Chemistry of peptides and proteins · than 50 amino acids are called “proteins”. Peptides and...
Transcript of Chemistry of peptides and proteins · than 50 amino acids are called “proteins”. Peptides and...
Chemistry of peptides and proteins
Amino acids
• Provide the monomer units from whichthe long polypeptide chains of proteins aresynthesized
Derived Amino Acids:
Derived and Incorporated in proteins:
• Some amino acids are modified after proteinsynthesis such as hydroxy proline and hydroxy lysinewhich are important component of collagen.
• Gamma carboxylation of glutamic acid residues ofproteins 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.
Derived Amino Acids
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Derived Amino Acids:
Derived and Incorporated in proteins:
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:
Derived Amino Acids
Derived but not incorporated in tissue proteins:
Homocysteineis biosynthesized from methionine by theremoval of its terminal C methyl group
Derived Amino Acids
Seleno cysteine - cysteine analogue with a selenium- in place of the sulfur-containing thiol group.Selenocysteine is present in several enzymes.
Non standard amino acids
Amino acids
L-amino acids and their derivativesparticipate in cellular functions as diverse as
nerve transmission
and the biosynthesis of
✓ porphyrins,
✓ purines,
✓ pyrimidines,
✓ and urea.
• Serotonin is synthesized from Tryptophan
• Serotonin and melatonin and niacin are synthesized from Tryptophan
(vitamin B3)
Catecholamines, Melanin, thyroid hormone
are synthesized from Tyrosine
Epinephrine (adrenaline)
Melanin, thyroid hormone, catecholamines
are synthesized from Tyrosine
First step Iodination
GABA (neurotransmitter) is synthesized from Glutamic acid
γ-amino butyric acid (GABA)
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. Reactions due to amino group
2. Reactions due to carboxyl group
3. Reactions due to side chain
4. 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 + -ketoglutarate oxaloacetate + glutamate
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. Examples-
Glutamic acid GABAHistidine HistamineTyrosine 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.
Glutamic 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.
tyrosine
decarboxylase
Reactions due to carboxyl group
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 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
Proteins are commonly modified at serine, tyrosine and threonine amino acids by adding a phosphate group. Phosphorylation is a common mode of activating or deactivating a protein as a form of regulation.
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
N-linkage: The nitrogen atom in the side chain of Asparagine is attached to the sugar.
Reactions due to side chains: Glycoproteins
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.
4)Reactions due to both amino & carboxyl groups
Formation of peptide bond
Reactions due to side chains
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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
20 amino acids are commonly found in protein.
These 20 amino acids are linked together through“peptide bond forming peptides and proteins (what’sthe difference?).
- The chains containing less than 50 amino acids arecalled “peptides”, while those containing greaterthan 50 amino acids are called “proteins”.
Peptides and 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).34
Peptides
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Peptides
• Amino acids linked by amide (peptide) bonds
Gly Lys Phe Arg Ser
H2N- -COOH
end Peptide bonds 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
Examples of Peptides:
➢Dipeptide ( 2 amino acids joined by one peptide bond):
Example: Aspartame which acts as sweetening agentbeing used in replacement of cane sugar. It is composedof aspartic acid and phenylalanine.
Cannot be intaken by people suffered fromphenylketonuria (phenylalanine hydroxylase iscompletely or nearly completely deficient, andPhe 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
Examples of Peptides:
➢Tripeptides ( 3 amino acids linked by two peptide bonds).
Example: GSH - glutathione which is formed from3 amino acids: glutamic acid, cysteine and glycine.
It protects against hemolysis of RBC (Red Blood Cell) bybreaking H2O2 which causes cell damage.
Glu-Cys-Gly41
Examples of Peptides:
➢nonapeptides (9 amino acids)Examples:Two hormones;oxytocine and vasopressin (ADH) also namedantidiuretic hormone.
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Examples of 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
Protein structure:Primary structure:
The primary structure of a protein isits unique sequence of amino acids.
Lysozyme, an enzyme that attacksbacteria, consists of a polypeptidechain of 129 amino acids.
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High orders of Protein structure
A functional protein is not just a polypeptide chain, butone or more polypeptides precisely twisted, folded andcoiled 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 - or β-structured of peptide bonds
• Looks like a coiled “telephone cord” ()
• or nearly fully extended polypeptide chain (β)
According to H-bondingthere are two main forms ofsecondary structure:α-helix: is a spiral structure
resulting from hydrogenbonding between one peptidebond and the fourth one
Secondary Structure
β-sheets
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Secondary Structure
β-sheets: is another form ofsecondary structure in which two ormore polypeptides (or segments ofthe same peptide chain) are linkedtogether by hydrogen bondbetween H- of NH- of one chain andcarbonyl oxygen of adjacent chain(or segment).
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Amino acids
Hydrogen
bond
Alpha helix Β-Pleated sheet
Secondary Structure
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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 -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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b-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–
Is determined by a variety of interactions (bond formation) among R groups and between R groups and the polypeptide backbone
Tertiary Structure
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Tertiary Structure
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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.
Strong covalent bonds
include disulfide bridges, that form between thesulfhydryl 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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•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)
Quaternary Structure
Quaternary structure: two or more polypeptidesubunits held together by non-covalentinteraction like H-bonds, ionic or hydrophobicinteractions.
Quaternary Structure
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• 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 inconnective tissue.
Quaternary Structure
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⅓ of structure is glycine, 10% proline, 10% hydroxyproline and 1% hydroxylysine.
• Examples on protein having quaternary structure:
– Hemoglobin is a globular protein with four polypeptide chains (tetrameric)
Quaternary Structure
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Hemoglobin tetramer
(a) Human oxyhemoglobin (b) Tetramer schematic
• Examples on proteinhaving quaternarystructure:
– Insulin : two polypeptide chains (dimeric) held together primarily by disulfide bonds between cysteine residues
Quaternary Structure
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Conjugated proteins
On hydrolysis, give protein part and non protein part and 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 anhydrased- Mg containing proteins:e.g. Kinases and phosphatases.
6-Chromoproteins: These are proteins conjugated withpigment. 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
H3N CH
CH3
C
O
N
H
CH C
OCH2
OH
OH
+
H3N CH
CH3
COH
O
+ CH C
OCH2
OH
OHH3N
H2O, H+
++
heat
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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 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)
a gel
coagulation
peptization
Reversible coagulation
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 ora few proteins from a complex mixture,usually cells, tissues
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
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Size-exclusion chromatography(gel filtration)
The column contains a cross-linked 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 cross-linked 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.
Electrophoretic 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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Electrophoretic Separation of Proteins
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Positively charged (cationic) amino acids are attracted to the negative electrode (the cathode), and negatively charged (anionic) amino acids are attracted to the positive electrode (the anode). An amino acid at its isoelectric point has no net charge, so it does not move.
pH 6 buffer solution
A pH of 6 is more acidic than the isoelectric pH for lysine (9.6),
so lysine is in the cationic form. Aspartic acid has an isoelectricpH of 2.8, so it is in the anionic form.
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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anode
•Proteins studied are strictly
being separated by size
Proteins
• SDS-PAGE– Visualization of proteins in
gel• Coomassie Blue
– Milligram amounts of protein.
• Silver stain
– Microgram amounts of protein.
– Size of unknown bands can be determined from comparison to protein molecular weight standard
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Proteins
Western blotting combines electrophoretic separation (SDS-PAGE) with
detection using specific antibodies
• for the analysis of the target proteins in a mixture.
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