27- 1 Amino Acids, Peptides, and Proteins. 27- 2ObjectivesObjectives Draw a general amino acid and...

27- 1

Amino Acids, Peptides, Amino Acids, Peptides, and Proteinsand Proteins

27- 2

Objectives Objectives Objectives Objectives

Draw a general amino acid and identify the Draw a general amino acid and identify the two functional groupstwo functional groups commoncommon to all. to all.

Classify each amino acid according to the Classify each amino acid according to the chemical naturechemical nature of its R group. of its R group.

Define the meaning of an Define the meaning of an essential amino acidessential amino acid..

Draw the reaction that joins two amino acids to form a Draw the reaction that joins two amino acids to form a peptide bondpeptide bond..

Describe and differentiate Describe and differentiate primary, secondary, tertiary, and quaternaryprimary, secondary, tertiary, and quaternary protein protein structures.structures.

Describe and differentiate Describe and differentiate co-enzymes and prostheticco-enzymes and prosthetic groups. groups.

List and discuss List and discuss four forcesfour forces that stabilize globular protein structure. that stabilize globular protein structure.

List important structural similarities and differences between List important structural similarities and differences between myoglobinmyoglobin and and hemoglobinhemoglobin..

Describe the mutation present in hemoglobin giving rise to Describe the mutation present in hemoglobin giving rise to sickle cell diseasesickle cell disease..

27- 3

What is an amino acid?

Twenty different kinds of amino acids are used by living organisms to produce proteins

An amino acid is a molecule containing an amine (-NH2) an acid (-COOH) and a third chemical group (-R) that defines the amino acid. In glycine, the simplest amino acid, R is –H, or a hydrogen atom. In alanine, R = -CH3. The R groups give specific properties to each amino acid, and to the proteins composed of amino acids.

R |

Structure of an amino acid: H2N – C – COOH H

27- 4

FundamentalsFundamentals

While their name implies that amino acids are While their name implies that amino acids are compounds that contain an —NHcompounds that contain an —NH2 2 group and a group and a

—CO—CO22H group, these groups are actually H group, these groups are actually

present as —NHpresent as —NH33++ and —CO and —CO22

–– respectively. respectively.

They are classified as They are classified as , , , , , , etcetc. amino acids . amino acids according the carbon that bears the nitrogen.according the carbon that bears the nitrogen.

27- 5

The 20 Key Amino AcidsThe 20 Key Amino Acids

More than 700 amino acids occur naturally, but More than 700 amino acids occur naturally, but 20 of them are especially important.20 of them are especially important.

These 20 amino acids are the building blocks of These 20 amino acids are the building blocks of proteins. All are proteins. All are -amino acids.-amino acids.

They differ in respect to the group attached to They differ in respect to the group attached to the the carbon. carbon.

These 20 are listed in Table 27.1.These 20 are listed in Table 27.1.

27- 6

Amino AcidsAmino Acids NNHH33

++

CCOO22––

an an -amino acid that is an-amino acid that is anintermediate in the biosynthesisintermediate in the biosynthesisof ethyleneof ethylene

++HH33NNCHCH22CHCH22CCOO22

––a a -amino acid that is one of-amino acid that is one ofthe structural units present inthe structural units present incoenzyme Acoenzyme A

++HH33NNCHCH22CHCH22CHCH22CCOO22

–– a a -amino acid involved in-amino acid involved inthe transmission of nervethe transmission of nerveimpulsesimpulses

27- 7

Classification of Amino AcidsClassification of Amino Acids

27- 8

CC CC

OO

OO––

RRRR

HH

HH33NN++

The amino acids obtained by hydrolysis of The amino acids obtained by hydrolysis of proteins differ in respect to proteins differ in respect to R R (the side chain).(the side chain).

The properties of the amino acid vary as the The properties of the amino acid vary as the structure of structure of RR varies. varies.

27- 9

CC CC

OO

OO––

RRRR

HH

HH33NN++

The major differences among the side chains The major differences among the side chains concern:concern:

Size and shapeSize and shapeElectronic characteristicsElectronic characteristics

27- 10

General categories of General categories of -amino acids-amino acids

nonpolar side chainsnonpolar side chainspolar but nonionized side chainspolar but nonionized side chainsacidic side chainsacidic side chainsbasic side chainsbasic side chains

27- 11

Non-PolarNon-Polar

PolarPolar

Amino Acid Amino Acid R-groupsR-groups

Amino Acid Amino Acid R-groupsR-groups

UnchargedUnchargedCysteineCysteineProlineProlineSerineSerine

GlutamineGlutamineAsparagineAsparagine

HydrophobicHydrophobicTryptophanTryptophan

PhenylalaninePhenylalanineIsoleucineIsoleucineTyrosineTyrosineLeucineLeucineValineValine

MethionineMethionine

AmbivalentAmbivalentGlycineGlycine

ThreonineThreonineAlanineAlanine

ChargedChargedArginine (+)Arginine (+)

Glutamic acid (-)Glutamic acid (-)Aspartic Acid (-)Aspartic Acid (-)

Lysine (+)Lysine (+)Histidine (+)Histidine (+)

27- 12

1. Hydrophobic (non-polar) residues1. Hydrophobic (non-polar) residues1. Hydrophobic (non-polar) residues1. Hydrophobic (non-polar) residues

Usually interior of proteins away from water.Usually interior of proteins away from water.

Hydrocarbon: do not contain polar atoms.Hydrocarbon: do not contain polar atoms.

27- 13

Charged Amino AcidsCharged Amino AcidsCharged Amino AcidsCharged Amino Acids

N

ON

N

N

N

ON

N

O

O

ON

OO

O

Arginine [Arg] Glutamate [Glu] Aspartate [Asp] Lysine [Lys]

+-

+

-

Histidine [His]

N

O

N

N

+

27- 14

Hydrophobic IndexesHydrophobic IndexesHydrophobic IndexesHydrophobic Indexes

ArginineArginine Arg [R]Arg [R] -11.2-11.2Glutamic Acid Glu [E]Glutamic Acid Glu [E] -9.9-9.9Aspartic AcidAspartic Acid Asp [D]Asp [D] -7.4-7.4LysineLysine Lys [K]Lys [K] -4.2-4.2HistidineHistidine His [H]His [H] -3.3-3.3CysteineCysteine Cys [C]Cys [C] -2.8-2.8

ProlineProline Pro [P]Pro [P] -0.5-0.5SerineSerine Ser [S]Ser [S] -0.3-0.3GlutamineGlutamine Gln [Q]Gln [Q] -0.3-0.3Asparagine Asparagine Asn [N]Asn [N] -0.2 -0.2

GlycineGlycine Gly [G]Gly [G] 00ThreonineThreonine Thr [T]Thr [T] 0.40.4AlanineAlanine Ala [A]Ala [A] 0.50.5

MethionineMethionine Met [M]Met [M] 1.31.3ValineValine Val [V]Val [V] 1.51.5LeucineLeucine Leu [L]Leu [L] 1.81.8TyrosineTyrosine Tyr [Y]Tyr [Y] 2.32.3IsoleucineIsoleucine Ile [I]Ile [I] 2.52.5PhenylalaninePhenylalanine Phe [F]Phe [F] 2.52.5TryptophanTryptophan Trp [W]Trp [W] 3.43.4

27- 15

Essential amino acidsEssential amino acidsEssential amino acidsEssential amino acids

Definition - Those amino acids that cannot be Definition - Those amino acids that cannot be synthesized in the body in sufficient quantities for synthesized in the body in sufficient quantities for anabolic needs.anabolic needs.

In humans, In humans,

IsoleucineIsoleucine Leucine Leucine ValineValine

Tryptophan Tryptophan Methionine Methionine Lysine Lysine

PhenylalaninePhenylalanine ThreonineThreonine HistidineHistidine

27- 16

20 20 Amino acidsAmino acids20 20 Amino acidsAmino acids

Glycine (G)

Glutamic acid (E)Asparatic acid (D)

Methionine (M)

Threonine (T)

Serine (S)

Glutamine (Q)

Asparagine (N)

Tryptophan (W)Phenylalanine (F)

Cysteine (C)

Proline (P)

Leucine (L)Isoleucine (I)Valine (V)

Alanine (A)

Histidine (H)Lysine (K)

Tyrosine (Y)

Arginine (R)

White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic

27- 17

CC CC

OO

OO––

HH

HH

HH33NN++

Glycine is the simplest amino acid. It is the only Glycine is the simplest amino acid. It is the only one in the table that is achiral.one in the table that is achiral.

In all of the other amino acids in the table the In all of the other amino acids in the table the carbon is a chirality center.carbon is a chirality center.

GlycineGlycine

(Gly or G)(Gly or G)

27- 18

CC CC

OO

OO––

CHCH33

HH

HH33NN++

AlanineAlanine

(Ala or A)(Ala or A)

Alanine, valine, leucine, and isoleucine have Alanine, valine, leucine, and isoleucine have alkyl groups as side chains, which are nonpolar alkyl groups as side chains, which are nonpolar and hydrophobic.and hydrophobic.

27- 19

CC CC

OO

OO––

CH(CHCH(CH33))22

HH

HH33NN++

ValineValine

(Val or V)(Val or V)

27- 20

CC CC

OO

OO––

CHCH22CH(CHCH(CH33))22

HH

HH33NN++

LeucineLeucine

(Leu or L)(Leu or L)

27- 21

CC CC

OO

OO––

CHCH33CHCHCHCH22CHCH33

HH

HH33NN++

IsoleucineIsoleucine

(Ile or I)(Ile or I)

27- 22

CC CC

OO

OO––

CHCH33SCHSCH22CHCH22

HH

HH33NN++

MethionineMethionine

(Met or M)(Met or M)

The side chain in methionine is nonpolar, but The side chain in methionine is nonpolar, but the presence of sulfur makes it somewhat the presence of sulfur makes it somewhat polarizable.polarizable.

27- 23

ProlineProline

CC CC

OO

OO––

CHCH22

HH

HH22NN++

HH22CCCCHH22

(Pro or P)(Pro or P)

Proline is the only amino acid that contains a Proline is the only amino acid that contains a secondary amine function. Its side chain is secondary amine function. Its side chain is nonpolar and cyclic.nonpolar and cyclic.

27- 24

PhenylalaninePhenylalanine

CC CC

OO

OO––

CHCH22

HH

HH33NN++

(Phe or F)(Phe or F)

The side chain in phenylalanine (a nonpolar The side chain in phenylalanine (a nonpolar amino acid) is a benzyl group.amino acid) is a benzyl group.

27- 25

CC CC

OO

OO––

CHCH22

HH

HH33NN++

NN

HH

TryptophanTryptophan

(Trp or W)(Trp or W) The side chain in The side chain in tryptophan (a nonpolar tryptophan (a nonpolar amino acid) is larger amino acid) is larger and more polarizable and more polarizable than the benzyl group than the benzyl group of phenylalanine.of phenylalanine.

27- 26

CC CC

OO

OO––

CHCH22OHOH

HH

HH33NN++

SerineSerine

(Ser or S)(Ser or S)

The —CHThe —CH22OH side chain in serine can be OH side chain in serine can be

involved in hydrogen bonding.involved in hydrogen bonding.

27- 27

CC CC

OO

OO––

CHCH33CHOHCHOH

HH

HH33NN++

ThreonineThreonine

(Thr or T)(Thr or T)

The side chain in threonine can be involved in hydrogen The side chain in threonine can be involved in hydrogen bonding, but is somewhat more crowded than in serine.bonding, but is somewhat more crowded than in serine.

27- 28

CC CC

OO

OO––

CHCH22SSHH

HH

HH33NN++

CysteineCysteine

(Cys or C)(Cys or C)

The side chains of two remote cysteines can be The side chains of two remote cysteines can be joined by forming a covalent S—S bond.joined by forming a covalent S—S bond.

27- 29

TyrosineTyrosineCC CC

OO

OO––

CHCH22

HH

HH33NN++

OHOH

(Tyr or Y)(Tyr or Y) The side chain of The side chain of tyrosine is similar to tyrosine is similar to that of phenylalanine that of phenylalanine but can participate in but can participate in hydrogen bonding.hydrogen bonding.

27- 30

AsparagineAsparagine

CC CC

OO

OO––

HH

HH33NN++

HH22NNCCCHCH22

OO(Asn or N)(Asn or N)

The side chains of asparagine and glutamine The side chains of asparagine and glutamine (next slide) terminate in amide functions that are (next slide) terminate in amide functions that are polar and can engage in hydrogen bonding.polar and can engage in hydrogen bonding.

27- 31

GlutamineGlutamine

CC CC

OO

OO––

HH

HH33NN++

HH22NNCCHCCH22CHCH22

OO(Gln or Q)(Gln or Q)

27- 32

Aspartic AcidAspartic Acid

CC CC

OO

OO––

HH

HH33NN++

OOCCCHCH22

OO

––

(Asp or D)(Asp or D)

Aspartic acid and glutamic acid (next slide) exist Aspartic acid and glutamic acid (next slide) exist as their conjugate bases at biological pH. They as their conjugate bases at biological pH. They are negatively charged and can form ionic are negatively charged and can form ionic bonds with positively charged species.bonds with positively charged species.

27- 33

Glutamic AcidGlutamic Acid

CC CC

OO

OO––

HH

HH33NN++

OOCCHCCH22CHCH22

OO

––

(Glu or E)(Glu or E)

27- 34

CC CC

OO

OO––

CHCH22CHCH22CHCH22CHCH22NNHH33

HH

HH33NN++

LysineLysine++(Lys or K)(Lys or K)

Lysine and arginine (next slide) exist as their Lysine and arginine (next slide) exist as their conjugate acids at biological pH. They are conjugate acids at biological pH. They are positively charged and can form ionic bonds positively charged and can form ionic bonds with negatively charged species.with negatively charged species.

27- 35

CC CC

OO

OO––

CHCH22CHCH22CHCH22NNHHCCNNHH22

HH

HH33NN++

ArginineArginine

++ NNHH22

(Arg or R)(Arg or R)

27- 36

HistidineHistidine CC CC

OO

OO––

HH

HH33NN++

CHCH22

NHNHNN

(His or H)(His or H) Histidine is a basic Histidine is a basic amino acid, but less amino acid, but less basic than lysine and basic than lysine and arginine. Histidine can arginine. Histidine can interact with metal ions interact with metal ions and can help move and can help move protons from one site protons from one site to another.to another.

27- 37

Stereochemistry of Amino Stereochemistry of Amino AcidsAcids

27- 38

Configuration of Configuration of -Amino Acids-Amino Acids

Glycine is achiral. All of the other amino acids Glycine is achiral. All of the other amino acids in proteins have the in proteins have the LL-configuration at their -configuration at their carbon.carbon.

HH33NN++

HH

RR

COCO22––

27- 39

CHO

H OH

CH2OH

CHO

HO H

CH2OH

D - L -

=

R

C CO2HH

H2N

(L) - Amino Acids(-) -

=

CHO

CH2OHH

HO

(S) - Glyceraldehyde(-) -

R

CHH2N CO2H

All DNA encoded aa are

All are chiral, except GlycineR = HAll DNA

encoded aa are usually L-

27- 40

Acid-Base Behavior of Amino Acid-Base Behavior of Amino AcidsAcids

27- 41

RecallRecall

While their name implies that amino acids are While their name implies that amino acids are compounds that contain an —NHcompounds that contain an —NH2 2 group and a group and a

—CO—CO22H group, these groups are actually H group, these groups are actually

present as —NHpresent as —NH33++ and —CO and —CO22

–– respectively. respectively.

How do we know this?How do we know this?

27- 42

aa are high melting point solids! Why?

Answer = aa are ionic compounds under normal conditions

C

O

OHR

NH3

C

O

OR

NH3

C

O

OR

NH2

LOW pH

Zwitterion

NEUTRAL

Carboxylate Form

HIGH pH

ammonium Form

Isoelectric Point = concentration of zwitterion is at a maximum and the concentration of cations and anions is equal

For aa with basic R-groups, we require higher pHs, and

for aa with acidic R-groups, we require lower pHs

to reach the Isoelectric Point

27- 43

(CH2)2

CHCO2H3N

CO2

Glu

(CH2)2

CHCO2H3N

NH3

Lys

pH 7

Isoelectric Point is the pH at which an aa or peptide carries no net charge. i.e. [RCOO-] = [RNH3

+]

So, for basic R-groups, we require higher pHs, and for acidic R-groups we require lower pHs

e.g. Isoelectric point for gly pH = 6.0 Asp pH = 3.0 Lys pH = 9.8 Arg pH = 10.8

27- 44

27- 45

27- 46

27- 47

Properties of GlycineProperties of Glycine

The properties of glycine:The properties of glycine:

high melting point:high melting point: (when heated to 233°C (when heated to 233°C it decomposes before it melts)it decomposes before it melts)solubility:solubility: soluble in water; not soluble in soluble in water; not soluble in nonpolar solventnonpolar solvent

OO

OHOHHH22NNCHCH22CC••••

••••

••••

•••• ••••

––••••

OO

OOHH33NNCHCH22CC ••••

••••

•••• ••••++

more consistent with thismore consistent with this than thisthan this

27- 48

Properties of GlycineProperties of Glycine

The properties of glycine:The properties of glycine:

high melting point:high melting point: (when heated to 233°C (when heated to 233°C it decomposes before it melts)it decomposes before it melts)solubility:solubility: soluble in water; not soluble in soluble in water; not soluble in nonpolar solventnonpolar solvent

––••••

OO


••••

•••• ••••++

more consistent with thismore consistent with this

called a called a zwitterionzwitterion or or dipolar iondipolar ion

27- 49

Acid-Base Properties of GlycineAcid-Base Properties of Glycine

The zwitterionic structure of glycine also follows The zwitterionic structure of glycine also follows from considering its acid-base properties.from considering its acid-base properties.

A good way to think about this is to start with the A good way to think about this is to start with the structure of glycine in strongly acidic solution, structure of glycine in strongly acidic solution, say pH = 1.say pH = 1.

At pH = 1, glycine exists in its protonated form At pH = 1, glycine exists in its protonated form (a monocation).(a monocation).

OO

OHOHHH33NNCHCH22CC++

••••

••••

•••• ••••

27- 50


Now ask yourself "As the pH is raised, which is Now ask yourself "As the pH is raised, which is the first proton to be removed? Is it the proton the first proton to be removed? Is it the proton attached to the positively charged nitrogen, or is attached to the positively charged nitrogen, or is it the proton of the carboxyl group?"it the proton of the carboxyl group?"

You can choose between them by estimating You can choose between them by estimating their respective ptheir respective pKKaas.s.

OO


••••

••••

•••• ••••

typical typical ammonium ammonium ion: pion: pKKaa ~9 ~9

typical typical carboxylic carboxylic acid: pacid: pKKaa ~5 ~5

27- 51


The more acidic proton belongs to the COThe more acidic proton belongs to the CO22H H

group. It is the first one removed as the pH is group. It is the first one removed as the pH is raised.raised.

OO


••••

••••

•••• ••••


27- 52


Therefore, the more stable neutral form of Therefore, the more stable neutral form of glycine is the zwitterion.glycine is the zwitterion.

OO


••••

••••

•••• ••••


––••••

OO


••••

•••• ••••++

27- 53

The measured pThe measured pKKaa of glycine is 2.34. of glycine is 2.34.

Glycine is stronger than a typical carboxylic acid Glycine is stronger than a typical carboxylic acid because the positively charged N acts as an because the positively charged N acts as an electron-withdrawing, acid-strengthening electron-withdrawing, acid-strengthening substituent on the substituent on the carbon. carbon.


OO


••••

••••

•••• ••••


27- 54


––••••

OO


••••

•••• ••••++

The pThe pKKaa for removal of this proton is 9.60. for removal of this proton is 9.60.

This value is about the same as that for NHThis value is about the same as that for NH44++

(9.3).(9.3).

HOHO––––••••

OO


••••

•••• ••••

••••

A proton attached to N in the zwitterionic form of A proton attached to N in the zwitterionic form of nitrogen can be removed as the pH is increased nitrogen can be removed as the pH is increased further. further.

27- 55

Isoelectric Point pIsoelectric Point pII

––••••

OO


••••

•••• ••••++

––••••

OO


••••

•••• ••••

••••

OO


••••

••••

•••• ••••

ppKKaa = 2.34 = 2.34

ppKKaa = 9.60 = 9.60

The pH at which the The pH at which the concentration of the concentration of the zwitterion is a zwitterion is a maximum is called the maximum is called the isoelectric pointisoelectric point. Its . Its numerical value is the numerical value is the average of the two average of the two ppKKaas.s.

The pThe pII of glycine is of glycine is 5.97.5.97.

27- 56

Acid-Base Properties of Amino AcidsAcid-Base Properties of Amino Acids

One way in which amino acids differ is in One way in which amino acids differ is in respect to their acid-base properties. This is the respect to their acid-base properties. This is the basis for certain experimental methods for basis for certain experimental methods for separating and identifying them.separating and identifying them.

Just as important, the difference in acid-base Just as important, the difference in acid-base properties among various side chains affects properties among various side chains affects the properties of the proteins that contain them.the properties of the proteins that contain them.

Table 27.2 gives pTable 27.2 gives pKKaa and p and pII values for amino values for amino

acids with neutral side chains.acids with neutral side chains.

27- 57

Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains

CC CC

OO

OO––

HH

HH

HH33NN++

GlycineGlycineppKKa1a1 = = 2.342.34

ppKKa2a2 == 9.609.60

ppI I == 5.975.97

27- 58


AlanineAlanineppKKa1a1 = = 2.342.34

ppKKa2a2 == 9.699.69

ppI I == 6.006.00

HH33NN CC CC

OO

OO––

CHCH33

HH++

27- 59


ValineValineppKKa1a1 = = 2.322.32

ppKKa2a2 == 9.629.62

ppI I == 5.965.96

HH33NN CC CC

OO

OO––

CH(CHCH(CH33))22

HH++

27- 60


LeucineLeucineppKKa1a1 = = 2.362.36

ppKKa2a2 == 9.609.60

ppI I == 5.985.98

HH33NN CC CC

OO

OO––

CHCH22CH(CHCH(CH33))22

HH++

27- 61


IsoleucineIsoleucineppKKa1a1 = = 2.362.36

ppKKa2a2 == 9.609.60

ppI I == 5.985.98

HH33NN CC CC

OO

OO––

CHCH33CHCHCHCH22CHCH33

HH++

27- 62


MethionineMethionineppKKa1a1 = = 2.282.28

ppKKa2a2 == 9.219.21

ppI I == 5.745.74

HH33NN CC CC

OO

OO––

CHCH33SCHSCH22CHCH22

HH++

27- 63


ProlineProlineppKKa1a1 = = 1.991.99

ppKKa2a2 == 10.6010.60

ppI I == 6.306.30

HH22NN CC CC

OO

OO––

HH++

CHCH22HH22CCCCHH22

27- 64


PhenylalaninePhenylalanineppKKa1a1 = = 1.831.83

ppKKa2a2 == 9.139.13

ppI I == 5.485.48

HH33NN CC CC

OO

OO––

HH++

CHCH22

27- 65


TryptophanTryptophanppKKa1a1 = = 2.832.83

ppKKa2a2 == 9.399.39

ppI I == 5.895.89

HH33NN CC CC

OO

OO––

HH++

CHCH22

HH

NN

27- 66


AsparagineAsparagineppKKa1a1 = = 2.022.02

ppKKa2a2 == 8.808.80

ppI I == 5.415.41

HH33NN CC CC

OO

OO––

HH++

HH22NNCCCHCH22

OO

27- 67


GlutamineGlutamineppKKa1a1 = = 2.172.17

ppKKa2a2 == 9.139.13

ppI I == 5.655.65

HH33NN CC CC

OO

OO––

HH++

HH22NNCCHCCH22CHCH22

OO

27- 68


SerineSerineppKKa1a1 = = 2.212.21

ppKKa2a2 == 9.159.15

ppI I == 5.685.68

HH33NN CC CC

OO

OO––

CHCH22OHOH

HH++

27- 69


ThreonineThreonineppKKa1a1 = = 2.092.09

ppKKa2a2 == 9.109.10

ppI I == 5.605.60

HH33NN CC CC

OO

OO––

CHCH33CHOHCHOH

HH++

27- 70


TyrosineTyrosineppKKa1a1 = = 2.202.20

ppKKa2a2 == 9.119.11

ppI I == 5.665.66

HH33NN CC CC

OO

OO––

HH++

CHCH22 OHOH

27- 71


CysteineCysteine HH33NN CC CC

OO

OO––

CHCH22SHSH

HH++ ppKKa1a1 = = 1.961.96

ppKKa2a2 == 8.188.18

ppI I == 5.075.07

27- 72

Amino Acids with Ionizable Side ChainsAmino Acids with Ionizable Side Chains

Aspartic acidAspartic acidppKKa1a1 = = 1.881.88

ppKKa2a2 == 3.653.65

ppKKa3a3 == 9.60 9.60

ppI I == 2.772.77

HH33NN CC CC

OO

OO––

HH++

OOCCCHCH22

OO

––

For amino acids with acidic side chains, pI is the For amino acids with acidic side chains, pI is the average of paverage of pKKa1a1 and p and pKKa2a2..

27- 73


Glutamic acidGlutamic acidppKKa1a1 = = 2.192.19

ppKKa2a2 == 4.254.25

ppKKa3a3 == 9.67 9.67

ppI I == 3.223.22

HH33NN CC CC

OO

OO––

HH++

OO

OOCCCHCH22CHCH22––

27- 74


LysineLysine

ppKKa1a1 = = 2.182.18

ppKKa2a2 == 8.958.95

ppKKa3a3 == 10.5310.53

ppI I == 9.749.74

HH33NN CC CC

OO

OO––

HH++

CHCH22CHCH22CHCH22CHCH22NNHH33

++

For amino acids with basic side chains, pI is the For amino acids with basic side chains, pI is the average of paverage of pKKa2a2 and p and pKKa3a3..

27- 75


ArginineArginine

ppKKa1a1 = = 2.172.17

ppKKa2a2 == 9.049.04

ppKKa3a3 == 12.4812.48

ppI I == 10.7610.76

HH33NN CC CC

OO

OO––

HH++

CHCH22CHCH22CHCH22NNHHCCNNHH22

++ NNHH22

27- 76


HistidineHistidine

ppKKa1a1 = = 1.821.82

ppKKa2a2 == 6.006.00

ppKKa3a3 == 9.17 9.17

ppI I == 7.597.59

HH33NN CC CC

OO

OO––

HH++

CHCH22

NHNHNN

27- 77

Reactions of Amino AcidsReactions of Amino Acids

27- 78

Acylation of Amino GroupAcylation of Amino Group

The amino nitrogen of an amino acid can be The amino nitrogen of an amino acid can be converted to an amide with the customary converted to an amide with the customary acylating agents.acylating agents.

OO

HH33NNCHCH22COCO––++++ CHCH33COCCHCOCCH33

OO OO

CHCH33CCNNHCHHCH22COHCOH

OO OO

(89-92%)(89-92%)

27- 79

Esterification of Carboxyl GroupEsterification of Carboxyl Group

The carboxyl group of an amino acid can be The carboxyl group of an amino acid can be converted to an ester. The following illustrates converted to an ester. The following illustrates Fischer esterification of alanine. Fischer esterification of alanine.

++ CHCH33CHCH22OHOH

HClHCl

OO

HH33NNCHCOCHCO––++

CHCH33

(90-95%)(90-95%)

OO

HH33NNCHCOCHCHCOCH22CHCH33

++

CHCH33

––ClCl

27- 80

Ninhydrin TestNinhydrin Test

Amino acids are detected by the formation of a purple Amino acids are detected by the formation of a purple color on treatment with color on treatment with ninhydrinninhydrin..

OHOH

OO

OO

OHOH++

OO

HH33NNCHCOCHCO––++

RR

OO OO

OO

NN

OO

––

OO

RCHRCH ++ COCO22 ++ HH22OO ++

27- 81

Some Biochemical ReactionsSome Biochemical Reactionsof Amino Acidsof Amino Acids

27- 82

27- 83

DecarboxylationDecarboxylation

Decarboxylation is a common reaction of Decarboxylation is a common reaction of --amino acids. An example is the conversion of amino acids. An example is the conversion of LL-histidine to histamine. Antihistamines act by -histidine to histamine. Antihistamines act by blocking the action of histamine.blocking the action of histamine.

CHCH22CHCOCHCO22

––

NNHH33++NNHH

NN

27- 84

DecarboxylationDecarboxylation

CHCH22CHCOCHCO22

––

NNHH33++NNHH

NN

––COCO22, enzymes, enzymes

CHCH22CHCH2 2 NNHH22

NNHH

NN

27- 85

NeurotransmittersNeurotransmitters

The chemistry of the The chemistry of the brain and central brain and central nervous system is nervous system is affected by affected by neurotransmitters.neurotransmitters.

Several important Several important neurotransmitters neurotransmitters are biosynthesized are biosynthesized from from LL-tyrosine.-tyrosine.

OOHH

COCO22––

HHHH

HHHH33NN

++

LL-Tyrosine-Tyrosine

27- 86


The common name The common name of this compound is of this compound is LL-DOPA. It occurs -DOPA. It occurs naturally in the naturally in the brain. It is widely brain. It is widely prescribed to reduce prescribed to reduce the symptoms of the symptoms of Parkinsonism.Parkinsonism.

OOHH

COCO22––

HHHH

HHHH33NN

++

LL-3,4-Dihydroxyphenylalanine-3,4-Dihydroxyphenylalanine

HHOO

27- 87


Dopamine is formed Dopamine is formed by decarboxylation by decarboxylation of of LL-DOPA.-DOPA.

OOHH

HH

HHHH

HHHH22NN

HHOO

DopamineDopamine

27- 88


OOHH

HH

HHHH

OHOHHH22NN

HHOO

NorepinephrineNorepinephrine

27- 89


OOHH

HH

HHHH

OHOHCHCH33NNHH

HHOO

EpinephrineEpinephrine

27- 90

PeptidesPeptides

27- 91

PeptidesPeptides

Peptides are compounds in which an amide Peptides are compounds in which an amide bond links the amino group of one bond links the amino group of one -amino acid -amino acid and the carboxyl group of another.and the carboxyl group of another.

An amide bond of this type is often referred to An amide bond of this type is often referred to as a peptide bond.as a peptide bond.

27- 92

Peptide Bond FormationPeptide Bond Formation

27- 93

Peptide bond formationPeptide bond formationPeptide bond formationPeptide bond formation

H HC

C

HH

H

H

N+

C-

O

O

C

-OO

A sp a rta te

C

HH

HH H

H

H

N+

C-

O

O

CA la n in e

H O2

condensation

27- 94

Peptide bond formationPeptide bond formationPeptide bond formationPeptide bond formation

H HC

C

HH

H

H

N+

C

O

C

H

H

H HH

N C-

O

O

C

C

-OO

P eptide bond

Primary Structure

27- 95

aa are covalently linked by amide bonds (Peptide Bonds)

The resulting molecules are called Peptides & Proteins

NC R

R'

O

NC R

R'

O

Features of a Peptide Bond;1. Usually inert2. Planar to allow delocalisation3. Restricted Rotation about the amide bond4. Rotation of Groups (R and R’) attached to the

amide bond is relatively free

27- 96

that are part of a peptide or aa protein are referred to as residues.

Peptides are made up of about 50 residues, and do not possess a well-defined 3D-structure

Proteins are larger molecules that usually contain at least 50 residues, and sometimes 1000. The most important feature of proteins is that they possess well-defined 3D-structure.Primary Structure is the order (or sequence) of amino acid residues

Peptides are always written and named with the amino terminus on the left and the carboxy terminus on the right

27- 97

27- 98

Alanine and GlycineAlanine and Glycine

CHCH33

OO

CC++

HH

CC OO––

HH33NN

OO

CC

HH

HH

CCHH33NN++

OO––

27- 99

AlanylglycineAlanylglycine

CHCH33

OO

CCHH33NN++

HH

CC

OO

CCNN

HH

HH

CC OO––

HH

Two Two -amino acids are joined by a peptide bond -amino acids are joined by a peptide bond in alanylglycine. It is a in alanylglycine. It is a dipeptidedipeptide..

27- 100


CHCH33

OO

CCHH33NN++

HH

CC

OO

CCNN

HH

HH

CC OO––

HH

Ala—GlyAla—Gly

AGAG

N-terminusN-terminus C-terminusC-terminus

27- 101

Alanylglycine and glycylalanine are Alanylglycine and glycylalanine are constitutional isomersconstitutional isomers

CHCH33

OO

CCHH33NN++

HH

CC

OO

CCNN

HH

HH

CC OO––

HH

HH

OO

CCHH33NN++

HH

CC

OO

CCNN

HH

CHCH33

CC OO––

HH

AlanylAlanylglycineglycineAlaAla——GlyGly

AAGG

GlycylGlycylalaninealanineGlyGly——AlaAla

GGAA

27- 102


CHCH33

OO

CCHH33NN++

HH

CC

OO

CCNN

HH

HH

CC OO––

HH

The peptide bond is The peptide bond is characterized by a characterized by a planar geometry.planar geometry.

27- 103

Strong Acid Required to hydrolyse peptide bonds

CH3

H3N CO

O

CH

H3N CO

O

CH2OH

H3N CO

O

Alanine SerineValine

CH3

H3N C

HN

O CH2OH

CNH

O

CO

O

Tripeptide : Ala . Ser. Val

- 2 H2O

27- 104

(CH2)4NH2

H2N C

HN

O

S

CNH

O

C

Ph

OH

O

S

CNH

O

OHC

O

HN

HOO

H2N

Ph

Lys. Cys. Phe

Phe. Ser. Cys

1. RSH

2. 6 M HCl hydrolysis

Lys + 2 Cys + 2 Phe + Ser

Cysteine residues create Disulfide Bridges between chains

This does not reveal Primary Structure

27- 105

Higher PeptidesHigher Peptides

Peptides are classified according to the number Peptides are classified according to the number of amino acids linked together.of amino acids linked together.

dipeptides, tripeptides, tetrapeptides, etc.dipeptides, tripeptides, tetrapeptides, etc.

Leucine enkephalin is an example of a Leucine enkephalin is an example of a pentapeptide.pentapeptide.

27- 106

Leucine EnkephalinLeucine Enkephalin

Tyr—Gly—Gly—Phe—LeuTyr—Gly—Gly—Phe—LeuYGGFLYGGFL

27- 107

OxytocinOxytocin

Oxytocin is a cyclic nonapeptide.Oxytocin is a cyclic nonapeptide.

Instead of having its amino acids linked in an Instead of having its amino acids linked in an extended chain, two cysteine residues are extended chain, two cysteine residues are joined by an joined by an S—S S—S bond.bond.

N-terminusN-terminus

C-terminusC-terminusIle—Gln—AsnIle—Gln—Asn

TyrTyr

CysCys SS SS

Cys—Pro—Leu—GlyNHCys—Pro—Leu—GlyNH22

11

22

3344 55

66 77 88 99

27- 108

OxytocinOxytocin

S—S bond

An S—S bond between two cysteines isAn S—S bond between two cysteines isoften referred to as a often referred to as a disulfide bridgedisulfide bridge..

27- 109

Introduction to Peptide Introduction to Peptide Structure DeterminationStructure Determination

27- 110

Primary StructurePrimary Structure

The primary structure is the amino acid The primary structure is the amino acid sequence plus any disulfide links.sequence plus any disulfide links.

27- 111

Proteins are linear polymers of amino acidsProteins are linear polymers of amino acidsProteins are linear polymers of amino acidsProteins are linear polymers of amino acids

R1

NH3＋

C CO

H

R2

NH C CO

H

R3

NH C CO

H

R2

NH3＋

C COOー

H＋

R1

NH3＋

C COOー

H＋

H2OH2O

Peptide bond

Peptide bond

The amino acid sequence is called

as primary structure A AF

NGG

S TS

DK

A carboxylic acid condenses with an amino group with the release of a water

27- 112

Classical StrategyClassical Strategy

1.1. Determine what amino acids are present and Determine what amino acids are present and their molar ratios.their molar ratios.

2. 2. Cleave the peptide into smaller fragments, and Cleave the peptide into smaller fragments, and determine the amino acid composition of these determine the amino acid composition of these smaller fragments.smaller fragments.

3. 3. Identify the N-terminus and C-terminus in the Identify the N-terminus and C-terminus in the parent peptide and in each fragment.parent peptide and in each fragment.

4.4. Organize the information so that the sequences Organize the information so that the sequences of small fragments can be overlapped to reveal of small fragments can be overlapped to reveal the full sequence.the full sequence.

27- 113

Amino Acid AnalysisAmino Acid Analysis

27- 114

Amino Acid AnalysisAmino Acid Analysis

Acid-hydrolysis of the peptide (6 M HCl, 24 hr) Acid-hydrolysis of the peptide (6 M HCl, 24 hr) gives a mixture of amino acids.gives a mixture of amino acids.

The mixture is separated by ion-exchange The mixture is separated by ion-exchange chromatography, which depends on the chromatography, which depends on the differences in pI among the various amino differences in pI among the various amino acids.acids.

Amino acids are detected using ninhydrin.Amino acids are detected using ninhydrin.

Automated method; requires only 10Automated method; requires only 10-5-5 to 10 to 10-7 -7 g g of peptide.of peptide.

27- 115

Partial Hydrolysis of ProteinsPartial Hydrolysis of Proteins

27- 116

Partial Hydrolysis of Peptides and ProteinsPartial Hydrolysis of Peptides and Proteins

Acid-hydrolysis of the peptide cleaves all of the Acid-hydrolysis of the peptide cleaves all of the peptide bonds.peptide bonds.

Cleaving some, but not all, of the peptide bonds Cleaving some, but not all, of the peptide bonds gives smaller fragments.gives smaller fragments.

These smaller fragments are then separated These smaller fragments are then separated and the amino acids present in each fragment and the amino acids present in each fragment determined.determined.

Enzyme-catalyzed cleavage is the preferred Enzyme-catalyzed cleavage is the preferred method for partial hydrolysis.method for partial hydrolysis.

27- 117

Partial Hydrolysis of Peptides and ProteinsPartial Hydrolysis of Peptides and Proteins

The enzymes that catalyze the hydrolysis of The enzymes that catalyze the hydrolysis of peptide bonds are called peptide bonds are called peptidasespeptidases, , proteasesproteases, , or or proteolyticproteolytic enzymesenzymes..

27- 118

R2 Pro

R1 Pro

ProteasesProteasesProteasesProteases

*

*

*

*

*

27- 119

TrypsinTrypsin

Trypsin is selective for cleaving the peptide bond Trypsin is selective for cleaving the peptide bond to the carboxyl group of lysine or arginine.to the carboxyl group of lysine or arginine.

NNHCHCHCHC

OO

R'R'

NNHCHCHCHC

OO

R"R"

NNHCHCHCHC

OO

RR

lysine or argininelysine or arginine

27- 120

ChymotrypsinChymotrypsin

Chymotrypsin is selective for cleaving the peptideChymotrypsin is selective for cleaving the peptidebond to the carboxyl group of amino acids withbond to the carboxyl group of amino acids withan aromatic side chain.an aromatic side chain.

NNHCHCHCHC

OO

R'R'

NNHCHCHCHC

OO

R"R"

NNHCHCHCHC

OO

RR

phenylalanine, tyrosine, tryptophanphenylalanine, tyrosine, tryptophan

27- 121

CarboxypeptidaseCarboxypeptidase

proteinproteinHH33NNCHCCHC

OO

RR

++NNHCHCOHCHCO

OO

RR

––CC

OO

Carboxypeptidase is selective for cleavingCarboxypeptidase is selective for cleavingthe peptide bond to the C-terminal amino acid.the peptide bond to the C-terminal amino acid.

27- 122

End Group AnalysisEnd Group Analysis

27- 123

End Group AnalysisEnd Group Analysis

Amino sequence is ambiguous unless we know Amino sequence is ambiguous unless we know whether to read it left-to-right or right-to-left.whether to read it left-to-right or right-to-left.

We need to know what the N-terminal and C-We need to know what the N-terminal and C-terminal amino acids are.terminal amino acids are.

The C-terminal amino acid can be determined The C-terminal amino acid can be determined by carboxypeptidase-catalyzed hydrolysis.by carboxypeptidase-catalyzed hydrolysis.

Several chemical methods have been Several chemical methods have been developed for identifying the N-terminus. They developed for identifying the N-terminus. They depend on the fact that the amino N at the depend on the fact that the amino N at the terminus is more nucleophilic than any of the terminus is more nucleophilic than any of the amide nitrogens.amide nitrogens.

27- 124

Sanger's MethodSanger's Method

The key reagent in Sanger's method for The key reagent in Sanger's method for identifying the N-terminus is 1-fluoro-2,4-identifying the N-terminus is 1-fluoro-2,4-dinitrobenzene.dinitrobenzene.

1-Fluoro-2,4-dinitrobenzene is very reactive 1-Fluoro-2,4-dinitrobenzene is very reactive toward nucleophilic aromatic substitution toward nucleophilic aromatic substitution (Section 23.5).(Section 23.5).

FFOO22NN

NONO22

27- 125


1-Fluoro-2,4-dinitrobenzene reacts with the 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid.amino nitrogen of the N-terminal amino acid.

FFOO22NN

NONO22

NNHCHHCH22CC NNHCHCOHCHCO

CHCH33

NNHCHCHCHC

CHCH22CC66HH55

HH22NNCHCCHC

OO OOOOOO

CH(CHCH(CH33))22

––++

OO22NN

NONO22


CHCH33

NNHCHCHCHC

CHCH22CC66HH55

NNHCHCHCHC

OO OOOOOO

CH(CHCH(CH33))22

––

27- 126


Acid hydrolysis cleaves all of the peptide bonds Acid hydrolysis cleaves all of the peptide bonds leaving a mixture of amino acids, only one of leaving a mixture of amino acids, only one of which (the N-terminus) bears a 2,4-DNP group.which (the N-terminus) bears a 2,4-DNP group.

OO22NN

NONO22


CHCH33

NNHCHCHCHC

CHCH22CC66HH55

NNHCHCHCHC

OO OOOOOO

CH(CHCH(CH33))22

––

HH33OO++

OO

OO22NN

NONO22

NNHCHCOHHCHCOH

CH(CHCH(CH33))22

HH33NNCHCOCHCO––

CHCH33

++HH33NNCHCH22COCO––

OO OO

++

OO

HH33NNCHCOCHCO––

CHCH22CC66HH55

++++ ++

++

27- 127

InsulinInsulin

27- 128

InsulinInsulin

Insulin is a polypeptide with 51 amino acids.Insulin is a polypeptide with 51 amino acids.

It has two chains, called the A chain (21 amino It has two chains, called the A chain (21 amino acids) and the B chain (30 amino acids).acids) and the B chain (30 amino acids).

The following describes how the amino acid The following describes how the amino acid sequence of the B chain was determined.sequence of the B chain was determined.

27- 129

Primary Structure of Bovine InsulinPrimary Structure of Bovine Insulin

N terminus N terminus of of A chainA chain

N terminus N terminus of B chainof B chain

C terminus C terminus of of B chainB chain

C terminus C terminus of of A chainA chain55

55

1515

1010

1515

2020

2020

25253030

SS SS

1010

SSSS

SSSSF

F F

F

V N Q H L

C C

C

C

C

VV

VVG

G

G

S

S

S

H L L

L

G A

A

AC

L Y

Y

E

E L E

R

Y

YI Q

K P T

QN

N

27- 130

The B Chain of Bovine InsulinThe B Chain of Bovine Insulin

Phenylalanine (F) is the N terminus.Phenylalanine (F) is the N terminus.

Pepsin-catalyzed hydrolysis gave the four peptides:Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHLFVNQHLCGSHL VGAL VGAL VCGERGF VCGERGF YTPKA YTPKA

27- 131


FVNQHLCGSHLFVNQHLCGSHL

VGALVGAL

VCGERGFVCGERGF

YTPKAYTPKA

27- 132



Pepsin-catalyzed hydrolysis gave the four peptides:Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL FVNQHLCGSHL VGAL VGAL VCGERGF VCGERGF YTPKA YTPKA

Overlaps between the above peptide sequences were Overlaps between the above peptide sequences were found in four additional peptides:found in four additional peptides:

SHLVSHLVLVGALVGAALTALTTLVCTLVC

27- 133



SHLVSHLVLVGALVGA

VGALVGAL

ALTALT

TLVCTLVCVCGERGFVCGERGF

YTPKAYTPKA

27- 134



Pepsin-catalyzed hydrolysis gave the four peptides:Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL FVNQHLCGSHL VGAL VGAL VCGERGF VCGERGF YTPKA YTPKA

Overlaps between the above peptide sequences were Overlaps between the above peptide sequences were found in four additional peptides:found in four additional peptides:

SHLVSHLVLVGALVGAALTALTTLVCTLVC

Trypsin-catalyzed hydrolysis gave GFFYTPK which Trypsin-catalyzed hydrolysis gave GFFYTPK which completes the sequence.completes the sequence.

27- 135



SHLVSHLVLVGALVGA

VGALVGAL

ALTALT


GFFYTPKGFFYTPK

YTPKAYTPKA

27- 136



SHLVSHLVLVGALVGA

VGALVGAL

ALTALT


GFFYTPKGFFYTPK

YTPKAYTPKA

FVNQHLCGSHLVGALTLVCGERGFFYTPKAFVNQHLCGSHLVGALTLVCGERGFFYTPKA

27- 137

InsulinInsulin

The sequence of the A chain was determined The sequence of the A chain was determined using the same strategy.using the same strategy.

Establishing the disulfide links between cysteine Establishing the disulfide links between cysteine residues completed the primary structure.residues completed the primary structure.

27- 138

The Edman Degradation and The Edman Degradation and Automated Sequencing of Automated Sequencing of

PeptidesPeptides

27- 139

Edman DegradationEdman Degradation

1. 1. Method for determining N-terminal amino Method for determining N-terminal amino acid.acid.

2.2. Can be done sequentially one residue at a Can be done sequentially one residue at a time on the same sample. Usually one can time on the same sample. Usually one can determine the first 20 or so amino acids from determine the first 20 or so amino acids from the N-terminus by this method.the N-terminus by this method.

3. 103. 10-10 -10 g of sample is sufficient.g of sample is sufficient.

4. Has been automated.4. Has been automated.

27- 140


The key reagent in the Edman degradation is The key reagent in the Edman degradation is phenyl isothiocyanate.phenyl isothiocyanate.

NN CC SS

27- 141


Phenyl isothiocyanate reacts with the amino Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid.nitrogen of the N-terminal amino acid.

peptidepeptideHH33NNCHCCHC

OO

RR

++NNHHCC66HH55NN CC SS ++

27- 142


peptidepeptideHH33NNCHCCHC

OO

RR

++NNHHCC66HH55NN CC SS ++

peptidepeptideCC66HH55NNHCHCNNHHCHCCHC

OO

RR

NNHH

SS

27- 143



OO

RR

NNHH

SS

The product is a phenylthiocarbamoyl (PTC)The product is a phenylthiocarbamoyl (PTC)derivative.derivative.

The PTC derivative is then treated with HCl in The PTC derivative is then treated with HCl in an anhydrous solvent. The N-terminal amino an anhydrous solvent. The N-terminal amino acid is cleaved from the remainder of the acid is cleaved from the remainder of the peptide.peptide.

27- 144



OO

RR

NNHH

SS

HClHCl

peptidepeptideHH33NN++

++CC66HH55NNHH CC

SSCC

NN CHCH

RR

OO

27- 145


CC66HH55NNHH CCSS

CC

NN CHCH

RR

OO

The product is a thiazolone. Under theThe product is a thiazolone. Under theconditions of its formation, the thiazoloneconditions of its formation, the thiazolonerearranges to a phenylthiohydantoin (PTH)rearranges to a phenylthiohydantoin (PTH)derivative.derivative.


++

27- 146


CC66HH55NNHH CCSS

CC

NN CHCH

RR

OO

CCCCNN

HNHN CHCH

RR

OOSS

CC66HH55The PTH derivative is The PTH derivative is isolated and identified. isolated and identified. The remainder of the The remainder of the peptide is subjected to peptide is subjected to a second Edman a second Edman degradation.degradation.


++

27- 147

Secondary StructuresSecondary Structuresof Peptides and Proteinsof Peptides and Proteins

27- 148

Levels of Protein StructureLevels of Protein Structure

Primary structure = the amino acid sequence Primary structure = the amino acid sequence plus disulfide linksplus disulfide links

Secondary structure = conformational Secondary structure = conformational relationship between nearest neighbor amino relationship between nearest neighbor amino acidsacids

helixhelixpleated pleated sheet sheet

27- 149

Levels of Protein StructureLevels of Protein Structure

planar geometry of peptide bondplanar geometry of peptide bondanti conformation of main chainanti conformation of main chainhydrogen bonds between N—H and O=Chydrogen bonds between N—H and O=C

The The -helix and pleated -helix and pleated sheet are both sheet are both characterized by:characterized by:

27- 150

-helixes-helixes-helixes-helixes

Intra-chain

H-bonds

Secondary Structure

27- 151

HelixHelix

Shown is an Shown is an helix of a protein helix of a protein in which all of the amino acids in which all of the amino acids are are LL-alanine.-alanine.

Helix is right-handed with 3.6 Helix is right-handed with 3.6 amino acids per turn.amino acids per turn.

Hydrogen bonds are within a Hydrogen bonds are within a single chain.single chain.

Protein of muscle (myosin) and Protein of muscle (myosin) and wool (wool (-keratin) contain large -keratin) contain large regions of regions of -helix. Chain can -helix. Chain can be stretched.be stretched.

27- 152

-strands-strands-strands-strands

Inter-chain

H-bonds

Secondary Structure

27- 153

Two Types of -Pleated Sheets

27- 154

Pleated Pleated Sheet Sheet

Shown is a Shown is a sheet of protein chains composed of sheet of protein chains composed of alternating glycine and alanine residues.alternating glycine and alanine residues.

Adjacent chains are antiparallel.Adjacent chains are antiparallel.

Hydrogen bonds between chains.Hydrogen bonds between chains.

van der Waals forces produce pleated effect.van der Waals forces produce pleated effect.

27- 155

Tertiary StructureTertiary Structureof Peptides and Proteinsof Peptides and Proteins

27- 156

Tertiary StructureTertiary Structure

Specific compact structure for one entire polypeptide Chain stabilizing primarily through weak bonds

27- 157

Tertiary StructureTertiary Structure

Refers to overall shape (how the chain is folded)Refers to overall shape (how the chain is folded)

Fibrous proteins (hair, tendons, wool) have Fibrous proteins (hair, tendons, wool) have elongated shapeselongated shapes

Globular proteins are approximately sphericalGlobular proteins are approximately spherical

most enzymes are globular proteinsmost enzymes are globular proteins

an example is carboxypeptidasean example is carboxypeptidase

27- 158

Tertiary Structure

This is the 3D structure resulting from further regular folding of the polypeptide chains using H-bonding, Van der Waals, disulfide bonds and electrostatic forces – Often detected by X-ray crystallographic methods

Globular Proteins – “Spherical Shape” , include Insulin, Hemoglobin, Enzymes, Antibodies---polar hydrophilic groups are aimed outwards towards water, whereas non-polar “greasy” hydrophobic hydrocarbon portions cluster inside the molecule, so protecting them from the hostile aqueous environment ----- Soluble Proteins

Fibrous Proteins – “Long thin fibres” , include Hair, wool, skin, nails – less folded ----- e.g. keratin - the -helix strands are wound into a “superhelix”. The superhelix makes one complete turn for each 35 turns of the -helix.

27- 159

In globular proteins this tertiary structure or macromolecular shape determines biological propertiesBays or pockets in proteins are called Active SitesEnzymes are Stereospecific and possess Geometric Specificity

Emil Fischer formulated the lock-and-key mechanism for enzymes

The range of compounds that an enzyme excepts varies from a particular functional group to a

specific compound

All reactions which occur in living cells are mediated by enzymes and are catalysed by 106-108

Some enzymes may require the presence of a Cofactor.This may be a metal atom, which is essential for its redox activity.Others may require the presence of an organic molecule, such as NAD+, called a Coenzyme.If the Cofactor is permanently bound to the enzyme, it is called a Prosthetic Group.

27- 160

For a protein composed of a single polypeptide molecule, tertiary structure is the highest level of structure that is attained

Myoglobin and hemoglobin were the first proteins to be successfully subjected to completely successful X-rays analysis by J. C. Kendrew and Max Perutz (Nobel Prize for Chemistry 1962)

Quaternary Structure

When multiple sub-units are held together in aggregates by Van der Waals and electrostatic forces (not covalent bonds)Hemoglobin is tetrameric myglobin

For example, Hemoglobin has four heme units, the protein globin surrounds the heme – Takes the shape of a giant tetrahedron – Two identical and globins.The and chains are very similar but distinguishable in both primary structure and folding

27- 161

TertiaryTertiarystructurestructureTertiaryTertiarystructurestructure

Hb monomer (or myoglobin)

Hb 22 tetramer

QuaternaryQuaternarystructurestructure

27- 162


Carboxypeptidase is an enzyme that catalyzes Carboxypeptidase is an enzyme that catalyzes the hydrolysis of proteins at their C-terminus.the hydrolysis of proteins at their C-terminus.

It is a metalloenzyme containing ZnIt is a metalloenzyme containing Zn2+2+ at its at its active site.active site.

An amino acid with a positively charged side An amino acid with a positively charged side chain (Arg-145) is near the active site.chain (Arg-145) is near the active site.

27- 163


Disulfide bondDisulfide bond


C-terminusC-terminus

Zn2+

Arg-145

tube modeltube model ribbon modelribbon model

27- 164

MyoglobinMyoglobin


C-terminusC-terminus Heme

Heme is the coenzyme that binds oxygen in myoglobin Heme is the coenzyme that binds oxygen in myoglobin (oxygen storage in muscles) and hemoglobin (oxygen (oxygen storage in muscles) and hemoglobin (oxygen transport).transport).

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Protein Quaternary Structure:Protein Quaternary Structure:HemoglobinHemoglobin

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Protein Quaternary StructureProtein Quaternary Structure

Some proteins are assemblies of two or more Some proteins are assemblies of two or more chains. The way in which these chains are chains. The way in which these chains are organized is called the quaternary structure.organized is called the quaternary structure.

Hemoglobin, for example, consists of 4 Hemoglobin, for example, consists of 4 subunits.subunits.

There are 2 There are 2 chains (identical) and 2 chains (identical) and 2 chains chains (also identical).(also identical).

Each subunit contains one heme and each Each subunit contains one heme and each protein is about the size of myoglobin. protein is about the size of myoglobin.

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PrimaryPrimary structure is the amino acid sequence. structure is the amino acid sequence.

Protein StructureProtein StructureProtein StructureProtein Structure

Tertiary Tertiary structure is the 3-dimensional folding of the secondary structure is the 3-dimensional folding of the secondary structural elements and connecting loops in space.structural elements and connecting loops in space.

SecondarySecondary structure is how the amino acids in sequence fold up structure is how the amino acids in sequence fold up

locally. Examples are locally. Examples are -helixes and -helixes and -strands and loops.-strands and loops.

Quaternary Quaternary structure is the association of multiple subunits, structure is the association of multiple subunits, each with a tertiary structure and each a unique gene product.each with a tertiary structure and each a unique gene product.

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Electrostatic interactions Electrostatic interactions involve the interaction of (+) and (-) involve the interaction of (+) and (-) charged side groups.charged side groups.

Stabilization of Protein StructureStabilization of Protein StructureStabilization of Protein StructureStabilization of Protein Structure

Van der Waal’s forcesVan der Waal’s forces are weak forces based on optimal are weak forces based on optimal overlap of adjacent electronic orbitals. Can be repulsive. overlap of adjacent electronic orbitals. Can be repulsive.

Hydrogen bonds Hydrogen bonds involve sharing of a hydrogen atom between involve sharing of a hydrogen atom between

two eletronegative atoms (e.g., O, N).two eletronegative atoms (e.g., O, N).

Hydrophobic interactions Hydrophobic interactions are, by far, the most powerful force are, by far, the most powerful force stabilizing protein structure. Basis of force is entropy gain stabilizing protein structure. Basis of force is entropy gain realized by burying hydrophobic residues.realized by burying hydrophobic residues.

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CofactorsCofactors are exogenous molecules that associate with are exogenous molecules that associate with proteins to yield full activity. In the absence of cofactor, proteins to yield full activity. In the absence of cofactor, protein is an protein is an apoproteinapoprotein..

CofactorsCofactorsCofactorsCofactors

Prosthetic groupsProsthetic groups are covalently attached to the protein. are covalently attached to the protein. Examples are heme, in hemoglobin, and riboflavin, in Examples are heme, in hemoglobin, and riboflavin, in flavoproteins.flavoproteins.

Co-enzymesCo-enzymes are soluble and associate transiently with enzyme are soluble and associate transiently with enzyme during catalytic cycle. An example is vitamin K in activation of during catalytic cycle. An example is vitamin K in activation of

blood clotting enzymes.blood clotting enzymes.

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Denaturation agentsDenaturation agentsHeat: Break apart hydrogen bonds and disrupt hydrophobic attractions Heat: Break apart hydrogen bonds and disrupt hydrophobic attractions between nonpolar side groups.between nonpolar side groups.Acids/Bases: Break hydrogen bonds between polar R groups and Acids/Bases: Break hydrogen bonds between polar R groups and disrupt the ionic bonds (salt bridges).disrupt the ionic bonds (salt bridges).Organic Compounds: Ethanol and isopropanol act as disinfectants by Organic Compounds: Ethanol and isopropanol act as disinfectants by forming their own hydrogen bonds with a protein and disrupting the forming their own hydrogen bonds with a protein and disrupting the hydrophobic interactions.hydrophobic interactions.Heavy metal ions: Heavy metal ions like AgHeavy metal ions: Heavy metal ions like Ag++, Pb, Pb2+2+ and Hg and Hg2+2+ React with React with S-S bonds to form solids.S-S bonds to form solids.Agitation: Stretches chains until bonds breakAgitation: Stretches chains until bonds break

Protein Denaturation

Definition: Disruption of any of the bonds that stabilize the secondary, tertiary or quaternary structure. However, the covalent amide bonds of the primary structure are not affected.

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Different Classification of ProteinsDifferent Classification of ProteinsDifferent Classification of ProteinsDifferent Classification of Proteins

On the basis of:On the basis of:

Shape:Shape:

GlobularGlobular

FibrilarFibrilar

Homo or heteroHomo or hetero

Function Function

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Immunoglobulin StructureImmunoglobulin StructureImmunoglobulin StructureImmunoglobulin Structure

Heavy & Light Heavy & Light ChainsChains

Disulfide bondsDisulfide bonds

Inter-chainInter-chain

Intra-chainIntra-chain

CH1

VL

CL

VH

CH2 CH3

Hinge Region

Carbohydrate

Disulfide bond

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Globular ProteinsGlobular Proteins

Some design principles Some design principles

Globular proteins fold so as to "bury" the hydrophobic side Globular proteins fold so as to "bury" the hydrophobic side chains, minimizing their contact with waterchains, minimizing their contact with water Most polar residues face the outside of the protein and interact Most polar residues face the outside of the protein and interact with solvent with solvent Most hydrophobic residues face the interior of the protein and Most hydrophobic residues face the interior of the protein and interact with each other interact with each other Packing of residues is close, but protein interiors contain some Packing of residues is close, but protein interiors contain some empty space empty space The empty space is in the form of small cavitiesThe empty space is in the form of small cavities

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Globular ProteinsGlobular Proteins

More design principlesMore design principles "Random coil" is not random "Random coil" is not random Structures of globular proteins are not static Structures of globular proteins are not static Various elements and domains of protein move to different Various elements and domains of protein move to different degrees degrees Some segments of proteins are very flexible and Some segments of proteins are very flexible and disordered.disordered.Myoglobin and hemoglobin are typical examples of globular Myoglobin and hemoglobin are typical examples of globular proteins.proteins.Both are heme-containing proteins and each is involved in Both are heme-containing proteins and each is involved in oxygen metabolism.oxygen metabolism.

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ObjectivesObjectivesObjectivesObjectives

Diagram and describe the effect of oxygen on the Diagram and describe the effect of oxygen on the position of ironposition of iron relative to the relative to the heme plane.heme plane.

Describe how Describe how cooperativecooperative binding of oxygen by hemoglobin improves its binding of oxygen by hemoglobin improves its effectiveness as an oxygen carrier.effectiveness as an oxygen carrier.

Describe the relationship between Hb Describe the relationship between Hb structurestructure to the to the Bohr effectBohr effect and explain its and explain its physiological significance..physiological significance..

Discuss how Discuss how carbon dioxidecarbon dioxide affects the affinity of Hb for oxygen and why this is affects the affinity of Hb for oxygen and why this is physiologically significant.physiologically significant.

Explain the effect of Explain the effect of bisphosphoglyceratebisphosphoglycerate (BPG) on the affinity of Hb for oxygen (BPG) on the affinity of Hb for oxygen and how this is related to and how this is related to altitudealtitude and and HbFHbF..

Explain how Explain how carbon monoxidecarbon monoxide (CO) binds to Hb and its affinity relative to that of (CO) binds to Hb and its affinity relative to that of oxygen..oxygen..

Describe the molecular basis of Describe the molecular basis of thalassemiasthalassemias and the aberrant Hb that are and the aberrant Hb that are produced in these diseases..produced in these diseases..

List three List three embryonic formsembryonic forms of Hb.. of Hb..

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Myoglobin: 2Myoglobin: 2oo and 3 and 3oo aspects aspectsMyoglobin: 2Myoglobin: 2oo and 3 and 3oo aspects aspects

Myoglobin is a single peptide chain of 153 residues Myoglobin is a single peptide chain of 153 residues

arranged in eight arranged in eight -helical regions labeled A-H.-helical regions labeled A-H.

The heme cofactor is the oxygen binding site so it is The heme cofactor is the oxygen binding site so it is necessary for myoglobin’s function, oxygen storage in necessary for myoglobin’s function, oxygen storage in mammalian muscle tissue.mammalian muscle tissue.

His E7 and F8 are important for binding the heme group His E7 and F8 are important for binding the heme group within the protein and for stabilizing bound oxygen.within the protein and for stabilizing bound oxygen.

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Myoglobin and HemoglobinMyoglobin and HemoglobinMyoglobin and HemoglobinMyoglobin and Hemoglobin

Mb is monomer, Hb is a tetramer (Mb is monomer, Hb is a tetramer (2222).).Hb subunits are structurally similar to Mb, with 8 Hb subunits are structurally similar to Mb, with 8 --helical regions, no helical regions, no -strands and no water.-strands and no water.Both contain heme prosthetic group Both contain heme prosthetic group Both Mb and Hb contain proximal and distal Both Mb and Hb contain proximal and distal histidines.histidines.Affinity of Mb for oxygen is high, affinity of Hb for Affinity of Mb for oxygen is high, affinity of Hb for oxygen is low.oxygen is low.

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Myoglobin &HemoglobinMyoglobin &HemoglobinMyoglobin &HemoglobinMyoglobin &Hemoglobin

Two related protein for OTwo related protein for O2 2 transportation.transportation.

Mb has one chainMb has one chain

Hb has four chainsHb has four chains

Each chain has two parts: a globin ( protein) and Each chain has two parts: a globin ( protein) and a heme ( non-protein)a heme ( non-protein)

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MyoglobinMyoglobinMyoglobinMyoglobin

• An O2 transport protein in muscle

• A Globin( globular soluble protein), 151 residues that contains 8 -helices (A,B,C,…..H)

•Contains a heme

•prosthetic group

Binds heme in hydrophobic Binds heme in hydrophobic pocket. pocket.

Polar groups exposed to Polar groups exposed to solvent, Non-polar groups solvent, Non-polar groups buriedburied..

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Myoglobin: 2o and 3o structure

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The Heme Prosthetic GroupThe Heme Prosthetic GroupThe Heme Prosthetic GroupThe Heme Prosthetic Group

• Protoporphyrin with Fe(II)

• Covalent attachment of Fe via His F8 side chain

• Additional stabilization via hydrophobic interaction

• Fe(II) state is active, Fe(III) [oxidized]

• Fe(II) atom in heme binds O2

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Binding of OBinding of O22 to Heme to HemeBinding of OBinding of O22 to Heme to Heme

• Binding of O2 to a free heme group is irreversible ( heme- heme sandwich)

• Enclosure in a protein( globin) allows reversible binding

O2 has only limited solubility (1 X 10-4 M) in water

Solubility problem overcome by binding to proteins

• Binding of O2 alters heme structure

Bright scarlet color of blood in arteries

Dark purple color of blood in veins

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The Heme GroupThe Heme GroupThe Heme GroupThe Heme Group

NN

NN

CH2CH2COO-

CH2CH2-OOC

CH3 CH3

CH

CH2

CH3

CH3 CH CH2

Fe(II)Pyrrole ring

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Oxygen binds to 6Oxygen binds to 6thth coordination site on coordination site on heme ironheme iron

N of His F8 binds to 5th coordination site on heme iron

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His E7 acts as a gate to favor oxygen binding over carbon monoxide.

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HemoglobinHemoglobinHemoglobinHemoglobin

A tetrameric proteinA tetrameric protein

two two -chains (141 AA)-chains (141 AA)

two two -chains (146 AA)-chains (146 AA)

four heme cofactors, one in each chainfour heme cofactors, one in each chain

The The and and chains are homologous to myoglobin. chains are homologous to myoglobin.

Oxygen binds to heme in hemoglobin with same Oxygen binds to heme in hemoglobin with same structure as in Mb but cooperatively: as one Ostructure as in Mb but cooperatively: as one O22 is is

bound, it becomes easier for the next to bind.bound, it becomes easier for the next to bind.

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HemoglobinHemoglobinHemoglobinHemoglobin

• Ubiquitous O2 transport protein

• A globular soluble protein, 2X2 chains (164 kDa)

• and chains 44% identical

• All helical secondary structure (like myoglobin)

• quaternary structure-subunit 141 residues-subunit 146 residues

• Extensive contacts between subunits Mix of hydrophobic, H-bond, and ionic

interactions 11 (22)- 35 residues, 12 (21)- 19 residues

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Hemoglobin: The Oxygen Transporter

OXYHEMOGLOBIN DEOXYHEMOGLOBIN

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Each chain is in Each chain is in ribbon form.ribbon form.

The heme The heme groups are in groups are in space filling space filling formform

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Oxygen Binding CurvesOxygen Binding CurvesOxygen Binding CurvesOxygen Binding Curves

Hemoglobin and myoglobin respond differently Hemoglobin and myoglobin respond differently to increase in Oto increase in O22 concentration. concentration.

Myoglobin shows normal saturation behavior Myoglobin shows normal saturation behavior while hemoglobin shows cooperative behavior. while hemoglobin shows cooperative behavior. Each oxygen added to a heme of Hb makes Each oxygen added to a heme of Hb makes addition of the next one easier. addition of the next one easier.

The myoglobin curve is hyperbolic.The myoglobin curve is hyperbolic.

The hemoglobin curve is sigmoidal. The hemoglobin curve is sigmoidal.

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Hemoglobin O2 Binding Curve

Binding curve is sigmoidal Artery: high pO2, loading of protein Vein: lower pO2, unloading from protein P50(hemoglobin) = 26 torr, adjusts as needed!!

*Drastic change in pO2 over physiological range*

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Oxygen Binding Curves-2Oxygen Binding Curves-2Oxygen Binding Curves-2Oxygen Binding Curves-2

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Hemoglobin EquilibriumHemoglobin EquilibriumHemoglobin EquilibriumHemoglobin Equilibrium

T(low affinity)

R (high affinity)

O2

H+,CO2,BPG

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A Quaternary Structure A Quaternary Structure Change Change

One alpha-beta pair moves One alpha-beta pair moves relative to the other by 15 relative to the other by 15 degrees upon oxygen binding degrees upon oxygen binding

This large change is caused by This large change is caused by movement of Fe by only 0.039 movement of Fe by only 0.039

nm when oxygen bindsnm when oxygen binds

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Oxygen binding by hemoglobinOxygen binding by hemoglobinOxygen binding by hemoglobinOxygen binding by hemoglobin

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Allosteric EffectorsAllosteric EffectorsAllosteric EffectorsAllosteric Effectors

• The R or T state can be stabilized by the binding of ligands other than O2.

1. H+. Lower pH favors the T state which causes Hb to release bound O2. This is known as the Bohr Effect.

2. CO2. Release of CO2 lowers pH via conversion to

HCO3-: CO2 + H2O HCO3

- + H+. Reinforces Bohr Effect

3. Bisphosphoglycerate (BPG). Regulation of activity via binding more strongly to T state, helps to release O2.

Increase in levels of BPG helps adaptation to high altitude- faster than making more hemoglobin. Also important in hypoxia diseases (e.g. anemia)

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The Bohr EffectThe Bohr Effect

Competition between oxygen and HCompetition between oxygen and H++

Discovered by Christian Bohr Discovered by Christian Bohr

Binding of protons diminishes oxygen binding Binding of protons diminishes oxygen binding

Binding of oxygen diminishes proton binding Binding of oxygen diminishes proton binding

Important physiological significance-OImportant physiological significance-O22 saturation saturation

of Hb responds to pH of Hb responds to pH

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The Bohr EffectThe Bohr Effect

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Bohr Effect IIBohr Effect II

Carbon dioxide diminishes oxygen binding Carbon dioxide diminishes oxygen binding

COCO22 produced in metabolically active tissue produced in metabolically active tissue

(requires oxygen)(requires oxygen)

Hydration of COHydration of CO22 in tissues and extremities leads to in tissues and extremities leads to

proton production proton production

COCO22 + H + H22O O HCO HCO33-- + H + H++

These protons are taken up by Hb forcing more These protons are taken up by Hb forcing more oxygen to dissociate oxygen to dissociate

The reverse occurs in the lungsThe reverse occurs in the lungs

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Carbon Monoxide PoisoningCarbon Monoxide PoisoningCarbon Monoxide PoisoningCarbon Monoxide Poisoning

• Heme Fe(II) binds many other small molecules with structures similar to O2 including: CO, NO, H2S

• O2 is actually binds to these other molecules, particularly CO.

• When exposed to CO, even at low concentrations, O2 transport proteins will be filled with CO limiting their vital O2 capacity.

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2,3-Bisphosphoglycerate2,3-Bisphosphoglycerate

An Allosteric Effector of HemoglobinAn Allosteric Effector of Hemoglobin

The sigmoid binding curve is only observed in the presence The sigmoid binding curve is only observed in the presence of 2,3-BPG of 2,3-BPG

Since 2,3-BPG binds at a site distant from the Fe where Since 2,3-BPG binds at a site distant from the Fe where oxygen binds, it is called an allosteric effectoroxygen binds, it is called an allosteric effector

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2,3-BPG

2,3-bisphosphoglycerate (2,3-BPG) is a negative allosteric effector of O2 binding to Hb - binds tighter to deoxyHb

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F e

CNO

===

P roxim alH is (F8)

D ista lH is (E 7)

Heme in hemoglobinHeme in hemoglobinHeme in hemoglobinHeme in hemoglobin

Side view of Hb tetramer

F e

CNO

===

P roxim alH is (F8)

D ista lH is (E 7)

Heme prosthetic group

F e

CNO

===

P roxim alH is (F8)

D ista lH is (E 7)

F e

CNO

===

P roxim alH is (F8)

D ista lH is (E 7)

F e

CNO

===

P roxim alH is (F8)

D ista lH is (E 7)

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Binding of oxygen to heme ironBinding of oxygen to heme ironBinding of oxygen to heme ironBinding of oxygen to heme iron

F e

CNO

===

P roxim alH is (F8)

D ista lH is (E 7)

e -

Ferrous is reduced and +2 charge

Ferric is oxidized and +3 charge

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Effect of oxygen on heme ironEffect of oxygen on heme ironEffect of oxygen on heme ironEffect of oxygen on heme iron

CNO

===

Plane of hem e

Dista lHis (E7)

Fe

Proxima lHis (F8)

F7

F6

FG 1

FG 2FG 3

NC

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Fe

Proxima lHis (F8)

F7

F6

FG 1

FG 2FG 3

NC

Plane of hem e

CNO

===

Dista lHis (E7)

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CNO

===

Plane of hem e

Dista lHis (E7)

Fe

Proxima lHis (F8)

F7

F6

FG 1

FG 2FG 3

NC

Fe

Proxima lHis (F8)

F7

F6

FG 1

FG 2FG 3

NC

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CooperativityCooperativityCooperativityCooperativity

Oxygen binding to one subunit of Hb, increases the affinity of the other subunits for additional oxygens. In other words, the first one is the hardest, the rest are easy.

Example: square of postage stamps.

Book of four stamps. To pull first stamp, you have to break two edges.

To pull second stamp, you have to break only one edge.

To pull third stamp, you have to break only one edge.

To pull fourth stamp, you don’t have to break any edges.

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CooperativityCooperativityCooperativityCooperativity

BPGEffect

pO2 (mm Hg)

0 20 40 60 80 100 120 140 160

0

20

40

60

80

100

pO2 vs p50=8pO2 vs p50=26

Hb alone

Hb + BPG

Mb

Hb

Sigmoid shape indicates positive

cooperativity

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Bohr EffectBohr EffectBohr EffectBohr Effect

Bohr Effect

pO2 (mm Hg)

0 20 40 60 80 100 120 140 160

0

20

40

60

80

100

7.6

7.2

O2 level in venous blood

O2 level in arterial blood

7.4

7.0

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Hb structural familiesHb structural familiesHb structural familiesHb structural families

Beta familyBeta family - - - found in adult hemoglobin HbA- found in adult hemoglobin HbA11.. - found in adult hemoglobin HbA- found in adult hemoglobin HbA22. . - found in fetal hemoglobin HbF.- found in fetal hemoglobin HbF. - found in embryonic hemoglobin Hb Gower 1 and - found in embryonic hemoglobin Hb Gower 1 and Hb Hb

Gower 2Gower 2

Alpha familyAlpha family - - - found in adult hemoglobins HbA- found in adult hemoglobins HbA11, HbA, HbA22..

- found in embryonic hemoglobins Hb Gower 1 - found in embryonic hemoglobins Hb Gower 1 and and Hb Portland.Hb Portland.

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COCO22 effect effectCOCO22 effect effect

CO2 Effect

pO2 (mm Hg)

0 20 40 60 80 100 120 140 160

0

20

40

60

80

100

pO2 vs p50=20pO2 vs p50=40

pCO2 20 mm

pCO2 80 mm

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Effect of BPGEffect of BPGEffect of BPGEffect of BPG

BPGEffect

pO2 (mm Hg)

0 20 40 60 80 100 120 140 160

0

20

40

60

80

100

pO2 vs p50=8pO2 vs p50=26

Hb alone

Hb + BPG High altitude increases BPG, pushing curve further to right

BPG is responsible for cooperativity.

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Side view (R)

Side view (T)

BPG

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His+His+Lys+

His+

Lys+

His+--- -BPG


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Hemoglobin EquilibriumHemoglobin EquilibriumHemoglobin EquilibriumHemoglobin Equilibrium

T(low affinity)

R (high affinity)

O2

H+,CO2,BPG

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α

β

HbA HbF HbA2

98% ~1% <3.5%

Hemoglobins in normal adultsHemoglobins in normal adultsHemoglobins in normal adultsHemoglobins in normal adults

α α

α

α

αβ γ

δ

δ

γ

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Globin gene clustersGlobin gene clustersGlobin gene clustersGlobin gene clusters

= duplicate genes, bo th expressed

G = fe ta l genes, G ly and A la at postion 136, bo th expressed

c luster, 16p 13.3

G A c luster, 11p 15.5

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Hb structural familiesHb structural familiesHb structural familiesHb structural families

Beta familyBeta family - - - found in adult hemoglobin HbA- found in adult hemoglobin HbA11.. - found in adult hemoglobin HbA- found in adult hemoglobin HbA22. . - found in fetal hemoglobin HbF.- found in fetal hemoglobin HbF. - found in embryonic hemoglobin Hb Gower 1 and - found in embryonic hemoglobin Hb Gower 1 and Hb Hb

Gower 2Gower 2

Alpha familyAlpha family - - - found in adult hemoglobins HbA- found in adult hemoglobins HbA11, HbA, HbA22..

- found in embryonic hemoglobins Hb Gower 1 - found in embryonic hemoglobins Hb Gower 1 and and Hb Portland.Hb Portland.

- (theta) newly discovered embryonic form. - (theta) newly discovered embryonic form.

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FETAL AND NEONATAL ERYTHROPOIESISFETAL AND NEONATAL ERYTHROPOIESISFETAL AND NEONATAL ERYTHROPOIESISFETAL AND NEONATAL ERYTHROPOIESIS

TABLE 1. Globin-chain development and compositionTABLE 1. Globin-chain development and composition

DevelopmentalDevelopmental

stagestage

HemoglobinHemoglobin

typetype

Globin-chainGlobin-chain

compositioncomposition

EmbryoEmbryo

EmbryoEmbryo

EmbryoEmbryo

Embryo to fetusEmbryo to fetus

Fetus to adultFetus to adult

AdultAdult

AdultAdult

Gower 1Gower 1

Gower 2Gower 2

PortlandPortland

FetalFetal

AA

AA22

FetalFetal

ZetaZeta2 2 , epsilon, epsilon22aa

AlphaAlpha22, epsilon, epsilon22

ZetaZeta22, gamma, gamma22

AlphaAlpha22, gamma, gamma22

AlphaAlpha22, beta, beta22

AlphaAlpha22, delta, delta22

AlphaAlpha22, gamma, gamma22bb

a This tetramer may be an epsilon tetrad.

b Fetal hemoglobin produced by adults has a different amino acid

heterogeneity of the gamma chain at the 136 position than fetal hemoglobin

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InheritedInherited Hemoglobin disorder Hemoglobin disorderInheritedInherited Hemoglobin disorder Hemoglobin disorder

Definition:Definition: An inherited mutation of the An inherited mutation of the globin genes leading to a globin genes leading to a qualitativequalitative or or quantitativequantitative abnormality of globin abnormality of globin synthesissynthesis

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The ThalassemiasThe Thalassemias ( (quantitativequantitative) )

The ThalassemiasThe Thalassemias ( (quantitativequantitative) )

Syndromes in which the rate of synthesis of a globin chain is reduced

beta thalassemia - reduced beta chain synthesis

alpha thalassemia – reduced alpha chain synthesis

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Alpha ThalassemiasAlpha ThalassemiasAlpha ThalassemiasAlpha Thalassemias

Rare, since Rare, since gene is duplicated (four genes per gene is duplicated (four genes per diploid).diploid).Usually more severe than beta thalassemia because Usually more severe than beta thalassemia because

there is no substitute for there is no substitute for gene in adults. gene in adults.Almost all Almost all thalassemias are deletions thalassemias are deletions

In In thalassemia major ( thalassemia major (00//0000) - occurrence of HbH ) - occurrence of HbH ((4) and Hb Bart’s (4) and Hb Bart’s (4).4).BPG is ineffective in HbH & Hb Bart’s.BPG is ineffective in HbH & Hb Bart’s.

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Beta thalassemiaBeta thalassemiaBeta thalassemiaBeta thalassemia

Impaired production of beta chainImpaired production of beta chain

beta thalassemia minor – heterozygous (or trait)beta thalassemia minor – heterozygous (or trait)

beta thalassemia major - homozygousbeta thalassemia major - homozygous

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Beta thalassemia - heterozygous (minor or trait)

Target cell

Oval cell

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Beta thalassemia major

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Beta ThalassemiasBeta ThalassemiasBeta ThalassemiasBeta Thalassemias

More common, since More common, since gene is present in gene is present in only one copy per chromosome.only one copy per chromosome.

Less severe than Less severe than thalassemia, since thalassemia, since chain can effectively substitute in adults.chain can effectively substitute in adults.

The The chain can also persist into adulthood chain can also persist into adulthood (HPFH).(HPFH).

In In thal major ( thal major (00//00) excess ) excess chains do chains do not form soluble homotetramers.not form soluble homotetramers.

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Beta thalassemia majorBeta thalassemia majorBeta thalassemia majorBeta thalassemia major

No beta chain produced (no HbA)No beta chain produced (no HbA)

Severe microcytic anemia occurs gradually in Severe microcytic anemia occurs gradually in the first year of lifethe first year of life

Marrow expansionMarrow expansion

Iron overloadIron overload

Growth failure and death Growth failure and death

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Alpha thalassemiaAlpha thalassemiaAlpha thalassemiaAlpha thalassemia

// NormalNormal

//-- Mild microcytosisMild microcytosis

/- -/- - Mild microcytosisMild microcytosis

-/- --/- - Hemoglobin H diseaseHemoglobin H disease

- -/- -- -/- - Hemoglobin Barts – Hydrops FetalisHemoglobin Barts – Hydrops Fetalis

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Structural hemoglobinopathyStructural hemoglobinopathy((qualitativequalitative))

Structural hemoglobinopathyStructural hemoglobinopathy((qualitativequalitative))

Amino acid substitution in the globin chain e.g. sickle

hemoglobin (HbS)

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Sickle cell hemoglobinSickle cell hemoglobinSickle cell hemoglobinSickle cell hemoglobin

G lu-

G lu-

H b A 1

G lu-

H b S (h e te ro z y g o u s)S ic k le ce ll tra it

H b S (h o m o zy g o u s)

S ic k le c e ll d isea se

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Red Blood Cells from Sickle Cell AnemiaRed Blood Cells from Sickle Cell AnemiaRed Blood Cells from Sickle Cell AnemiaRed Blood Cells from Sickle Cell Anemia

OXY-STATE DEOXY-STATE

Deoxygenation of SS erythrocytes leads to intracellular Deoxygenation of SS erythrocytes leads to intracellular hemoglobin polymerization, loss of deformability and changes in hemoglobin polymerization, loss of deformability and changes in cell morphology.cell morphology.

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Sickle Cells

Erythroblasts

Howell-Jolly Body

Sickle Cell Anemia – blood filmSickle Cell Anemia – blood filmSickle Cell Anemia – blood filmSickle Cell Anemia – blood film

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Fibres of Sickle HemoglobinFibres of Sickle HemoglobinFibres of Sickle HemoglobinFibres of Sickle Hemoglobin

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Fibres of Sickle Fibres of Sickle Hemoglobin – cross Hemoglobin – cross sectionsection

Fibres of Sickle Fibres of Sickle Hemoglobin – cross Hemoglobin – cross sectionsection

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Hemoglobin SHemoglobin SHemoglobin SHemoglobin S

Valine is exposed in deoxy-Hemoglobin

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Polymerization of HbSPolymerization of HbSPolymerization of HbSPolymerization of HbS

27- 1 Amino Acids, Peptides, and Proteins. 27- 2ObjectivesObjectives Draw a general amino acid and...

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