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2016-11-14
Chemistry of amino acids, peptides
and proteins
Amino acids
• Provide the monomer units from which
the long polypeptide chains of proteins are
synthesized
Derived Amino Acids
Derived Amino Acids:
Derived and Incorporated in proteins:
•
Some amino acids are modified after protein
synthesis such as hydroxy proline and hydroxy lysine
which are important component of collagen.
• Gamma carboxylation of glutamic acid residues of
proteins is important for clotting process.
Coagulation (also known as clotting) is the process by which
blood changes from a liquid to a gel, forming a blood clot.
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Derived Amino Acids:
Derived and Incorporated in proteins:
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Derived Amino Acids
Derived Amino Acids:
Derived but not incorporated in tissue proteins:
e.g.: Ornithine, Citrulline, Homocysteine
No-protein amino acids.
L-Ornithine and citrulline
• natural amino acid not found in proteins,
• play a role in the urea cycle
Derived Amino Acids
Derived but not incorporated in tissue proteins:
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Derived Amino Acids
Derived but not incorporated in tissue proteins:
Homocysteine
is biosynthesized from methionine by the
removal of its terminal Cε methyl group
Non standard amino acids
Seleno cysteine - cysteine analogue with a
selenium- in place of the sulfur-containing thiol
group.
Selenocysteine is present in several enzymes.
Amino acids
L-amino acids and their derivatives
participate in cellular functions as diverse as
nerve transmission
and the biosynthesis of
porphyrins,
purines,
pyrimidines,
and urea.
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• Niacin, Serotonin and melatonin are
synthesized from Tryptophan
(vitamin B3)
Melanin, thyroid hormone, catecholamines
are synthesized from Tyrosine
Epinephrine (adrenaline)
GABA (neurotransmitter) is synthesized from
Glutamic acid
γ-amino butyric acid (GABA)
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Nitric oxide, a smooth muscle relaxant is
synthesized from Arginine.
Aminoacids are precursors for haem, creatine
and glutathione, porphyrins, purines and
pyrimidines.
haem
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Reactions of amino acids
1.
2.
3.
4.
Reactions due to amino group
Reactions due to carboxyl group
Reactions due to side chain
Reaction due to both amino and carboxyl
groups
Reactions due to amino group
Oxidative deamination-α amino group is removed and
corresponding α-keto acid is formed. α-keto acid produced
is either converted to glucose or ketone bodies or is
completely oxidized.
Reactions due to amino group
Transamination-Transfer of an α amino group from an
amino acid to an α keto acid to form a new amino acid and
a corresponding keto acid.
aspartate + α-ketoglutarate ⇔ oxaloacetate + glutamate
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Reactions due to amino group
Formation of carbamino compound
• CO2 binds to α amino acid on the globin chain of
hemoglobin to form carbamino hemoglobin
• The reaction takes place at alkaline pH and serves as a
mechanism for the transfer of Carbon dioxide from the
tissues to the lungs by hemoglobin.
Reactions due to carboxyl group
1) Decarboxylation- Amino acids undergo alpha
decarboxylation to form corresponding amines.
ExamplesGlutamic acid
GABA
Histidine
Histamine
Tyrosine
Tyramine
2) Formation of amide linkage
• Non α carboxyl group of an acidic amino acid reacts
with ammonia by condensation reaction to form
corresponding amides
Aspartic acid
Asparagine
Glutamic acid
Glutamine
Reactions due to carboxyl group
1) Decarboxylation- Amino acids undergo alpha
decarboxylation to form corresponding amines.
ExamplesGlutamic acid
GABA
Histidine
Histamine
Tyrosine
Tyramine
GABA is an inhibitory neurotransmitter whose receptors
lower muscle tone, promote relaxation, diminish
anxiety, and stimulate digestion.
Histamine is involved in many allergic reactions.
Tyramine acts as a catecholamine releasing agent.
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Reactions due to side chains
1) Ester formation
• OH containing amino acids e.g. serine,
threonine can form esters with phosphoric
acid in the formation of phosphoproteins.
Phospho-serine
Reactions due to side chains: Glycoproteins
1) Ester formation
•
OH group containing amino acid can also form:
Glycosides – by forming O- glycosidic bond with
carbohydrate residues.
O-linkage: The oxygen
atom in the side chain
of serine or threonine
amino acids is
attached to the sugar
Reactions due to side chains: Glycoproteins
N-linkage: The
nitrogen atom in the
side chain of
Asparagine is attached
to the sugar.
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Reactions due to side chains
2) Reactions due to SH group
(Formation of disulphide bonds)
• Cysteine has a sulfhydryl group( SH) group and
can form a disulphide (S-S) bond with another
cysteine residue.
• The dimer is called Cystine
• Two cysteine residues can connect two
polypeptide chains by the formation of
interchain disulphide chains.
Formation of disulphide bond
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Reactions due to side chains
3) Transmethylation
The methyl group of Methionine can be
transferred after activation to an acceptor for
the formation of important biological
compounds.
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Reactions due to side chains
4) Reactions due to both amino & carboxyl groups
Formation of peptide bond
Peptide Bonds Link Amino Acids in Proteins
• Peptide bond - linkage between amino acids is
a secondary amide bond
• Formed by condensation of the α-carboxyl of one
amino acid with the α-amino of another amino acid
(loss of H2O molecule)
Ala-Ser
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Peptides and Proteins
20 amino acids are commonly found in protein.
These 20 amino acids are linked together through
“peptide bond forming peptides and proteins (what’s
the difference?).
- The chains containing less than 50 amino acids are
called “peptides”, while those containing greater
than 50 amino acids are called “proteins”.
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Peptide bond formation:
- Each polypeptide chain starts on the left side by free amino group
of the first amino acid enter in chain formation . It is N- terminus.
- Each polypeptide chain ends on the right side by free COOH group
of the last amino acid and termed (C-terminus).
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Peptides
Peptides
• Amino acids linked by amide (peptide) bonds
Gly
H2Nend
Lys
Phe
Peptide bonds
Arg
Ser
-COOH
end
Glycyllysylphenylalanylarginylserine
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Resonance structure
of the peptide bond
(a) Peptide bond shown as a
C-N single bond
(b) Peptide bond shown as a
double bond (40%)
(c) Actual structure is a hybrid
of the two resonance
forms. Electrons are
delocalized over three
atoms: O, C, N
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Trans and cis conformations
of a peptide group
• Nearly all peptide groups in proteins are
in the trans conformation
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Examples on Peptides:
Dipeptide ( 2 amino acids joined by one peptide bond):
Example: Aspartame which acts as sweetening agent
being used in replacement of cane sugar. It is composed
of aspartic acid and phenylalanine.
Cannot be intaken by people suffered from
phenylketonuria (phenylalanine hydroxylase is
completely or nearly completely deficient, and
Phe isn’t metabolised to Tyr)
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Aspartame, an artificial sweetener
• Aspartame is a dipeptide methyl ester
(aspartylphenylalanine methyl ester)
• About 200 times sweeter than table sugar
• Used in diet drinks
• Shouldn’t be used with hot solutions and decomposes
during heating
Asp-Phe-OCH3
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Examples on Peptides:
Tripeptides ( 3 amino acids linked by two peptide bonds).
Example: GSH - glutathione which is formed from
3 amino acids: glutamic acid, cysteine and glycine.
It protects against hemolysis of RBC (Red Blood Cell) by
breaking H2O2 which causes cell damage.
Glu-Cys-Gly
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Examples on Peptides:
Octapeptides: (8 amino acids)
Examples:
Two hormones;
oxytocine and vasopressin (ADH)
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Examples on Peptides:
Polypeptides:
10- 50 amino acids:
e.g. Insulin hormone,
Chain B and Chain A
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There Are Four Levels of Protein Structure
•Primary structure - amino acid linear sequence
•Secondary structure – the type and the shape of the
peptide chain, such as a-helices and b-sheets
•Tertiary structure - describes the shape of the fully
folded polypeptide chain in space
•Quaternary structure - arrangement of two or more
polypeptide chains into multisubunit molecule
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Primary Structure of Proteins
The particular sequence of amino acids that is
the backbone of a peptide chain or protein
• ca
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Protein structure:
Primary structure:
The primary structure of a protein is
its unique sequence of amino acids.
Lysozyme, an enzyme that attacks
bacteria, consists of a polypeptide
chain of 129 amino acids.
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High orders of Protein structure
A functional protein is not just a polypeptide chain, but
one or more polypeptides precisely twisted, folded and
coiled into a molecule of unique shape (conformation).
This conformation is essential for some protein function
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Secondary Structure
• Results from hydrogen bond between hydrogen of
–NH group of peptide bond and the –O of C=O
(carbonyl oxygen) of another peptide bond.
• Three-dimensional arrangement of amino acids
in a form of α- or β-structured of peptide bonds
• Looks like a coiled “telephone cord” (α
α)
or nearly fully extended polypeptide chain (β)
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Secondary Structure
According to H-bonding
there are two main forms of
secondary structure:
α-helix: is a spiral structure
resulting
from
hydrogen
bonding between one peptide
bond and the fourth one
β-sheets
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Secondary Structure
β-sheets: is another form of
secondary structure in which two or
more polypeptides (or segments of
the same peptide chain) are linked
together by hydrogen bond
between H- of NH- of one chain and
carbonyl oxygen of adjacent chain
(or segment).
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Secondary Structure
Amino acids
Hydrogen
bond
Alpha helix
Β-Pleated sheet
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The α Helix Is a Common Protein Secondary
Structure
• The α helix is a common type of secondary
structure in proteins.
• It is the predominant structure in α-keratins.
• In globular proteins, about one-fourth of all amino
acid residues are found in α helices
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Stereo view of right-handed a-helix
• All side chains project outward from helix axis
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Secondary Structure – Beta Pleated Sheet
• Polypeptide chains are arranged side by side
• Hydrogen bonds between chains
• R groups of extend above and below the sheet
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Secondary Structure – Beta Pleated Sheet
The adjacent polypeptide chains
in a β pleated sheet can be
either
antiparallel
(having
the
opposite
amino-to-carboxyl
orientation)
or parallel (having the same
amino-to-carboxyl polypeptide
orientation).
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β -Sheets (a) parallel, (b) antiparallel
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Secondary Structure – Beta Pleated Sheet
• Typical of fibrous proteins such as
silk (produced by the larva of the
silkworm moth, to make cocoons) is
almost pure antiparallel beta
pleated sheet.
• Elements of beta pleated sheet are
found in many protein domains.
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Tertiary Structure
• Specific overall shape of a protein
• Cross links between R groups of amino acids in
chain
disulfide –S–S–
+
ionic –COO–
H3N–
H bond C=O
HO–
hydrophobic –CH3 H3C–
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Tertiary Structure
Is determined by a
variety of interactions
(bond formation)
among R groups and
between R groups
and the polypeptide
backbone
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Tertiary Structure
The weak interactions include:
Hydrogen bonds
among polar side chains
Ionic bonds
between charged R groups (basic
and acidic amino acids)
Hydrophobic interactions
among hydrophobic ( non polar)
R groups.
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Strong covalent bonds
include disulfide bridges, that form between the
sulfhydryl groups (SH) of cysteine monomers,
stabilize the structure.
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Formation of cystine (disulfide bridge)
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Tertiary Structure
Amino acids
Hydrogen
bond
Alpha helix
Pleated sheet
Polypeptide
(single subunit
of transthyretin)
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Quaternary Structure
•Refers to the organization of subunits in a protein
with multiple subunits (2 or more chains)
•Subunits (may be identical or different)
•Subunits are held together by many weak,
noncovalent interactions (hydrophobic,
electrostatic)
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Quaternary Structure
Quaternary structure: two or more polypeptide
subunits held together by non-covalent
interaction like H-bonds, ionic or hydrophobic
interactions.
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Quaternary Structure
• Examples on protein having quaternary structure:
– Collagen is a fibrous protein of three polypeptides
(trimeric) that are supercoiled like a rope.
• This provides the structural strength for their role in
connective tissue.
⅓ of structure is glycine, 10% proline,
10% hydroxyproline and 1% hydroxylysine.
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Quaternary Structure
• Examples on protein having quaternary structure:
– Hemoglobin is a globular protein with four
polypeptide chains (tetrameric)
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Hemoglobin tetramer
(a) Human oxyhemoglobin
(b) Tetramer schematic
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Quaternary Structure
• Examples on protein
having quaternary
structure:
– Insulin : two
polypeptide
chains (dimeric)
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Conjugated proteins
On hydrolysis, give protein part and non protein part
are subclassified into:
1- Phosphoproteins: These are proteins conjugated with
phosphate group. Phosphorus is attached to OH group of
serine or threonine.
e.g. Casein of milk and vitellin of yolk.
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2- Lipoproteins:
These are proteins conjugated with lipids.
Functions: help lipids to transport in blood
Enter in cell membrane structure helping lipid soluble
substances to pass through cell membranes.
3- Glycoproteins:
proteins conjugated with sugar (carbohydrate)
e.g. – Mucin
- Some hormones such as erythropoeitin
- present in cell membrane structure
- blood groups.
4- Nucleoproteins: These are basic proteins ( e.g. histones)
conjugated with nucleic acid (DNA or RNA).
e.g. a- chromosomes: are proteins conjugated with DNA
b- Ribosomes: are proteins conjugated with RNA
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5- Metalloproteins: These are proteins conjugated with metal like
iron, copper, zinc.
a- Iron-containing proteins: Iron may present in heme such as in
- hemoglobin (Hb)
- myoglobin (protein of skeletal muscles and cardiacmuscle),
- cytochromes,
- catalase, peroxidases (destroy H2O2)
- tryptophan pyrrolase (desrtroy indole ring of tryptophan).
Iron may be present in free state ( not in heme) as in:
- Ferritin: Main store of iron in the body. ferritin is present in liver,
spleen and bone marrow.
- Hemosidrin: another iron store.
- Transferrin: is the iron carrier protein in plasma.
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b- Copper containing proteins:
e.g. - Ceruloplasmin which oxidizes ferrous ions into ferric
ions.
- Oxidase enzymes such as cytochrome oxidase.
c- Zn containing proteins: e.g. Insulin and carbonic anhydrase
d- Mg containing proteins:e.g. Kinases and phosphatases.
6-Chromoproteins: These are proteins conjugated with
pigment. e.g.
- All proteins containing heme (Hb, myoglobin)
- Melanoprotein: e.g proteins of hair which contain melanin.
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Protein Hydrolysis
•
•
•
•
•
Break down of peptide bonds
Requires acid or base, or enzymes, water and heat
Gives smaller peptides and amino acids
Similar to digestion of proteins using enzymes
Occurs in cells to provide amino acids to synthesize
other proteins and tissues
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Hydrolysis of a Dipeptide
OH
+
CH 3 O
CH 2 O
H 2O, H
+
H 3N CH C N CH C OH
heat
H
OH
CH 3 O
+
H 3N CH COH
CH 2 O
+
+
H 3N CH C OH
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Denaturation - irreversible coagulation
Disruption of secondary, tertiary and quaternary protein
structure by
heat/organics (formaline, detergents)
Break apart H bonds and disrupt hydrophobic
attractions
acids/ bases
Break H bonds between polar R groups and
ionic bonds
heavy metal ions
React with S-S bonds to form solids: S-Pb-S
agitation
Stretches chains until bonds break
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Reversible coagulation
Reversible coagulation of proteins – is caused by aqueous
solutions of Na+, K+ and NH4+ salts and diluted alcohols, when
proteins precipitate out of a solution.
After adding water to precipitated proteins, the original protein
form is restored (it is called peptization).
a protein
(a coloid)
coagulation
a gel
peptization
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Biuret reaction – detection of peptide bond
All proteins, peptides with the chain length
of at least 3 amino acids give a positive result
in this test.
This is a typical reaction for identification of
peptide bonds.
Biuret reaction – detection of peptide bond
The Biuret reagent is made of
sodium hydroxide (NaOH) and
CuSO4 solution.
The reaction of the cupric ions
with the nitrogen atoms
involved in peptide bonds
leads to the displacement of
the peptide hydrogen atoms
under the alkaline conditions.
Protein purification
a processes intended to isolate one or
a few proteins from a complex mixture,
usually cells, tissues
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Protein purification based
on physico-chemical properties
Differences in
•
•
•
•
•
size, shape, and solubility
binding affinity
isoelectric point
charged surface residues
and biological activity
Protein purification strategies
• Size exclusion chromatography
• Affinity chromatography (Metal binding,
Immunoaffinity chromatography)
• Separation based on charge or hydrophobicity
• Electrophoresis
Size-exclusion
chromatography
(gel filtration)
The column contains a crosslinked polymer with pores of
selected size.
Larger proteins migrate faster
than smaller ones, because
they are too large to enter the
pores in the beads.
The smaller proteins enter the
pores and and their path
through the column is longer.
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Affinity chromatography
Separates proteins by their
binding specificities.
The proteins retained on the
column are those that bind
specifically to a ligand crosslinked to the beads.
After nonspecific proteins are
washed through the column,
the
bound
protein
of
particular interest is eluted by
a solution containing free
ligand.
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Separation of Proteins
Proteins are amphoteric compounds
Their net charge therefore is determined by pH of
medium in which they are suspended
In a solution with a pH above its isoelectric point,
a protein has a net negative charge and migrates
towards anode in an electrical field
Below its isoelectric point, protein is positively
charged and migrates towards cathode
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Electrophoresis of Proteins
– Gel electrophoresis allows for the separation of
proteins based on charge, size, and shape.
– Polyacrylamide gel electrophoresis (PAGE).
• Allows for better resolution
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Proteins are usually analyzed by sodium
dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE)
• Proteins are usually denatured in presence of a
detergent such as sodium dodecyl sulfate (SDS)
• In denaturing SDS-PAGE separations migration is
determined not by intrinsic electrical charge of
polypeptide, but by molecular weight
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Proteins
SDS-PAGE
Use of sodium dodecyl sulfate (SDS)
• Denatures proteins into
polypeptide strands
• Gives each polypeptide strand
an overall negative charge
• Proteins studied are strictly
being separated by size
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Proteins
SDS-PAGE
Visualization of
proteins in gel
• Coomassie Blue
• Silver stain
– Size of unknown bands can be determined from comparison
to protein molecular weight standard
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