chem 40 (1) amino acids, proteins_fs10-11
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Three Principal Areas of Biochemistry
1. structural chemistry of thecomponents of living matter
2. metabolism or the chemical reactionsthat occur in living matter
3. the chemistry of processes and
substances that store and transmit
biological information; molecular
genetics
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To the -carbon of every amino acid is
attached a hydrogen atom, a carboxylic
acid, and a side chain.
AMINO ACIDS
-are the basic structural units of proteins
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All 20 amino acids, except
glycine, contain an asymmetric -
carbon and haveL andD
enantiomers (exhibit chirality).
Only theL-enantiomers are found in
proteins.
GlycineAlanine
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Ten Essential Amino Acids
- the amino acids that human body
cant synthesize in adequate amounts
1. Arginine 6. Methionine2. Histidine 7. Phenylalanine
3. Isoleucine 8. Threonine
4. Leucine 9. Tryptophan5. Lysine 10. Valine
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Limiting Amino Acids in Some Foods:
Wheat and grainslysine, threonine
Peas, beans, legumesmethionine,
tryptophan Nuts, seedslysine
Leafy green vegetables - methionine
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Classes of -Amino Acids:
1. Amino acid with aliphatic sidechains (Gly, Ala, Val, Leu, Ile)
- hydrophobicity of the amino acid
increases as R group gets bigger
-pro has also an aliphatic chain but its
side chain is bonded to both the N andthe -C atoms; has a 2o rather than a 1o
amino group
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2. Amino acid with hydroxyl- or sulfur-
containing side chain (Ser, Thr, Cys,
Met)
- more hydrophilic than their aliphatic
analogs, althoughmet is morehydrophobic.
- Cys side chain can ionize at
moderately high pH and oxidation canoccur between the pairs ofcys side
chains to form a disulfide bond.
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3. Acidic amino acids and their amides
(Asp, Asn, Glu, Gln)
-asp andglu contains acidic side chains;
usually called aspartate and glutamate
because they are negatively charges atphysiological pH
-asn andgln are the uncharged
derivatives of aspartate and glutamate;both contain a terminal amide in place
of a carboxylate
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4. Basic amino acids (His, Lys, Arg)
- have very polar side chains which
render them highly hydrophilic
- lys andarg are positively charged atneutral pH whilehis can be uncharged
or positively charged, depending on itsenvironment
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Amino acid with aliphatic side chains
Gly Ala
Val
Leu
Ile
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Acidic amino acids and their amides
Asp Asn
Glu
Gln
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Basic amino acids
HisLys
Arg
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Aromatic amino acids
Phe Tyr
Trp
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Hydrophobic Amino Acids
-tend to repel the aqueousenvironment and, therefore, reside
predominantly in the interior of
proteins
- do not ionize nor participate in the
formation of H-bonds
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Hydrophilic Amino Acids
- tend to interact with the aqueousenvironment
- are often involved in the formation
of H-bonds and are predominantly
found on the exterior surfaces
proteins or in the reactive centers ofenzymes
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Uncommon Amino Acids
- derived from the common amino acids
and are synthesized by modification of
the parent amino acid in a process called
posttranslational modificationHO
4-hydroxyproline
(proline)
NH2CH
2CHCH
2
OH
-hydroxylysine (lysine)
thyroxine (tyrosine)
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Acid-Base Properties of Amino Acids
Amino acids are difunctional.They contain both a basicamino
group and an acidiccarboxyl
group.
C
R
- COOH
NH2H -
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The -COOH and -NH2 groups in
amino acids are capable of ionizingin aqueous environment (as well as
the acidic and basic R-groups).
R-COOH R-COO- + H+
R-NH3+ R-NH2 + H
+
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At physiological pH (around 7.4) the
carboxyl group will be unprotonated
and the amino group will be
protonated.
The carboxyl group (-COOH) is a far
stronger acid than the amino group
(-NH2).
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An amino acid with no ionizable R-
group, e.g. glycine, would be
electrically neutral at pH 7.4. This
species is termed as zwitterion.
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Amino acids have many properties
associated with inorganic salts:
-are crystalline
-have high melting points-are soluble in water but insoluble
in hydrocarbons
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Acts as the base
and accepts the
proton, H+, inacid solution.
Acts as the acid and donates the
proton, H+
, in base solution.
Amino acids areamphoteric: can act
either as acids or as bases.
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In aqueousacidsolution, an amino
acid zwitterionaccepts a proton toyield a cation:
+ H3O+ + H2O
H
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In aqueousbasic solution, an amino
acid zwitterion loses a proton toyield an anion:
+ OH- + H2O
H
H
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Exercises:
1. Write an equation for the reaction of
aspartic acid with:a. 1 equiv. NaOH b. 2 equiv. NaOH
2. Draw phenylalanine in its
zwitterionic form.
3. Complete and write the structural
formulas for the following equations:a. Phenylalanine + 1 equiv NaOH
b. Product of (a) + 1 equiv HCl
c. Product of (a) + 2 equiv HCl
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Isoelectric Points, pI
-pI is the pH when the algebraic sumof all the charged groups present in an
amino acid or protein is zero.
- pI values are not necessarily neutral(pH 7) because theCOOH groups are
stronger acids in aqueous solution than
the basicNH2 groups.
- pI of an amino acid depends on its
structure
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Amino acids with neutral side chains have pI
values near neutrality, in the pH range 5.0-
6.5.
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If additional acidic or basic groups are
present as side-chain functions, the pI is the
average of the pKa's of thetwo most similaracids.
Amino acids with acidic side chains have pIvalues at lower pH, which suppresses
dissociation of extra COOH.
Amino acid with basic side chains have pI
values at higher pH, which suppresses
protonation of the extra amino group.
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Example:
Aspartic acid
The similar acids are the alpha-carboxylfunction (pKa = 2.1) and the side-chain
carboxyl function (pKa = 3.9)
pI = (2.1 + 3.9)/2 = 3.0
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Example:
Arginine
The similar acids are the guanidiniumspecies on the side-chain (pKa = 12.5)
and the alpha-ammonium function (pKa
= 9.0)
pI = (12.5 + 9.0)/2 = 10.75
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Amino Acid -carboxylic acid -amino Side chain
Alanine 2.35 9.87
Arginine 2.01 9.04 12.48
Asparagine 2.02 8.80
Aspartic Acid 2.10 9.82 3.86
Cysteine 2.05 10.25 8.00
Glutamic Acid 2.10 9.47 4.07
Glutamine 2.17 9.13
Glycine 2.35 9.78
Histidine 1.77 9.18 6.10
Isoleucine 2.32 9.76
Amino Acid pKa Values
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E i
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Exercises:
1. Draw the structure of the predominant
form of each of the following:a. Lys at pH 2.0 c. Asp at pH 6.0
b.Lys at pH 11.0 d. Ala at pH 3.0
2. Give the pI values of the following
amino acids:a. Thr c. His
b.Cys d. Gln
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Peptide Bonds
Amino acids can be linked linearly
by formation of covalent bond via a
dehydration synthesis reaction
between the -carboxyl group ofthe first amino acid with the -
amino group of the second aminoacid.
Water is eliminated in the process.
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The bond formed is calledpeptide bond.
Formation of Peptide Bonds
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Stability of Peptide Bond
1. Peptide bond is metastable;hydrolysis at physiological pH and
temperature is exceedingly slow.
2. Hydrolysis is rapid only under
extreme conditions e.g. boiling in
strong mineral acid (6 M HCl) or
when catalysts/ enzymes are present.
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Peptides-are also called amides
(Why?)
Dipeptide
- a linkage of two amino acids- example is aspartame or
NutraSweetTM (L-aspartyl-L-
phenylalanine), a sugar substitute
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Polypeptides are made of:
1. the main chain (backbone)
regularly repeating part
2. the side chaindistinct variable part
Polypeptidea linkage of amino acids
from three to several dozen units
Th li k d i id i i
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The linked amino acid moieties are
calledamino acid residues.
The amino acid residues are named by
replacing the ending -ine or -ate to -yl.
The -ylending indicates that the residue
is an acyl unit (a structure that lacks the
hydroxyl of the carboxyl group).Ex. glu-gly-ala-lys
glutamyl-glycyl-alanyl-lysine.
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The amino end (N-terminus) is the
beginning of a polypeptide chain whilecarboxylic end (C-terminus) is the end
of the chain.
Polypeptide sequences are written left
to right from the N- to the C-terminus.
Ex. glu-gly-ala-lys or E-G-A-K
(Glu contains the N-terminus while lys
contains the C-terminus.)
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Exercise:
Give the structure of leucine
enkephalin (Y-G-G-F-L, a
naturally occuring analgesic)
Indicate where the peptide bondsare.
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Th l l i ht f
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The average molecular weight of
an amino acid residue is about 110
so the molecular weights of mostpolypeptides are between 5500
220,000Since onedalton is equal to one
atomic mass unit, a protein with a
molecular weight of 50,000 has amass of 50,000 daltons or 50 kd
(kilodaltons)
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Peptide Bonds
- have partial double bond characterdue to delocalization of thepi
electron orbitals
- the resonance structures makes
the amide group planar
- the peptide bond can be written as
a resonance hybrid of two structures
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The bond between the -C atom and the carbonyl-
C atom is a pure single bond, likewise the bond
between the -C atom and the peptide nitrogen isa pure single bond.
Due to the specific electronic structure of the
peptide bond, the atoms on its two ends cannot
rotate around the bond. Hence, the atoms of the
group, O=C-N-H, are fixed on the same plane,
known as the peptide plane.
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P l tid h i f ld i t
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Polypeptide chains can fold into
regular structures: the helix and
the pleated sheets.
helix pleated sheets
A h li i ti ht h li f d t f
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An helix is a tight helix formed out of
the polypeptide chain.
helices are commonly made up of
hydrophobic amino acids, because H
bonds (the strongest attraction) arepossible between such amino acids.
The polypeptide main chain makes up
the central structure, and the side
chains extend out and away from the
helix.
Helical Structure
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-Helical Structure
H d B di i H li
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Hydrogen Bonding in Helix
In an helix, the CO group of one amino
acid is hydrogen bonded to the NH group.
Every CO and NH group of the backbone
ishydrogen bonded.
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Hydrogen Bonding in Helix
The CO group of one amino acid (n) is
hydrogen bonded to the NH group of the
amino acid four residues away (n+4).
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pleated conformation consists of pairs
of chains lying side-by-side and
stabilized byH bonds between the
carbonyl oxygen on one chain and the
amide hydrogens on the adjacent chain.
l t d f ti
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pleated conformation
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pleated sheets can be either
parallel or anti-parallel.
Parallel pleated sheetsrun in
the same N- to C-terminal
direction.
Antiparallel pleated sheets
run
in opposite N- to C-terminal
direction.
P ll l l t d h t
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Parallel pleated sheets
A i
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Anti-parallel -pleated sheets
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P ll l l t d h t l
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Parallel pleated sheets are less
stable than antiparallel sheets,
possibly because the hydrogen
bonds are distorted in the parallel
arrangement.
pleated sheets may contain 2 to
15 strands, with an average of 6residues per strand.
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PROTEINS
- are polypeptides ofdefined amino
acid sequence . The amino acid
sequences of proteins are
genetically determined; alterationsin amino acid sequence can
produce abnormal function anddisease.
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Biologically active proteins are
linked by covalent peptide bonds.
Some proteins are held together
by noncovalent or covalent forces.
S Bi l i l F ti f
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Some Biological Functions of
Proteins
Enzymes - act as biological
catalyst; chymotrypsin
Hormones - regulate body
processes; insulin
Protective Proteins - fightinfection; antibodies
Storage Proteins casein stores
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Storage Proteins - casein stores
nutrients; myoglobin stores
oxygen in muscles
Structural Proteins - form the
structure of the body; keratin,elastin, collagen
Transport Proteins - transport
oxygen and other substances
through the body; hemoglobin
C j t d P t i
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Conjugated Proteins1.Glycoproteinsproteins bonded
to carbohydrates; cell membranes
have glycoprotein coating
2.Lipoproteinsproteins bonded tofats and oils (lipids); these proteins
transport cholesterol and other
fats through the body
3.Metalloproteins proteins bonded to a
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3.Metalloproteins proteins bonded to a
metal ion; example is cytochrome
oxidase, an enzyme necessary forbiological production
4.Nucleoproteinsproteins bonded to
RNA (ribonucleic acids); found in cellribosomes
5.Phosphoproteinsproteins bonded toa phosphate group; example is milk
casein, which stores nutrients for
growing embryos
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Structures of Proteins
1.Primary Structure
2.Secondary Structure
3.Tertiary Structure4.Quaternary Structure
Structures of Proteins
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Structures of Proteins
Structures of Proteins
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Structures of Proteins
Primary Structurethe amino acid
sequence or order in which the aminoacids are linked together and the
location of disulfides, if any, that make
up a protein
Protein primary sequences can be
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Protein primary sequences can be
written with a 3-letter code for the
or with a 1-letter code.
Example: bovine insulinA-Chain:
GIVEQCCTSICSLYQLENYCN
B-Chain:FVNQHLCGSHLVEALYLVCGERGF
FYTPKT
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Secondary Structure
-refers to the way in whichsegments of the peptide bonds
orient into a regular pattern;
Examples:alpha helix and thebeta
sheet
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Helices and sheets could be in the
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same proteins
Tertiary Structure
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y
-refers to the way in
which the entire proteinmolecule coils into an
overall three-dimensional
shape-final shapes of proteins
are determined and
stabilized by chemical
bonds, including weak
bonds
Rib l
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Alpha helices,
beta sheets,and turns,
contribute to
the
ribonuclease
tertiarystructure
Ribonuclease
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Myoglobin
Quaternary Structure
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Quaternary Structure
- refers to the spatial arrangement of
polypeptide chains (subunits) and thenature of their contacts
- the arrangement of the individual
subunit of a protein with multiple
polypeptide subunits (ex. hemoglobin
has two alpha and two beta subunits)
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Hemoglobin
Hemoglobin, a protein with four polypeptides;
two alpha globins and two beta globins. The
red patches are the heme group.
Factors that Determines
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Factors that Determines
Secondary and Tertiary Structure
1.Amino acid sequence - plays a majorrole, because subtle changes in the
sequence can easily change thesecondary and tertiary structures of
proteins
2. Thermodynamic Factors - Folding is a
thermodynamically favored process.
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3.Disulfide Bonds - Bonds between
cysteine residues in a protein help tostabilize it once it has folded.
Bovine pancreatic trypsin inhibitor
(BPTI), which has 3 disulfide bonds,
is one of the stablest proteins
known; can only be denatured at100oC in very acid solutions.
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S-S bonds: Cys5-Cys55, Cys14-Cys38 and Cys30-Cys51
RPDFC LEPPY TGPCK ARIIR YFYNA KAGLC5 14 30
QTFVY GGCRA KRNNF KSAEDCMRTCGGA38 51 55
BPTI
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Ribonuclease (RNase A)
Protein
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noncovalent
interactions helpstabilize tertiary
structure of
protiensNoncovalent
interactions are
individually weak
but collectively
strong. Three principal kinds of noncovalent
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forces:
1. Ionic interactions2. Hydrophobic interactions
3. Hydrogen bonds
Ionic Interaction
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Ionic InteractionIonic interactions are
highly sensitive to pHchanges and salt
concentration.
As the pH drops, H+ binds to the carboxylgroups (COO-) of Asp and Glu,
neutralizing their negative charge, and H+
bind to the unoccupied pair of electrons onthe N atom of the amino (NH2 ) groups of
Lys and Arg giving them a positive charge.
Hydrophobic Interaction
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Hydrophobic Interaction
The side chain-R groups such
as phe and leu are nonpolar andthus interact poorly with polar
molecules like water. Nonpolar residues
in proteins are directed toward theinterior of the molecule and have
hydrophobic interactions.
The strength of hydrophobic interactions is
not appreciably affected by changes in pH
or in salt concentration.
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Hydrogen Bonds
Hydrogen bonds can form when a
strongly electronegative atom (e.g. O
and N) approaches a hydrogen
atom which is covalently attached toa second strongly electronegative
atom.
T f H d B d i
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Types of Hydrogen Bonds in
Proteins:
Hydroxyl-hydroxyl
Hydroxyl-carbonylAmide-carbonyl
Amide-hydroxylAmide-imidazole N
Forces responsible for protein
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Forces responsible for protein
folding:
1. hydrogen bonding
2. salt bridges - ionic attraction3. hydrophobic effect
4. crosslinking - e.g. disulfidebridges
Two major classes of proteins
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1. Fibrous proteinsconsist of polypeptide
chains arranged side by side in longfilaments such as collagen and keratin
- are tough and insoluble in water
- structural materials of animal cells andtissues
- include the major proteins of skin and
connective tissues and of animal fiberslike hair and silk
- favors the secondary structure
Some Common Fibrous Proteins
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Collagens: animal hides, tendons,
connective tissues
Elastins: blood vessels, ligaments
Fibrinogen: necessary for bloodclotting
Keratins: skin, wool, feathers, hooves,
silk, nails Myosins: muscle tissues
helical keratins
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helical-keratins
are the major proteins of hair and
fingernails and compose a major
fraction of animal skin;
play important structural roles inthe nuclei, cytoplasm, and surfaces
of many cell types;doesnt stretch due to
crosslinking of disulfide bonds
Structure of
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typical -keratin
in hair
-keratin is built
on a coiled- -
helical structure
Collagens
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- make up bones, skin, tendons, and
cartilage; the most abundant proteinfound in vertebrates
-usually contain three very longpolypeptide chains, each with about
1000 amino acids, that twist into a
regularly repeating triple helix and
give tendons and skin their great
tensile strength
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Collagen Structure
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C ll
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When long collagen fibrils aredenatured by boiling, their chains
are shortened to form gelatin.
Collagens
Fibroin
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-a sheet protein
-almost half of its residues areglyand between them lie eitherala or
ser residues this allows the sheetsto fit together and pack on top of
one another which results in a
strong and relatively inextensible
fiber.
Structure of Silk Fibroin
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Fibroin
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Fibroin
- The covalently bonded chains arestretched to nearly their maximum
possible length.
- Bonding between the sheetsinvolves van der Waals force of
attraction which provide littleresistance to bending
Silk Fibroin
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Silk Fibroin
Elastin
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-forms elastic fibers found in
ligaments and blood vessels-rich in glycine, alanine, and valine,
and is very flexible and easilyextended
-its conformation approximates that
of a random coil, with little
secondary structure
Elastin
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-the glycine, alanine, and valine
sequence also contains frequentlysine side chains, which can be
involved in cross-links-these cross-links prevent the elastin
fibers from being extended
indefinitely, causing the fibers to
snap back when tension is removed
Globular Proteins
f b fib i
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-far outnumber fibrous proteins
-perform most of the chemical "work"of the cell: synthesis, transport, and
catabolism
-are folded into compact structures,nearly spherical shapes, unlike the
extended, filamentous forms of the
fibrous proteins
- are generally soluble in water and are
mobile within cells
Globular Proteins
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- have no systematic structures but are
relatively spherical in shape- there may be single, two or more
chains; the chains could be helical,
pleated, or completely random
structures
- examples are egg albumin,hemoglobin, myoglobin, insulin, serum
globulins in blood, and many enzymes
helical (blue)
sheet (orange) structures
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Examples of globular protein structures
sheet (orange) structures
Common Globular Protein
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Hemoglobin: involved in oxygen
transport
Immunoglobulins: involved in
immune system Insulin: hormone for controlling
glucose metabolism
Ribonuclease: enzyme controllingRNA synsthesis
General rules that have been
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observed in globular proteins:
1. Folds favor orientation of aminoacid residues in specific ways that
pack hydrophobic amino acidresidues on the inside of the
protein (away from water) and
hydrophilic amino acid residues on
the outside of the protein.
2. sheets are usually twisted, or
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y ,
wrapped into barrel structures
e.g. immunoglobulin and
prealbumin
3. Turns (interruptions between
d t t ) i
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secondary structures) can occur in a
number of ways: occur at the surfaceof proteins via hydrogen bonding
between residues 1 and 4 ( turns).
A tighter turn, called the turn (or
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hairpin loop), can also occur in only 3
amino residues.
Myoglobin (Mb)
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-consists of a single polypeptide chain of
153 amino acid residues and includes aprosthetic group, the heme which binds
the oxygen
-has eight -helical regions
-H-bond stabilizes the -helical region
-present in skeletal muscles as an extrastorage protein to enable muscle cells to
have a readily available supply of O2
heme
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heme
Each myoglobin molecule contains one heme prostheticgroup inserted into a hydrophobic cleft in the protein. Each
heme residue contains one bound iron atom that is normally
in the Fe2+oxidation state.
Heme is made of a
series of nitrogen
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series of nitrogen
cyclic rings andjoined to each other
by more rings. At
the center of theheme group is the
Fe2+. The N atoms
bind to Fe2+ throughcoordinate covalent
bonds.
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Skeletal structure Molecular structure
Protoporphyrin IX
Hemoglobin (Hb)
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- consists of four polypeptide chains;
two -chains and two -chains- both - and -chains are very similar
to myoglobin; the -chain has 141 a.a.
residues while the -chain has 146; the
-chains and -chains of hemoglobin
and the myoglobin are homologous;each subunit contains a heme, the
protoporphyrin IX and Fe (II) complex.
Comparison of Myoglobin and Hemoglobin Structures
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MyoglobinHemoglobin
Hemoglobin
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Hemoglobin
f ti i h bl d i
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- function in human blood is oxygen
transport from the lungs to the tissuesof the body
- each hemoglobin molecule can bind to
a total of four oxygen molecules- coordination of Fe (II) in a porphyrin
within a hydrophobic globin pocketallows O2 binding without iron
oxidation
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O2 binding of Hb and Mb
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Oxygenationthe reversible
binding of O2 to Hb or Mb
Oxymyoglobinoxygen-bearingmyoglobin
Deoxymyoglobinoxygen-freemyoglobin
Fe(II) Coordination in Oxymyoglobin
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Heme pocket
showing the
proximal (F8)
and distal (E7)
histidine side
chains (His 93
and 64 resp.).
His 64 forms Hbond with O2and His 93 is
complexed to
Fe2+
.
O2-binding site in oxymyoglobinHis-64
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N
N
O
H
O
N
N
NN
NFe2+
HN His-93
Six ligands are coordinatedto Fe2+, with the ligands in
octahedral geometry
around iron. Four of the
ligands are the N atoms ofthe tetrapyrrole ring
system, the fifth is an
imidazole ring from His-93
(proximal) and the sixth is
the O2 bound between the
Fe and the imidazole side
chain of His-64 (distal).
heme
The heme group is
nonplanar when it is not
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nonplanar when it is not
bound to oxygen; the iron
atom is pulled out of theplane of the porphyrin,
toward the histidine
residue to which it is
attached. This nonplanar
configuration is
characteristic of the
deoxygenated heme group,and is commonly referred
to as a "domed" shape.Deoxygenated heme group
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When a single heme group in the hemoglobin protein becomes
oxygenated, the whole protein changes its shape. In the new shape,it is easier for the other three heme groups to become oxygenated.
Thus, the binding of one molecule of O2 to hemoglobin enhances
the ability of hemoglobin to bind more O2 molecules. This
property of hemoglobin is known as "cooperative binding."
Comparison of the O2 binding properties of Mb and Hb
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Satura
tion
Mb
Hb
O2 partial pressure, mm Hg
sigmoidal
hyperbolic
The curve of oxygen binding to
h l bi i i id l t i l f
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hemoglobin is sigmoidal typical of
allosteric proteins in which thesubstrate, in this case oxygen, is a
positive homotropic effector.
In contrast the oxygen binding curve
for myoglobin is hyperbolic incharacter indicating the absence of
allosteric interactions in this process.
Hemoglobin (Hb) Saturation Curve
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Normal pO2 (40
mm Hg) in the
capillaries in
resting tissues.
Normal pO2(100 mm Hg)
in the
capillaries in
the lungs. This
is constant and
does not
change under
normal
circumstances.
The Effect of CO2 and H+ on O2 Binding
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Increased concentrations of CO2 and
H+ promote the release of O2 fromHb in the blood (Bohr Effect).
CO2 and H
+
are produced frommetabolic activities of the body. The
tissues that perform the most
metabolic activity produce largequantities of CO2 and H
+, facilitating
the release of O2 from the blood.
Bohr Effect
As O is utilized in tissues CO is
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As O2 is utilized in tissues, CO2 is
produced. Accumulation of CO2 lowersthe pH in erythrocytes (red blood cells)
through the bicarbonate reaction:
CO2 + H2O HCO3-
+ H+
This is catalyzed by carbonic anhydrase.
A drop in pH in tissues and in venousblood signals a demand in more oxygen.
Bohr EffectA pH drop in the blood in the capillaries
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A pH drop in the blood in the capillaries
lowers the oxygen affinity of hemoglobinand allows more efficient release of oxygen.
The overall reaction may be written,Hb4O2 +nH+ HbnH+ + 4O2
wheren is about > 2.
The response of hemoglobin to changes in
pH is called theBohr effect.
Effect of pH on O2 Binding
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Bohr EffectThe reverse reaction occurs in the lungs or
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The reverse reaction occurs in the lungs or
gills where there is high O2
conc. This favors
oxygenation (oxy state). This releasesH+ by
shifting the equilibrium to the left.
Hb4O2 +nH
+
HbnH
+
+ 4O2This tends to release CO2 from the HCO3- by
the reversal of the bicarbonate reaction. The
free CO2 can then be exhaled.CO2 + H2O HCO3
- + H+
Release ofCO2 from respiring
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2 p g
tissues lowers the O2 affinity ofHb in two ways:
1.Some of the CO2 becomes HCO3-,CO2 + H2O HCO3
- + H+
releasing protons that contributeto the Bohr effect.
2. Some of the HCO3- is transported out
of the erythrocytes and is carried
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of the erythrocytes and is carried
dissolved in the blood serum.A portion reacts directly with Hb,
binding to theN-terminal amino
groups of the chains to formcarbamates (carbamation reaction):
NH3
+ + HCO3- -NHCOO- + H+ + H2O
or
CO2 + Hb-NH2 H+ + Hb-NH-COO-
Carbamation Reaction
1 Aid i th t t f CO f
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1. Aids in the transport of CO2 from
tissues to lungs or gills2. Releases H+ which contributes to the
Bohr effect
3. Introduces a negatively charged group
at the N-terminus of the chains,
stabilizing salt bridge formationbetween the and chains, a
characteristic of thedeoxy state.
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Summary of the effects of H+
and CO2 in the respiratory
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2
cycle:1. In the lungs or gills, oxygenation
favors the oxy conformation of
hemoglobin which stimulates
release of CO2.2. As the blood travels via arteries
into tissue capillaries, the lower
pH and high CO2 content favorsthe deoxy form, promoting O2release from hemoglobin and
binding of CO2
.
Mb and Hb protect the oxygen-binding
Fe2+ from irreversible oxidation. How?
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Fe from irreversible oxidation. How?
O2 will normally oxidize Fe2+
to Fe3+
inclose contact. The heme does not
protect Fe2+ since heme if dissolved
free in solution is readily oxidized byO2.
The hydrophobic interior of Mb and Hb
protects the heme and Fe2+ to become
easily oxidized.
When O2 is bound, a temporary
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2 , p y
electron rearrangement occurs.When O2 is released, the iron
remains in the ferrous state.
Mb and Hb provide environments in
which binding of O2 is permittedbut oxidation is blocked.
Why is CO toxic?
The heme pocket can also accept other
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The heme pocket can also accept other
small molecules like CO which hasapproximately the same size and
electron configuration as O2.
However, CO is bound with muchgreater affinity to Mb and Hb than is
O2, and the binding is not readily
reversible. CO ties up O2 binding sites
and thereby blocks respiration.
Protein breakdown: Hydrolysis vs
Denaturation
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Denaturation
Hydrolysis-involves breaking of the peptide bonds
through the addition of water
-requires high temperatures and either
strong acid or strong base
-takes place in the cells at bodytemperature when catalyzed by an
enzyme
Denaturation of Proteins
-involves the disruption and possible
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involves the disruption and possible
destruction of both the secondary andtertiary structures of native proteins
-disrupts the normal -helix and -sheets in a protein and uncoils it into a
random shape
-results in loss of biological activity
-Denaturation reactions are not strong
enough to break the peptide bonds; the
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enough to break the peptide bonds; the
primary structure or the sequence ofamino acids remains the same after a
denaturation.
-Most common observation in the
denaturation process is the
precipitation or coagulation of theprotein.
Causes of Protein Denaturation
1. Heat - disrupts hydrogen bonds and non-
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p y g
polar hydrophobic interactions
- occurs because heat increases the
kinetic energy and causes the molecules
to vibrate so rapidly and violently thatthe bonds are disrupted.
Examples:
- proteins in eggs coagulate upon heating- sterilization by heating denature proteins
in bacteria and thus destroy the bacteria
2. AlcoholAlcohol disrupts the hydrogen bonding
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p y g g
between amide groups in the 2
o
structure and those between side
chains in the 3o structure.
Alcohol denatures proteins by formingnew hydrogen bonds between the
alcohol molecule and the protein side
chains.
Example: 70% alcohol solution is
used as a disinfectant on the skin
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3. Acids and Bases
acids and bases disrupt salt bridges
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- acids and bases disrupt salt bridges
held together by ionic charges; saltbridges result from the neutralization
of an acid and amine on side chains
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- salt bridge has the effect of straight-
ening an alpha helix; denaturation
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ening an alpha helix; denaturation
reaction on the salt bridge by theaddition of an acid results in a further
straightening effect on the protein chain
Example: reaction in the digestive
system, when the acidic gastric juicescause the curdling (coagulating) of milk.
4. Heavy Metal Salts
-heavy metal salts denature proteins in
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-heavy metal salts denature proteins in
much the same manner as acids andbases
-salts are ionic thus disrupt salt bridges
in proteins. The reaction of a heavy
metal salt with a protein usually leads
to an insoluble metal protein salt
Heavy metal salts usually contain Hg2+,
Pb2+, Ag+ ,Tl+, Cd2+ and other metals
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Pb , Ag ,Tl , Cd and other metals
with high atomic weights.AgNO3, used to prevent gonorrhea
infections in the eyes of new born
infants, is also used in the treatment ofnose and throat infections.
Heavy metal salts may also disruptdisulfide bonds because of their high
affinity and attraction for sulfur.
5. Reducing
-Disulfide bonds are formed by
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y
oxidation of the sulfhydryl groups oncysteine.
-Different protein chains or loops within
a single chain are held together by thestrong covalent disulfide bonds.
-Reducing agents add hydrogen atoms
to make the thiol group, -SH.
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Disulfide bonds in Insulin
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Determining Protein Denaturation
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1. Loss of Solubility
- one of the oldest methods utilized to
follow the course of denaturation was
to measure changes in solubility
2.Increased Proteolysis
- most native proteins are quite
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os a ve p o e s a e qu e
resistant to the action of proteolyticenzymes;
During digestion, hydrochloric acid in
the stomach denatures proteins.Pepsin, a protease that functions
optimally in acidic environment,
catalyzes hydrolysis of proteins.
3.Loss of Biological Activity
-For enzymes, denaturation can be
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y ,
defined as the loss of enough structureto render the enzyme inactive.
-Changes in the rate of the reaction,
the affinity for substrate, pH optimum,temperature optimum, specificity of
reaction, etc., may be affected by
denaturation of enzyme molecules.
Loss of biological activity of an enzyme
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4. Spectroscopic Procedures
Both ultraviolet and fluorescence
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Both ultraviolet and fluorescence
spectroscopy have been utilized tofollow changes in the environments of
various groups within protein
molecules.
Such changes in environment reflect
changes in protein structure and thusdenaturation.
Protein Sequencing
Is accomplished using several
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Is accomplished using several
methods performed in
combination.
Enzymaticvery specificChemical
Instrumental
Specificities of Several Endoproteases
Enzyme
Source Specificity
Additional
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Enzyme Source SpecificityPoints
TrypsinBovine
pancreas
peptide bond
C-terminal to
R, K, H but
not if next to
P
highly
specific for
positively
charged
residues
ChymotrypsinBovine
pancreas
peptide bond
C-terminal toF, Y, W but
not if next to
P
prefers
bulky
hydrophobicresidues,
cleaves
slowly at N,
H, M, L
ElastaseBovine
pancreas
peptide bondC-terminal to
A, G, S, V, but
not if next to
-
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pnot if next to
P
Thermolysin
Bacillus
thermoproteolyt
icus
peptide bond
N-terminal to
I, M, F, W, Y,
V, but not ifnext to P
prefers
small
neutral
residues,
can cleaveat A, D,
H, T
PepsinBovine gastric
mucosa
peptide bondN-terminal to
L, F, W, Y, but
when next to
P
exhibitslittle
specificity,
requires
low pH
Carboxy-Terminal Sequence
Determination
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Enzymes, exopeptidases, cleavespeptides at the C-terminal residue
which can then be analyzed
chromatographically and compared tostandard amino acids. This class of
exopeptidases are called,
carboxypeptidases.
Chemical Digestion
C b id (CNB ) l
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Cyanogen bromide (CNBr) cleaves
specifically at the C-terminal side of M
residues. The number of peptide
fragments that result from CNBrcleavage is equivalent to more than the
number of M residues in a protein.
This method is only used on
carboxypeptidase resistant peptides.
Sanger's MethodFrederick Sangerdetermined the
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complete sequence of insulin- 2,4-dinitrofluorobenzene (DNF)
reacts with the N-terminal residue
under alkaline conditions.- The derivatized amino acid can be
hydrolyzed and will be labeled with a
dinitrobenzene group that imparts a
yellow color to the amino acid.
(Sanger's Method)
- the modified amino acids (DNP-
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t e od ed a o ac ds ( N
derivative) is separated byelectrophoresis and comparison
with the migration of DNP-derivative standards allows for the
identification of the N-terminal
amino acid.
Dansyl chloride,5-(dimethylamino)-1-
naphthalenesulfonyl chloride)
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- Dansyl chloride reacts with the N-terminal residue under alkaline
conditions.
- Analysis of the modified amino acids iscarried out similarly as the Sanger method
except that the dansylated amino acids are
detected by fluorescence.- Has higher sensitivity than the Sanger
method.
Edman Degradation Technique
Pehr Edman developed a technique that
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permits removal and identifi-cation ofone residue at a time from the N-
terminus of a protein.
- The technique utilizes phenyliso-thiocyanate (PITC) to react with the N-
terminal residue under alkaline
conditions.
(Edman Degradation Technique)
-cleaves the N terminal and allows for
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additional amino acid sequence to beobtained from the N-terminus inward
because it leaves the remaining peptide
intact-the entire sequence of reactions can be
repeated over and over to obtain the
sequences of the peptide
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Protein Sequencing
Basic strategy to determine the
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gy
sequence of proteins:1. Determine the amino acid
composition.
This can be accomplished withstrong acids (i.e. 6N HCl) or strong
bases or by exhaustive enzymaticdigestion; minimum length for the
polypeptide will be known.
2.Break all Disulfide Bonds
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Disulfide cross-links usually are
cleaved by reduction or oxidation
before sequence analysis.
3.Perform an Initial N-terminal and
C-terminal Sequence Determination.
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C terminal Sequence Determination.
Use end group,aminopeptidase and
Edman degradation experiments todetermine the N-terminal sequence,
and usecarboxypeptidase to
determine the C-terminal amino
acids.
4.Divide and Conquer
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Break the polypeptide into
fragments by cleaving at specific
amino acids. Several cleavagemethods are available, each of
which have different specificity (i.e.
cleave at different amino acids).
5.Repeat steps 3 and 4 to determine sub-
sequences and create overlappings
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From the overlapping peptides and
information gained from the original
protein, a unique sequence for theprotein or polypeptide of interest can be
constructed. The overlaps should be at
least two amino acids in length.
6.Locate the Disulfide Bonds
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No primary structure analysis of acysteine-containing protein can be
regarded as complete before thepresence and location of disulfide
bonds has been established.
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