Chem 40 (1) Amino Acids, Proteins_fs10-11

7/31/2019 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



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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.

Documents

Chem 40 (1) Amino Acids, Proteins_fs10-11