Upload
clifton-bailey
View
215
Download
2
Embed Size (px)
Citation preview
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
OOHH33NNCHCH22CC ••••
••••
•••• ••••++
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
Acid-Base Properties of GlycineAcid-Base Properties of Glycine
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
OHOHHH33NNCHCH22CC++
••••
••••
•••• ••••
typical typical ammonium ammonium ion: pion: pKKaa ~9 ~9
typical typical carboxylic carboxylic acid: pacid: pKKaa ~5 ~5
27- 51
Acid-Base Properties of GlycineAcid-Base Properties of Glycine
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
OHOHHH33NNCHCH22CC++
••••
••••
•••• ••••
typical typical carboxylic carboxylic acid: pacid: pKKaa ~5 ~5
27- 52
Acid-Base Properties of GlycineAcid-Base Properties of Glycine
Therefore, the more stable neutral form of Therefore, the more stable neutral form of glycine is the zwitterion.glycine is the zwitterion.
OO
OHOHHH33NNCHCH22CC++
••••
••••
•••• ••••
typical typical carboxylic carboxylic acid: pacid: pKKaa ~5 ~5
––••••
OO
OOHH33NNCHCH22CC ••••
••••
•••• ••••++
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.
Acid-Base Properties of GlycineAcid-Base Properties of Glycine
OO
OHOHHH33NNCHCH22CC++
••••
••••
•••• ••••
typical typical carboxylic carboxylic acid: pacid: pKKaa ~5 ~5
27- 54
Acid-Base Properties of GlycineAcid-Base Properties of Glycine
––••••
OO
OOHH33NNCHCH22CC ••••
••••
•••• ••••++
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
OOHH22NNCHCH22CC ••••
••••
•••• ••••
••••
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
OOHH33NNCHCH22CC ••••
••••
•••• ••••++
––••••
OO
OOHH22NNCHCH22CC ••••
••••
•••• ••••
••••
OO
OHOHHH33NNCHCH22CC++
••••
••••
•••• ••••
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
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
AlanineAlanineppKKa1a1 = = 2.342.34
ppKKa2a2 == 9.699.69
ppI I == 6.006.00
HH33NN CC CC
OO
OO––
CHCH33
HH++
27- 59
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
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
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
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
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
IsoleucineIsoleucineppKKa1a1 = = 2.362.36
ppKKa2a2 == 9.609.60
ppI I == 5.985.98
HH33NN CC CC
OO
OO––
CHCH33CHCHCHCH22CHCH33
HH++
27- 62
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
MethionineMethionineppKKa1a1 = = 2.282.28
ppKKa2a2 == 9.219.21
ppI I == 5.745.74
HH33NN CC CC
OO
OO––
CHCH33SCHSCH22CHCH22
HH++
27- 63
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
ProlineProlineppKKa1a1 = = 1.991.99
ppKKa2a2 == 10.6010.60
ppI I == 6.306.30
HH22NN CC CC
OO
OO––
HH++
CHCH22HH22CCCCHH22
27- 64
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
PhenylalaninePhenylalanineppKKa1a1 = = 1.831.83
ppKKa2a2 == 9.139.13
ppI I == 5.485.48
HH33NN CC CC
OO
OO––
HH++
CHCH22
27- 65
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
TryptophanTryptophanppKKa1a1 = = 2.832.83
ppKKa2a2 == 9.399.39
ppI I == 5.895.89
HH33NN CC CC
OO
OO––
HH++
CHCH22
HH
NN
27- 66
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
AsparagineAsparagineppKKa1a1 = = 2.022.02
ppKKa2a2 == 8.808.80
ppI I == 5.415.41
HH33NN CC CC
OO
OO––
HH++
HH22NNCCCHCH22
OO
27- 67
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
GlutamineGlutamineppKKa1a1 = = 2.172.17
ppKKa2a2 == 9.139.13
ppI I == 5.655.65
HH33NN CC CC
OO
OO––
HH++
HH22NNCCHCCH22CHCH22
OO
27- 68
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
SerineSerineppKKa1a1 = = 2.212.21
ppKKa2a2 == 9.159.15
ppI I == 5.685.68
HH33NN CC CC
OO
OO––
CHCH22OHOH
HH++
27- 69
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
ThreonineThreonineppKKa1a1 = = 2.092.09
ppKKa2a2 == 9.109.10
ppI I == 5.605.60
HH33NN CC CC
OO
OO––
CHCH33CHOHCHOH
HH++
27- 70
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
TyrosineTyrosineppKKa1a1 = = 2.202.20
ppKKa2a2 == 9.119.11
ppI I == 5.665.66
HH33NN CC CC
OO
OO––
HH++
CHCH22 OHOH
27- 71
Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains
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
Amino Acids with Ionizable Side ChainsAmino Acids with Ionizable Side Chains
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
Amino Acids with Ionizable Side ChainsAmino Acids with Ionizable Side Chains
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
Amino Acids with Ionizable Side ChainsAmino Acids with Ionizable Side Chains
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
Amino Acids with Ionizable Side ChainsAmino Acids with Ionizable Side Chains
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
NeurotransmittersNeurotransmitters
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
NeurotransmittersNeurotransmitters
Dopamine is formed Dopamine is formed by decarboxylation by decarboxylation of of LL-DOPA.-DOPA.
OOHH
HH
HHHH
HHHH22NN
HHOO
DopamineDopamine
27- 88
NeurotransmittersNeurotransmitters
OOHH
HH
HHHH
OHOHHH22NN
HHOO
NorepinephrineNorepinephrine
27- 89
NeurotransmittersNeurotransmitters
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
AlanylglycineAlanylglycine
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
AlanylglycineAlanylglycine
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
Sanger's MethodSanger's Method
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
NNHCHHCH22CC NNHCHCOHCHCO
CHCH33
NNHCHCHCHC
CHCH22CC66HH55
NNHCHCHCHC
OO OOOOOO
CH(CHCH(CH33))22
––
27- 126
Sanger's MethodSanger's Method
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
NNHCHHCH22CC NNHCHCOHCHCO
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
The B Chain of Bovine InsulinThe B Chain of Bovine Insulin
FVNQHLCGSHLFVNQHLCGSHL
VGALVGAL
VCGERGFVCGERGF
YTPKAYTPKA
27- 132
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: 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
The B Chain of Bovine InsulinThe B Chain of Bovine Insulin
FVNQHLCGSHLFVNQHLCGSHL
SHLVSHLVLVGALVGA
VGALVGAL
ALTALT
TLVCTLVCVCGERGFVCGERGF
YTPKAYTPKA
27- 134
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: 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
The B Chain of Bovine InsulinThe B Chain of Bovine Insulin
FVNQHLCGSHLFVNQHLCGSHL
SHLVSHLVLVGALVGA
VGALVGAL
ALTALT
TLVCTLVCVCGERGFVCGERGF
GFFYTPKGFFYTPK
YTPKAYTPKA
27- 136
The B Chain of Bovine InsulinThe B Chain of Bovine Insulin
FVNQHLCGSHLFVNQHLCGSHL
SHLVSHLVLVGALVGA
VGALVGAL
ALTALT
TLVCTLVCVCGERGFVCGERGF
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
Edman DegradationEdman Degradation
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
Edman DegradationEdman Degradation
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
Edman DegradationEdman Degradation
peptidepeptideHH33NNCHCCHC
OO
RR
++NNHHCC66HH55NN CC SS ++
peptidepeptideCC66HH55NNHCHCNNHHCHCCHC
OO
RR
NNHH
SS
27- 143
Edman DegradationEdman Degradation
peptidepeptideCC66HH55NNHCHCNNHHCHCCHC
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
Edman DegradationEdman Degradation
peptidepeptideCC66HH55NNHCHCNNHHCHCCHC
OO
RR
NNHH
SS
HClHCl
peptidepeptideHH33NN++
++CC66HH55NNHH CC
SSCC
NN CHCH
RR
OO
27- 145
Edman DegradationEdman Degradation
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.
peptidepeptideHH33NN++
++
27- 146
Edman DegradationEdman Degradation
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.
peptidepeptideHH33NN++
++
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
CarboxypeptidaseCarboxypeptidase
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
CarboxypeptidaseCarboxypeptidase
Disulfide bondDisulfide bond
N-terminusN-terminus
C-terminusC-terminus
Zn2+
Arg-145
tube modeltube model ribbon modelribbon model
27- 164
MyoglobinMyoglobin
N-terminusN-terminus
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).
27- 165
Protein Quaternary Structure:Protein Quaternary Structure:HemoglobinHemoglobin
27- 166
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.
27- 167
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.
27- 168
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.
27- 169
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.
27- 170
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.
27- 171
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
27- 172
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
27- 173
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
27- 174
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.
27- 175
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..
27- 176
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.
27- 177
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.
27- 178
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)
27- 179
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..
27- 180
Myoglobin: 2o and 3o structure
27- 181
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
27- 182
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
27- 183
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
27- 184
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
27- 185
His E7 acts as a gate to favor oxygen binding over carbon monoxide.
27- 186
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.
27- 187
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
27- 188
Hemoglobin: The Oxygen Transporter
OXYHEMOGLOBIN DEOXYHEMOGLOBIN
27- 189
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
27- 190
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.
27- 191
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*
27- 192
Oxygen Binding Curves-2Oxygen Binding Curves-2Oxygen Binding Curves-2Oxygen Binding Curves-2
27- 193
Hemoglobin EquilibriumHemoglobin EquilibriumHemoglobin EquilibriumHemoglobin Equilibrium
T(low affinity)
R (high affinity)
O2
H+,CO2,BPG
27- 194
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
27- 195
Oxygen binding by hemoglobinOxygen binding by hemoglobinOxygen binding by hemoglobinOxygen binding by hemoglobin
27- 196
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)
27- 197
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
27- 198
The Bohr EffectThe Bohr Effect
27- 199
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
27- 200
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.
27- 201
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
27- 202
2,3-BPG
2,3-bisphosphoglycerate (2,3-BPG) is a negative allosteric effector of O2 binding to Hb - binds tighter to deoxyHb
27- 203
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)
27- 204
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
27- 205
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
27- 206
Effect of oxygen on heme ironEffect of oxygen on heme ironEffect of oxygen on heme ironEffect of oxygen on heme iron
Fe
Proxima lHis (F8)
F7
F6
FG 1
FG 2FG 3
NC
Plane of hem e
CNO
===
Dista lHis (E7)
27- 207
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
Fe
Proxima lHis (F8)
F7
F6
FG 1
FG 2FG 3
NC
27- 208
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.
27- 209
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
27- 210
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
27- 211
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.
27- 212
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
27- 213
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.
27- 214
Effect of BPGEffect of BPGEffect of BPGEffect of BPG
Side view (R)
Side view (T)
BPG
27- 215
His+His+Lys+
His+
Lys+
His+--- -BPG
Effect of BPGEffect of BPGEffect of BPGEffect of BPG
27- 216
Hemoglobin EquilibriumHemoglobin EquilibriumHemoglobin EquilibriumHemoglobin Equilibrium
T(low affinity)
R (high affinity)
O2
H+,CO2,BPG
27- 217
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
27- 218
27- 219
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
27- 220
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.
27- 221
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
27- 222
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
27- 223
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
27- 224
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.
27- 225
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
27- 226
Beta thalassemia - heterozygous (minor or trait)
Target cell
Oval cell
27- 227
Beta thalassemia major
27- 228
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.
27- 229
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
27- 230
27- 231
Alpha thalassemiaAlpha thalassemiaAlpha thalassemiaAlpha thalassemia
// NormalNormal
//-- Mild microcytosisMild microcytosis
/- -/- - Mild microcytosisMild microcytosis
-/- --/- - Hemoglobin H diseaseHemoglobin H disease
- -/- -- -/- - Hemoglobin Barts – Hydrops FetalisHemoglobin Barts – Hydrops Fetalis
27- 232
Structural hemoglobinopathyStructural hemoglobinopathy((qualitativequalitative))
Structural hemoglobinopathyStructural hemoglobinopathy((qualitativequalitative))
Amino acid substitution in the globin chain e.g. sickle
hemoglobin (HbS)
27- 233
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
27- 234
27- 235
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.
27- 236
Sickle Cells
Erythroblasts
Howell-Jolly Body
Sickle Cell Anemia – blood filmSickle Cell Anemia – blood filmSickle Cell Anemia – blood filmSickle Cell Anemia – blood film
27- 237
Fibres of Sickle HemoglobinFibres of Sickle HemoglobinFibres of Sickle HemoglobinFibres of Sickle Hemoglobin
27- 238
Fibres of Sickle Fibres of Sickle Hemoglobin – cross Hemoglobin – cross sectionsection
Fibres of Sickle Fibres of Sickle Hemoglobin – cross Hemoglobin – cross sectionsection
27- 239
Hemoglobin SHemoglobin SHemoglobin SHemoglobin S
Valine is exposed in deoxy-Hemoglobin
27- 240
Polymerization of HbSPolymerization of HbSPolymerization of HbSPolymerization of HbS