specific regular coiled structure or irregular
random coiling held together by hydrogen,
ionic and disulfide bonds.
It is due to the interaction of amino acids
located very close to each other.

3. Tertiary structure:
structure
It is the final three-dimensional form due to
the more complicated course folding and
super-folding of the polypeptide chain in its
secondary level into globular or fibrous form
of different size.
It is due to interaction of amino acids located
far apart (away from each other).
It is the biologically active conformation of
the polypeptide and therefore, is the most
liable to denaturation.
The bond stabilizing the tertiary structure are
disulfide bonds, hydrogen bonds and ionic
bonds

4. Quaternary structures:
structures
Proteins consist of two or more polypeptide
chains in their tertiary structure united by
forces other than peptide bonds are said to
possess a quaternary structure.
Quaternary structure therefore, is the
positional relationship between individual
polypeptides associating to form one protein
molecule.
The bonds responsible for the quaternary
structure are not disulphid or peptide bonds

Subunit

-Sheet

-helix

Random coils

-helix

-Sheet

Random coils
Subunit
Quaternary protein structure

Classification of Proteins
I. According to the biological importance of the
protein:
1) Proteins of high biological value:
These are all proteins of animal origin (with a
few exceptions) and some proteins of plant
origin that contain all the 10 essential amino
acids in well balanced amounts and are
easily digestible.
Examples of animal proteins include; milks
and its products, egg, liver, fishes, red and
while meats.
Examples of the few plant proteins of high
biological value are lentils and broad beans.

2) Proteins of low biological value:


These are proteins that are deficient in one
or more of the essential amino acids or
containing very little amount of one of them
or are indigestible.
Most of plant protein are of low biological
value and a very few animal proteins are also
of low biological value such are collagen
because is deficient in tryptophan and
cysteine and keratins because they are
indigestible.
This does not imply that a person should eat
only a protein of high biological value to
avoid deficiency of essential amino acids,
but this can be also avoided by eating two or
more proteins of low biological value that
complete each others deficiency.
deficiency

II. According to the axial ratio of the


protein molecule:
Studies on the shape of the protein
molecule using ultramicroscope
indicates that there are two types of
proteins in nature:
1. Fibrous proteins.
2. Globular proteins.

1. Fibrous proteins:
They have an axial ratio of more than 10.
Axial ratio = Length/Width of the protein
molecule. They are fairly stable proteins.
Examples,
a. Keratin proteins in hairs, wool, skin, and
most cells. In its native state, it is present in
the form of coiled polypeptide chains called
-keratin. It can be stretched by
denaturation forming -keratin.
b. Myosin is the major protein of muscles.
During muscle relaxation it is called myosin but during muscle contraction, it
undergoes a change in its structure and it
becomes -myosin

2. Globular proteins:
proteins
Their axial ratio is less than 10.
Their peptide chains are folded or
coiled on themselves in a very compact
manner.
They are less stable than fibrous
proteins. Examples are albumin,
globulins, and insulin

III. According to the composition of the


Protein:
There are 3 main groups:
A) Simple proteins.
B) Conjugated or conjugated proteins.
C) Derived proteins.

A) Simple Proteins
These are proteins which on hydrolysis
produce amino acids only.
Simple proteins are subdivided according to
their physical properties, solubility,
molecular weight and amino acid
composition into:
1-Albumin and globulins .
2-Basic proteins (Histones & Protamines).
3-Acidic proteins (Glutelins & Gliadins ).
4-Scleroproteins(Keratin&Collagen&Elastin)

1-Albumin and globulins .

Albumin

Globulin

- Soluble in water and salt solution

- Soluble in salt solution.

- Coagulated by heat.

- The same as albumin

-They are of high biological value:

-The same as albumin.

- Contain all essential amino acids.

-The same as albumin.

- Easily digested.

-The same as albumin.

- M.W.: 68 KDa.

- M.W.: 150 KDa.

-Precipitated by full saturation with


ammonium sulfate.

-Precipitated by half saturation


with ammonium sulfate.

-They are present in: serum


albumin,egg albumin, milk:
lactalbumin.

They are present in: serum


globulin, milk Lactglobulin, egg
globulin.

- It functions as transporting
protein for elements, vitamins, and
hormones other than keeping blood
osmosis.

- It functions in transport also


but its major function is being
antibodies.

2-Basic proteins(rich in basic amino acids)


A-Protamines:
Protamines are the simplest natural proteins.
Their molecule is small and contains not
more than 20 amino acid residues
Protamines are present in sperms and ova
Protamines are water-soluble and ammonia
soluble proteins, non-coagulable .
They also exist in combination with nucleic
acids in the nucleus. In plants, protamines
are present in pollen grains.
Protamines are strongly basic, due to the
presence of large amount of basic amino
acids specially arginine.

B-Histones: (compare to protamines)


Histones are basic proteins, which are characterized,
by being soluble in water, dilute acids, and insoluble
in dilute ammonia.
Their insolubility in ammonia differentiates them
from protamines.
They are usually present associated with nucleic
acid and porphyrins.
They are not present in plants.
The most important amino acids entering in the
structure of histones are arginine, histidine and less
lysine.
The amount of their basic charges is less than
protamines.
Histones are present in glandular tissues as liver
and spleen. The protein globin, which enters in the
formation of the hemoglobin of blood, is similar to
histones.

3-Acidic proteins
A-Gliadins or Prolamines:

Gliadins are never present in animals but


only present in plant kingdom.
They are present in high concentrations in
cereals and have been obtained from all
grains except rice.
Gliadins are also called prolamines due to
the presence of a high percentage of the
amino acids proline (10-14%) and glutamine.
The amino acid lysine is deficient in gliadins.
Gliadins are proteins, which are insoluble in
water and saline, but soluble in 70-80%
alcohol due to the presence of excess
proline.
The examples of prolamines are gliadin of
wheat and zein of maize.

B-Glutelins:
They are plant proteins.
They are soluble in diluted acids but
not in water or diluted salt solutions.
They are very rich in glutamic acid.
They have very large molecular weight
and are heat coagulable.
Examples are oryzenin of rice and
glutelin of wheat.

4-Scleroproteins (Albuminoids)
Scleroproteins are characterized by their
extreme insolubility in water, dilute acids and
the most common reagents.
They are strong fibrous structural proteins
that are rich in sulfur containing amino acids
and hence disulfide bonds.
Their main function is the protection of the
body.
Hairs, nail and connective tissues, contain
scleroproteins and are never present in
plants. The main important groups of
scleroproteins are:
1. Elastins.
2. Collagens.
3. Keratin

A-Elastins:
They are present in the yellow fibers of the
connective tissues in lungs, uterine wall
during pregnancy, tendons and ligaments.
Elastins are also present in the elastic
tissues of tendons and big arteries.
It is rich in alanine, leucine, valine and
proline but deficient in cysteine, methionine,
lysine and histidine.
Boiling scleroproteins with strong acids or
strong alkalis or their digestion by elastase
leads to their hydrolysis to free amino acids

B-Collagens:

They are present in white fibrous connective


tissues, tendons and bones.
Collagen is insoluble in water, dilute acids and
alkalis. From this point of view it is similar to
elastins.
Collagen is resistant to peptic and trypsin digestion,
but when boiled for a long time with water, dilute
acids or alkalis, it changes to gelatin.
Thus, gelatin is a derived protein obtained from the
partial hydrolysis of collagen.
Both gelatin and collagen are of little nutritive value
because they contain about 40% of the non-essential
amino acid glycine.
Collagen is rich in glycine, proline and hydroxy
proline but low in sulfur containing amino acids,
tryptophan and cysteine

Gelatins:
It is the product of prolonged boiling of collagen in
water.
It is easily digested and has the property of
forming a gel on cooling (gel formation).
Gelatin is a very good diet for patients because it
is an appetizer and easily digested.
Gelatin is deficient in certain essential amino
acids. So it is not an adequate protein diet, as it is
deficient in tryptophan and cysteine and contains
very small amounts of methionine (protein of low
biological value).

C-Keratins
Keratins are highly insoluble compounds. They
are insoluble in all protein solvents, and are not
digestible by proteolytic enzymes (e.g pepsin and
trypsin).
Keratins are hydrolyzed by prolonged boiling
with alkalis.
The sulfur content of keratin is high. It is present
in the form of cystine, which is responsible for the
stability and insolubility of keratins.
Most keratins yield histidine, lysine and arginine
amino acids on hydrolysis

Keratins are present in hairs, nails and


superficial layer of the skin.
Barium sulfide is one of the important
substances that dissolve keratins. For
this reason this material enters in the
formation of cosmetics dealing with
removal of hair.
Keratin is a typical fibrous protein. It
consists of long peptide chains. The
peptide chains may be coiled or reset
in spiral or helix types.

B) Conjugated proteins
On hydrolysis, they give amino acids and
prosthetic group (i.e., a non-protein group).

They include:
1. Phosphoproteins:
These are proteins conjugated with
phosphate. Phosphate is attached to
OH group of serine, tyrosine or
threonine present in protein.
They are found in:
a. Casein: milk protein.
b. Vitellin: Egg yolk protein.

2. Lipoproteins:
These are proteins conjugated with lipids
converting them into water soluble
substances. Present in blood, brain and egg.
Cell membrane is of lipoprotein.
Examples: Plasma lipoproteins: see lipid
chemistry.
3. Glycoproteins:
These are proteins conjugated with
carbohydrates in varying amounts attached
as short or long chains.
Examples: Mucous secretion of
gastrointestinal tract and Glycoproteins of
cell wall.

4. Metalloproteins:
These are proteins conjugated with
metals such as;
Iron: e.g., ferritin is intracellular ironbinding protein and Transferrin is an
iron-binding transport protein in the
blood.
Zinc: e.g., Insulin hormone present in
crystals containing zinc.
Copper: e.g., Ceruloplasmin: is a
protein present in blood. It is
responsible for the oxidation of Fe2+
ions to Fe3+ ions.

5. Chromoproteins:
These are proteins conjugated with colored
pigment.
Example; Hemoglobin and cytochrome enzyme
present in mitochondria contains haem pigment,
which is red in color.
6. Nucleoproteins:
These are proteins (protamines or histones)
conjugated with nucleic acids (DNA or RNA).
Examples; Chromosomes: These are proteins
conjugated with DNA.
Ribosomes: They are proteins conjugated with
RNA.
RNA

C) Derived Proteins
They include:
A- Denatured protein: e.g., coagulated albumin
or globulin.
B. Hydrolytic product of protein: e.g.,

Protein Proteoses Peptone


Polypeptide.
1-Proteoses, they are soluble in water, not
coagulable and precipitated by saturated salt
solution.
2-Peptones: are soluble in water and not
coagulable by heat, not precepitated by
saturated salt solution.

3-Peptides: are soluble in water and salt


solution, not coagulable by heat. They are
formed from 2 or more amino acids.
The amino acids in any polypeptide chain
are arranged so that the first amino acid
has a free amino group (N-terminal end)
and the last one has a free carboxyl group
(C-terminal end). So the tripeptide
alanine-cysteine-tryptophan is different
from the tripeptide tryptophan-cysteinealanine.

Bonds responsible for protein structure


I-Strong bonds:
1. Peptide bonds (primary bond):
A peptide bond is a covalent bond formed by
a reaction between amino group of one
amino acid and a carboxylic group of the
next amino acid with the loss of H2O that
required ATP.
O
H2N CH C OH

H2N CH C HN CH C OH

+ H2N CH C OH

an amino acid

an amino acid

H2O

Dipeptide

2. Disulfide bonds (secondary bond):


The disulfide bond is formed between the SH groups
of two cysteine residues within same (intra-chain) or
two different polypeptide chains (inter-chain).
It maintain secondary structure of a peptide chain or
connects two polypeptide chains together in the
tertiary structure.
It follows the peptide bond in strength but liable to
denaturation.
O
R

HN

CH

O
R

CH2

HN

CH

peptide chain
disulfide bond

S
CH2

CH2
HN

two cysteine
residues
SH
R

CH2

2H

SH

CH

C
O

HN

CH

C
O

peptide chain

II- Weak bonds:


1. Hydrogen bonds: Hydrogen bond is a weak
bond formed between the hydrogen atom of
NH of a peptide bond on one peptide chain
and the oxygen of C=O of another peptide
bond on an adjacent peptide chain or a loop
belongs to same peptide chain.
R
C

CH

peptide chain

hydrogen bond
H

CH
R

peptide chain

2. Hydrophobic bonds:
The non polar side chains of neutral amino
acids tend to associate in hidden core of
protein molecule away from solvent.
O

HN CH C HN CH C
R

HN CH
R

HN CH C
O

3. Electrostatic bonds:
These are salt bonds formed between
oppositely charged groups in the side chains
of amino acids e.g. -amino group of lysine
and the carboxyl group of asparatic acid.
O
H2N

Lysine

CH

O
OH

H2N

CH

CH2

CH2

CH2

CH2
CH2
NH3+

O-

Electrostatic
attraction

OH

Asparatic acid

Denaturation of Proteins
Definition:
It is the loss of the native form of the protein
leading to disruption of its secondary,
tertiary and quaternary structure with the
changes in their physical and chemical
characteristics and loss of their biological
activity.

Causes of Denaturation
Physical causes:
As shaking (mechanical effect), high
temperature, X-rays and atomic
radiations.
Chemical causes:
As organic solvents (e.g. acetone), strong
alkalis and acids and agents that irreversibly
precipitate proteins.

Effects of Denaturation
Physical changes:
changes Increase in viscosity,
decreased solubility and decreased
diffusiblility.
Chemical changes: leads to loss of hydrogen,
hydrophobic and electrostatic bonds but
not peptide and disulfide bonds.The result
are loss of secondary, tertiary and quaternary
structures but not of the primary structure.
Biological changes: which include loss of
enzymatic, hormonal and other biological
properties of proteins. Denatured proteins
(e.g cooking), are easily digested than native
proteins.

Significance and Application of denaturation:


Denatured proteins, e.g., cooked meat are easily digested.
Avoidance of denaturation is important for biological
samples used for determination of enzymatic, hormonal
or protein contents. This is done by proper sample
collection and storage because if denaturation occurs
false results will be obtained.
Blood samples to be analyzed for small molecules, e.g.,
uric acid and glucose are first treated with acid such as
trichloroacetic acid or phosphotungestic acid to
precipitate the plasma proteins (by denaturation).
Detection of albumin in urine by heat coagulation test is
based on denaturation by heat.
Several approaches for stoppage of bleeding and
treatment of burns is based on precipitation and
denaturation of a superficial protein layer.

Protein Separation
Protein separation is based on
1) Protein solubility
2) Size of protein molecule
3) Charge of the molecule
Methods of protein separation
Chromatography.
Salting out
Electrophoresis.
Dialysis.
Ultracentrifugation.

1- Salting out
It depends on elemination of water from the solution. e.g.
albumin by full saturation with Amm. sulphate
globulin by half saturation with Amm. Sulphate
2-Chromatography
Chromatography is a group of separation techniques, where a
mixture of molecules is separated.
The separated molecules are divided between a stationary
sold phase and liquid mobile phase.
The separation process depends on the tendency of one
type of molecules in the mixture to associate more strongly
with one phase than the other.

3- Electrophoresis
It is movement of charged particles in an
electric field towards the oppositely charged
electrode.
By electrophoresis a mixture of amino acids,
polypeptides or proteins can be separated
into distinct bands by using electric current.
1-Globulin

Albumin

-Globulin

2-Globulin

-Globulin

Fibrinogen

Origin

4-Dialysis
Dialysis means separation of colloids from
crystalloids. Proteins have a high molecular weight
that forms a colloidal solution.
If there is a mixture of proteins (colloids) and salts
(crystalloids) they can be separated by dialysis, i.e.,
by using a semi-permeable membrane. Crystalloids
with very small molecular weight can pass through
this membrane, while colloids can not due to the
large size of their partic
Dialysis membrane
Crystalloids

Colloids

5-Ultracentrifugation:
Using high speed centrifuge, a mixture
of proteins is separated into different
fractions according to their densities.
Lipoproteins can be separated by
ultracenrifugation to chylomicrons,
VLDL, LDL, and HDL.