Transcript Chapter 27 Amino acid

Amino Acids, Peptides,
and Proteins
Dr. Wolf's CHM 424
27- 1
Objectives
Draw a general amino acid and identify the two functional groups common to all.
Classify each amino acid according to the chemical nature of its R group.
Define the meaning of an essential amino acid.
Draw the reaction that joins two amino acids to form a peptide bond.
Describe and differentiate primary, secondary, tertiary, and quaternary protein
structures.
Describe and differentiate co-enzymes and prosthetic groups.
List and discuss four forces that stabilize globular protein structure.
List important structural similarities and differences between myoglobin and
hemoglobin.
Describe the mutation present in hemoglobin giving rise to sickle cell disease.
Dr. Wolf's CHM 424
27- 2
Classification of Amino Acids
Dr. Wolf's CHM 424
27- 3
Fundamentals
While their name implies that amino acids are
compounds that contain an —NH2 group and a
—CO2H group, these groups are actually
present as —NH3+ and —CO2– respectively.
They are classified as , , , etc. amino acids
according the carbon that bears the nitrogen.
Dr. Wolf's CHM 424
27- 4
Amino Acids
+
NH3

CO2–
+
–
H3NCH2CH2CO2

+
–
H3NCH2CH2CH2CO2

Dr. Wolf's CHM 424
an -amino acid that is an
intermediate in the biosynthesis
of ethylene
a -amino acid that is one of
the structural units present in
coenzyme A
a -amino acid involved in
the transmission of nerve
impulses
27- 5
The 20 Key Amino Acids
More than 700 amino acids occur naturally, but
20 of them are especially important.
These 20 amino acids are the building blocks of
proteins. All are -amino acids.
They differ in respect to the group attached to
the  carbon.
These 20 are listed in Table 27.1.
Dr. Wolf's CHM 424
27- 6
H
+
H3N
C
O
C
O
–
R
The amino acids obtained by hydrolysis of
proteins differ in respect to R (the side chain).
The properties of the amino acid vary as the
structure of R varies.
Dr. Wolf's CHM 424
27- 7
H
+
H3N
C
O
C
O
–
R
The major differences among the side chains
concern:
Size and shape
Electronic characteristics
Dr. Wolf's CHM 424
27- 8
General categories of -amino acids
nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains
Dr. Wolf's CHM 424
27- 9
General categories of -amino acids
nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains
Dr. Wolf's CHM 424
27- 10
Amino Acid
R-groups
Non-Polar
Hydrophobic
Polar
Charged
Uncharged
Arginine (+)
Glutamic acid (-)
Aspartic Acid (-)
Lysine (+)
Histidine (+)
Cysteine
Proline
Serine
Glutamine
Asparagine
Tryptophan
Phenylalanine
Isoleucine
Tyrosine
Leucine
Valine
Methionine
Ambivalent
Glycine
Threonine
Alanine
Dr. Wolf's CHM 424
27- 11
Charged Amino Acids
+
+
N
N
O
N
-
O
O
O
O
N
Arginine [Arg]
Glutamate [Glu]
O
Aspartate [Asp]
N
N
Lysine [Lys]
O
+N
N
O
O
-
N
N
N
Histidine [His]
Dr. Wolf's CHM 424
27- 12
Hydrophobic Indexes
Arginine Arg [R] -11.2
Glutamic Acid Glu [E]-9.9
Aspartic Acid
Asp [D] -7.4
Lysine Lys [K] -4.2
Histidine
His [H] -3.3
Cysteine Cys [C] -2.8
Proline Pro [P] -0.5
Serine Ser [S] -0.3
Glutamine Gln [Q] -0.3
Asparagine Asn [N] -0.2
Dr. Wolf's CHM 424
Glycine Gly [G] 0
Threonine Thr [T] 0.4
Alanine Ala [A] 0.5
Methionine Met [M] 1.3
Valine Val [V] 1.5
Leucine Leu [L] 1.8
Tyrosine Tyr [Y] 2.3
Isoleucine Ile [I] 2.5
Phenylalanine
Phe [F] 2.5
Tryptophan Trp [W] 3.4
27- 13
Essential amino acids
Definition - Those amino acids that cannot be
synthesized in the body in sufficient quantities for
anabolic needs.
In humans,
Isoleucine Leucine
Valine
Tryptophan
Methionine
Lysine
Phenylalanine
Threonine Histidine
Dr. Wolf's CHM 424
27- 14
H
Glycine
(Gly or G)
+
H3N
C
O
C
O
–
H
Glycine is the simplest amino acid. It is the only
one in the table that is achiral.
In all of the other amino acids in the table the 
carbon is a chirality center.
Dr. Wolf's CHM 424
27- 15
H
+
H3N
C
O
C
O
–
CH3
Alanine
(Ala or A)
Alanine, valine, leucine, and isoleucine have
alkyl groups as side chains, which are nonpolar
and hydrophobic.
Dr. Wolf's CHM 424
27- 16
H
+
H3N
C
O
C
O
–
CH(CH3)2
Valine
(Val or V)
Dr. Wolf's CHM 424
27- 17
H
+
H3N
C
O
C
O
–
CH2CH(CH3)2
Leucine
(Leu or L)
Dr. Wolf's CHM 424
27- 18
H
+
H3N
C
O
C
O
–
CH3CHCH2CH3
Isoleucine
(Ile or I)
Dr. Wolf's CHM 424
27- 19
H
+
H3N
C
O
C
O
–
CH3SCH2CH2
Methionine
(Met or M)
The side chain in methionine is nonpolar, but
the presence of sulfur makes it somewhat
polarizable.
Dr. Wolf's CHM 424
27- 20
H
+
H2N
C
O
C
CH2
H2C
C
H2
O
–
Proline
(Pro or P)
Proline is the only amino acid that contains a
secondary amine function. Its side chain is
nonpolar and cyclic.
Dr. Wolf's CHM 424
27- 21
H
+
H3N
C
CH2
O
C
O
–
Phenylalanine
(Phe or F)
The side chain in phenylalanine (a nonpolar
amino acid) is a benzyl group.
Dr. Wolf's CHM 424
27- 22
H
+
H3N
C
O
C
O
–
Tryptophan
CH2
(Trp or W)
N
H
Dr. Wolf's CHM 424
The side chain in
tryptophan (a nonpolar
amino acid) is larger
and more polarizable
than the benzyl group
of phenylalanine.
27- 23
General categories of -amino acids
nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains
Dr. Wolf's CHM 424
27- 24
H
+
H3N
C
O
C
O
–
CH2OH
Serine
(Ser or S)
The —CH2OH side chain in serine can be
involved in hydrogen bonding.
Dr. Wolf's CHM 424
27- 25
H
+
H3N
C
O
C
O
–
CH3CHOH
Threonine
(Thr or T)
The side chain in threonine can be involved in
hydrogen bonding, but is somewhat more
crowded than in serine.
Dr. Wolf's CHM 424
27- 26
H
+
H3N
C
O
C
O
–
CH2SH
Cysteine
(Cys or C)
The side chains of two remote cysteines can be
joined by forming a covalent S—S bond.
Dr. Wolf's CHM 424
27- 27
H
+
H3N
C
O
C
O
–
Tyrosine
(Tyr or Y)
CH2
The side chain of
tyrosine is similar to
that of phenylalanine
but can participate in
hydrogen bonding.
OH
Dr. Wolf's CHM 424
27- 28
H
+
H3N
C
H2NCCH2
O
C
O
–
Asparagine
(Asn or N)
O
The side chains of asparagine and glutamine
(next slide) terminate in amide functions that are
polar and can engage in hydrogen bonding.
Dr. Wolf's CHM 424
27- 29
H
+
H3N
C
H2NCCH2CH2
O
C
O
–
Glutamine
(Gln or Q)
O
Dr. Wolf's CHM 424
27- 30
General categories of -amino acids
nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains
Dr. Wolf's CHM 424
27- 31
H
+
H3N
–
C
OCCH2
O
C
O
–
Aspartic Acid
(Asp or D)
O
Aspartic acid and glutamic acid (next slide) exist
as their conjugate bases at biological pH. They
are negatively charged and can form ionic
bonds with positively charged species.
Dr. Wolf's CHM 424
27- 32
H
+
H3N
–
C
OCCH2CH2
O
C
O
–
Glutamic Acid
(Glu or E)
O
Dr. Wolf's CHM 424
27- 33
General categories of -amino acids
nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains
Dr. Wolf's CHM 424
27- 34
H
Lysine
(Lys or K)
+
H3N
C
O
C
O
–
+
CH2CH2CH2CH2NH3
Lysine and arginine (next slide) exist as their
conjugate acids at biological pH. They are
positively charged and can form ionic bonds
with negatively charged species.
Dr. Wolf's CHM 424
27- 35
H
Arginine
(Arg or R)
+
H3N
C
O
C
O
–
CH2CH2CH2NHCNH2
+ NH2
Dr. Wolf's CHM 424
27- 36
H
Histidine
+
H3N
(His or H)
CH2
N
Dr. Wolf's CHM 424
C
NH
O
C
O
–
Histidine is a basic
amino acid, but less
basic than lysine and
arginine. Histidine can
interact with metal ions
and can help move
protons from one site
to another.
27- 37
Stereochemistry of Amino
Acids
Dr. Wolf's CHM 424
27- 38
Configuration of -Amino Acids
Glycine is achiral. All of the other amino acids
in proteins have the L-configuration at their
carbon.
–
CO2
+
H3N
H
R
Dr. Wolf's CHM 424
27- 39
R
H2N
CH
CO2H
All DNA encoded aa are 
CHO
All are chiral,
except Glycine
R=H
H
OH
HO
CH2OH
H
CH2OH
D-
All DNA
encoded aa
are usually L-
LR
CHO
=
HO
CH2OH
H
(S) - Glyceraldehyde
(-) Dr. Wolf's CHM 424
CHO
=
H2N
C
CO2H
H
(L) - Amino Acids
(-) 27- 40
Acid-Base Behavior of Amino
Acids
Dr. Wolf's CHM 424
27- 41
Recall
While their name implies that amino acids are
compounds that contain an —NH2 group and a
—CO2H group, these groups are actually
present as —NH3+ and —CO2– respectively.
How do we know this?
Dr. Wolf's CHM 424
27- 42
aa are high melting point solids! Why?
Answer = aa are ionic compounds under normal conditions
LOW pH
NEUTRAL
C
O
O
O
R
HIGH pH
R
C
OH
NH3
ammonium Form
R
C
O
NH3
Zwitterion
O
NH2
Carboxylate Form
Isoelectric Point = concentration of zwitterion is at a
maximum and the concentration of cations and anions is equal
r aa with basic R-groups, we require higher pHs, and
for aa with acidic R-groups, we require lower pHs
Dr. Wolf's CHM 424 to reach the Isoelectric Point
27- 43
NH 3
CO2
(CH2)2
pH 7
CH
CH
H3 N
(CH2)2
CO2
Glu
H3 N
CO2
Lys
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
Dr. Wolf's CHM 424
27- 44
Properties of Glycine
The properties of glycine:
high melting point: (when heated to 233°C
it decomposes before it melts)
solubility: soluble in water; not soluble in
nonpolar solvent
more consistent with this
than this
•• O ••
+
H3NCH2C
Dr. Wolf's CHM 424
•• O ••
•• •–
O•
••
••
H2NCH2C
••
OH
••
27- 45
Properties of Glycine
The properties of glycine:
high melting point: (when heated to 233°C
it decomposes before it melts)
solubility: soluble in water; not soluble in
nonpolar solvent
more consistent with this
•• O ••
+
H3NCH2C
Dr. Wolf's CHM 424
•• •–
O•
••
called a zwitterion or
dipolar ion
27- 46
Acid-Base Properties of Glycine
The zwitterionic structure of glycine also follows
from considering its acid-base properties.
A good way to think about this is to start with the
structure of glycine in strongly acidic solution,
say pH = 1.
At pH = 1, glycine exists in its protonated form
(a monocation).
•• O ••
+
H3NCH2C
Dr. Wolf's CHM 424
••
OH
••
27- 47
Acid-Base Properties of Glycine
Now ask yourself "As the pH is raised, which is
the first proton to be removed? Is it the proton
attached to the positively charged nitrogen, or is
it the proton of the carboxyl group?"
You can choose between them by estimating
their respective pKas.
typical
ammonium
ion: pKa ~9
Dr. Wolf's CHM 424
•• O ••
+
H3NCH2C
••
typical
carboxylic
acid: pKa ~5
OH
••
27- 48
Acid-Base Properties of Glycine
The more acidic proton belongs to the CO2H
group. It is the first one removed as the pH is
raised.
•• O ••
+
H3NCH2C
Dr. Wolf's CHM 424
••
typical
carboxylic
acid: pKa ~5
OH
••
27- 49
Acid-Base Properties of Glycine
Therefore, the more stable neutral form of
glycine is the zwitterion.
•• O ••
+
H3NCH2C
•• •–
O•
••
•• O ••
+
H3NCH2C
Dr. Wolf's CHM 424
••
typical
carboxylic
acid: pKa ~5
OH
••
27- 50
Acid-Base Properties of Glycine
The measured pKa of glycine is 2.34.
Glycine is stronger than a typical carboxylic acid
because the positively charged N acts as an
electron-withdrawing, acid-strengthening
substituent on the  carbon.
•• O ••
+
H3NCH2C
Dr. Wolf's CHM 424
••
typical
carboxylic
acid: pKa ~5
OH
••
27- 51
Acid-Base Properties of Glycine
A proton attached to N in the zwitterionic form of
nitrogen can be removed as the pH is increased
further.
•• O ••
+
H3NCH2C
•• •–
O•
••
HO
–
•• O ••
••
H2NCH2C
•• •–
O•
••
The pKa for removal of this proton is 9.60.
This value is about the same as that for NH4+
(9.3).
Dr. Wolf's CHM 424
27- 52
Isoelectric Point pI
•• O ••
+
H3NCH2C
••
OH
••
pKa = 2.34
•• O ••
+
H3NCH2C
•• •–
O•
••
pKa = 9.60
•• O ••
••
Dr. Wolf's CHM 424
H2NCH2C
•• •–
O•
••
The pH at which the
concentration of the
zwitterion is a
maximum is called the
isoelectric point. Its
numerical value is the
average of the two
pKas.
The pI of glycine is
5.97.
27- 53
Acid-Base Properties of Amino Acids
One way in which amino acids differ is in
respect to their acid-base properties. This is the
basis for certain experimental methods for
separating and identifying them.
Just as important, the difference in acid-base
properties among various side chains affects
the properties of the proteins that contain them.
Table 27.2 gives pKa and pI values for amino
acids with neutral side chains.
Dr. Wolf's CHM 424
27- 54
Amino Acids with Neutral Side Chains
H
Glycine
+
H3N
C
O
C
O
–
pKa1 = 2.34
pKa2 = 9.60
pI = 5.97
H
Dr. Wolf's CHM 424
27- 55
Amino Acids with Neutral Side Chains
H
Alanine
+
H3N
C
O
C
O
–
pKa1 = 2.34
pKa2 = 9.69
pI = 6.00
CH3
Dr. Wolf's CHM 424
27- 56
Amino Acids with Neutral Side Chains
H
Valine
+
H3N
C
O
C
O
–
pKa1 = 2.32
pKa2 = 9.62
pI = 5.96
CH(CH3)2
Dr. Wolf's CHM 424
27- 57
Amino Acids with Neutral Side Chains
H
Leucine
+
H3N
C
O
C
O
–
pKa1 = 2.36
pKa2 = 9.60
pI = 5.98
CH2CH(CH3)2
Dr. Wolf's CHM 424
27- 58
Amino Acids with Neutral Side Chains
H
Isoleucine
+
H3N
C
O
C
O
–
pKa1 = 2.36
pKa2 = 9.60
pI = 5.98
CH3CHCH2CH3
Dr. Wolf's CHM 424
27- 59
Amino Acids with Neutral Side Chains
H
Methionine
+
H3N
C
O
C
O
–
pKa1 = 2.28
pKa2 = 9.21
pI = 5.74
CH3SCH2CH2
Dr. Wolf's CHM 424
27- 60
Amino Acids with Neutral Side Chains
H
Proline
+
H2N
C
O
C
O
–
pKa1 = 1.99
pKa2 = 10.60
pI = 6.30
CH2
H2C
C
H2
Dr. Wolf's CHM 424
27- 61
Amino Acids with Neutral Side Chains
H
Phenylalanine
+
H3N
C
CH2
Dr. Wolf's CHM 424
O
C
O
–
pKa1 = 1.83
pKa2 = 9.13
pI = 5.48
27- 62
Amino Acids with Neutral Side Chains
H
Tryptophan
+
H3N
C
CH2
O
C
O
–
pKa1 = 2.83
pKa2 = 9.39
pI = 5.89
N
Dr. Wolf's CHM 424
H
27- 63
Amino Acids with Neutral Side Chains
H
Asparagine
+
H3N
C
O
C
O
–
pKa1 = 2.02
pKa2 = 8.80
pI = 5.41
H2NCCH2
O
Dr. Wolf's CHM 424
27- 64
Amino Acids with Neutral Side Chains
H
Glutamine
+
H3N
C
H2NCCH2CH2
O
C
O
–
pKa1 = 2.17
pKa2 = 9.13
pI = 5.65
O
Dr. Wolf's CHM 424
27- 65
Amino Acids with Neutral Side Chains
H
Serine
+
H3N
C
O
C
O
–
pKa1 = 2.21
pKa2 = 9.15
pI = 5.68
CH2OH
Dr. Wolf's CHM 424
27- 66
Amino Acids with Neutral Side Chains
H
Threonine
+
H3N
C
O
C
O
–
pKa1 = 2.09
pKa2 = 9.10
pI = 5.60
CH3CHOH
Dr. Wolf's CHM 424
27- 67
Amino Acids with Neutral Side Chains
H
Tyrosine
+
H3N
C
CH2
Dr. Wolf's CHM 424
OH
O
C
O
–
pKa1 = 2.20
pKa2 = 9.11
pI = 5.66
27- 68
Amino Acids with Neutral Side Chains
H
Cysteine
+
H3N
C
O
C
O
–
pKa1 = 1.96
pKa2 = 8.18
pI = 5.07
CH2SH
Dr. Wolf's CHM 424
27- 69
Amino Acids with Ionizable Side Chains
H
Aspartic acid
+
H3N
–
C
OCCH2
O
C
O
–
pKa1 =
pKa2 =
pKa3 =
pI =
1.88
3.65
9.60
2.77
O
For amino acids with acidic side chains, pI is the
average of pKa1 and pKa2.
Dr. Wolf's CHM 424
27- 70
Amino Acids with Ionizable Side Chains
H
+
H3N
Glutamic acid
–
C
OCCH2CH2
O
C
O
–
pKa1 =
pKa2 =
pKa3 =
pI =
2.19
4.25
9.67
3.22
O
Dr. Wolf's CHM 424
27- 71
Amino Acids with Ionizable Side Chains
H
+
H3N
C
O
C
O
–
+
CH2CH2CH2CH2NH3
pKa1 =
pKa2 =
pKa3 =
pI =
2.18
8.95
10.53
9.74
Lysine
For amino acids with basic side chains, pI is the
average of pKa2 and pKa3.
Dr. Wolf's CHM 424
27- 72
Amino Acids with Ionizable Side Chains
H
+
H3N
C
O
C
O
–
CH2CH2CH2NHCNH2
pKa1 =
pKa2 =
pKa3 =
pI =
2.17
9.04
12.48
10.76
+ NH2
Arginine
Dr. Wolf's CHM 424
27- 73
Amino Acids with Ionizable Side Chains
H
Histidine
+
H3N
C
CH2
N
Dr. Wolf's CHM 424
O
C
O
–
pKa1 =
pKa2 =
pKa3 =
pI =
1.82
6.00
9.17
7.59
NH
27- 74
Synthesis of Amino Acids
Dr. Wolf's CHM 424
27- 75
From -Halo Carboxylic Acids
O
CH3CHCOH + 2NH3
Br
H2O
O
–
CH3CHCO + NH4Br
+NH3
(65-70%)
Dr. Wolf's CHM 424
27- 76
Strecker Synthesis
O
CH3CH
NH4Cl
NaCN
CH3CHC
N
NH2
1. H2O, HCl, heat
2. HO–
O
–
CH3CHCO
Dr. Wolf's CHM 424
+NH3
(52-60%)
27- 77
Using Diethyl Acetamidomalonate
O
O
C
C
C
CH3CH2O
CH3CNH
H
OCH2CH3
O
Can be used in the same manner as diethyl
malonate (Section 21.7).
Dr. Wolf's CHM 424
27- 78
Example
O O
CH3CH2OCCCOCH2CH3
H
CH3CNH
O
1. NaOCH2CH3
2. C6H5CH2Cl
O O
CH3CH2OCCCOCH2CH3
CH3CNH
Dr. Wolf's CHM 424
O
CH2C6H5
(90%)
27- 79
O O
Example
HOCCCOH
–CO2
CH2C6H5
H3 N
+
O
HBr, H2O, heat
HCCOH
H3N
+
CH2C6H5
O O
(65%)
CH3CH2OCCCOCH2CH3
CH3CNH
Dr. Wolf's CHM 424
O
CH2C6H5
27- 80
Reactions of Amino Acids
Dr. Wolf's CHM 424
27- 81
Acylation of Amino Group
The amino nitrogen of an amino acid can be
converted to an amide with the customary
acylating agents.
O
O O
+
– +
H3NCH2CO
CH3COCCH3
O
O
CH3CNHCH2COH
Dr. Wolf's CHM 424
(89-92%)
27- 82
Esterification of Carboxyl Group
The carboxyl group of an amino acid can be
converted to an ester. The following illustrates
Fischer esterification of alanine.
O
+
– +
H3NCHCO
CH3CH2OH
CH3
HCl
O
Cl
Dr. Wolf's CHM 424
–
+
H3NCHCOCH2CH3
CH3
(90-95%)
27- 83
Ninhydrin Test
Amino acids are detected by the formation of a purple
color on treatment with ninhydrin.
O
O
OH
+
+ H3NCHCO–
OH
R
O
O
O
RCH + CO2 + H2O +
Dr. Wolf's CHM 424
O–
N
O
O
27- 84
Some Biochemical Reactions
of Amino Acids
Dr. Wolf's CHM 424
27- 85
Biosynthesis of L-Glutamic Acid
O
HO2CCH2CH2CCO2H + NH3
enzymes and
reducing coenzymes
–
HO2CCH2CH2CHCO2
+ NH3
This reaction is the biochemical analog of reductive
amination (Section 22.10).
Dr. Wolf's CHM 424
27- 86
Transamination via L-Glutamic Acid
O
–
HO2CCH2CH2CHCO2
+
CH3CCO2H
+ NH3
L-Glutamic acid acts as a source of the amine
group in the biochemical conversion of -keto
acids to other amino acids. In the example to be
shown, pyruvic acid is converted to L-alanine.
Dr. Wolf's CHM 424
27- 87
Transamination via L-Glutamic Acid
O
–
HO2CCH2CH2CHCO2
+
CH3CCO2H
+ NH3
enzymes
O
HO2CCH2CH2CCO2H
–
+ CH3CHCO2
+ NH3
Dr. Wolf's CHM 424
27- 88
Mechanism
O
–
HO2CCH2CH2CHCO2
+
CH3CCO2H
+ NH3
The first step is imine formation between the
amino group of L-glutamic acid and pyruvic
acid.
Dr. Wolf's CHM 424
27- 89
Mechanism
O
–
HO2CCH2CH2CHCO2
+
CH3CCO2H
+ NH3
–
HO2CCH2CH2CHCO2
N
CH3CCO2–
Dr. Wolf's CHM 424
27- 90
Formation of the imine is followed by proton
removal at one carbon and protonation of
another carbon.
H
–
HO2CCH2CH2CCO2
N
CH3CCO2–
Dr. Wolf's CHM 424
27- 91
–
HO2CCH2CH2CCO2
N
–
CH3CCO2
H
H
–
HO2CCH2CH2CCO2
N
CH3CCO2–
Dr. Wolf's CHM 424
27- 92
–
HO2CCH2CH2CCO2
N
–
CH3CCO2
H
Hydrolysis of the imine function gives
-keto glutarate and L-alanine.
Dr. Wolf's CHM 424
27- 93
–
HO2CCH2CH2CCO2
N
–
CH3CCO2
H
H2O
+NH3
–
HO2CCH2CH2CCO2
O
Dr. Wolf's CHM 424
+
CH3CCO2–
H
27- 94
Biosynthesis of L-Tyrosine
L-Tyrosine is biosynthesized from L-phenylalanine.
A key step is epoxidation of the aromatic ring to
give an arene oxide intermediate.
–
CH2CHCO2
+ NH3
Dr. Wolf's CHM 424
27- 95
Biosynthesis of L-Tyrosine
–
CH2CHCO2
O
+ NH3
O2, enzyme
–
CH2CHCO2
+ NH3
Dr. Wolf's CHM 424
27- 96
Biosynthesis of L-Tyrosine
–
CH2CHCO2
O
+ NH3
enzyme
HO
–
CH2CHCO2
+ NH3
Dr. Wolf's CHM 424
27- 97
Biosynthesis of L-Tyrosine
Conversion to L-tyrosine is one of the major
metabolic pathways of L-phenylalanine.
Individuals who lack the enzymes necessary to
convert L-phenylalanine to L-tyrosine can suffer
from PKU disease. In PKU disease, Lphenylalanine is diverted to a pathway leading
to phenylpyruvic acid, which is toxic.
Newborns are routinely tested for PKU disease.
Treatment consists of reducing their dietary
intake of phenylalanine-rich proteins.
Dr. Wolf's CHM 424
27- 98
Decarboxylation
Decarboxylation is a common reaction of amino acids. An example is the conversion of
L-histidine to histamine. Antihistamines act by
blocking the action of histamine.
N
–
CH2CHCO2
N
H
Dr. Wolf's CHM 424
+ NH3
27- 99
Decarboxylation
N
CH2CH2 NH2
N
H
–CO2, enzymes
N
–
CH2CHCO2
N
H
Dr. Wolf's CHM 424
+ NH3
27- 100
Neurotransmitters
–
The chemistry of the
brain and central
nervous system is
affected by
neurotransmitters.
Several important
neurotransmitters
are biosynthesized
from L-tyrosine.
+
H3N
H
CO2
H
H
OH
L-Tyrosine
Dr. Wolf's CHM 424
27- 101
Neurotransmitters
–
The common name
of this compound is
L-DOPA. It occurs
naturally in the
brain. It is widely
prescribed to reduce
the symptoms of
Parkinsonism.
+
H3N
H
CO2
H
H
HO
OH
L-3,4-Dihydroxyphenylalanine
Dr. Wolf's CHM 424
27- 102
Neurotransmitters
Dopamine is formed
by decarboxylation
of L-DOPA.
H
H2N
H
H
H
HO
OH
Dopamine
Dr. Wolf's CHM 424
27- 103
Neurotransmitters
H
H2N
H
H
OH
HO
OH
Norepinephrine
Dr. Wolf's CHM 424
27- 104
Neurotransmitters
H
CH3NH
H
H
OH
HO
OH
Epinephrine
Dr. Wolf's CHM 424
27- 105
Peptides
Dr. Wolf's CHM 424
27- 106
Peptides
Peptides are compounds in which an amide
bond links the amino group of one -amino acid
and the carboxyl group of another.
An amide bond of this type is often referred to
as a peptide bond.
Dr. Wolf's CHM 424
27- 107
Peptide bond formation
O
Aspartate C
H
H
C
O
H
Alanine
H
H
C
C
H
C
H
+
- H N
H N H C O
H C O
H
H
O
O
+
condensation
Dr. Wolf's CHM 424
H2O
27- 108
Peptide bond formation
O
O
C
H
H
+
C
H
C
H
C
H N
H C
H
O
H
H
H C
N H C O
O
Peptide bond
Primary Structure
Dr. Wolf's CHM 424
27- 109
Alanine and Glycine
H
+
H3N
C
CH3
Dr. Wolf's CHM 424
H
O
C
–
O
+
H3N
C
O
C
–
O
H
27- 110
Alanylglycine
H
+
H3N
C
CH3
H
O
C
N
C
H
H
O
C
–
O
Two -amino acids are joined by a peptide bond
in alanylglycine. It is a dipeptide.
Dr. Wolf's CHM 424
27- 111
Alanylglycine
H
+
H3N
N-terminus
C
CH3
H
O
C
N
C
H
H
O
C
–
O
C-terminus
Ala—Gly
AG
Dr. Wolf's CHM 424
27- 112
Alanylglycine and glycylalanine are
constitutional isomers
H
+
H3N
C
C
CH3
H
+
H3N
C
H
Dr. Wolf's CHM 424
H
O
N
C
H
H
H
O
C
N
C
H
CH3
O
C
–
O
Alanylglycine
Ala—Gly
AG
–
O
Glycylalanine
Gly—Ala
GA
O
C
27- 113
Alanylglycine
H
+
H3N
C
CH3
H
O
C
N
C
H
H
O
C
–
O
The peptide bond is
characterized by a
planar geometry.
Dr. Wolf's CHM 424
27- 114
Higher Peptides
Peptides are classified according to the number
of amino acids linked together.
dipeptides, tripeptides, tetrapeptides, etc.
Leucine enkephalin is an example of a
pentapeptide.
Dr. Wolf's CHM 424
27- 115
Leucine Enkephalin
Tyr—Gly—Gly—Phe—Leu
YGGFL
Dr. Wolf's CHM 424
27- 116
Oxytocin
3
2
4
5
Ile—Gln—Asn
Tyr
1
Cys
N-terminus
C-terminus
Cys—Pro—Leu—GlyNH2
6
S
7
8
9
S
Oxytocin is a cyclic nonapeptide.
Instead of having its amino acids linked in an
extended chain, two cysteine residues are
joined by an S—S bond.
Dr. Wolf's CHM 424
27- 117
Oxytocin
S—S bond
An S—S bond between two cysteines is
often referred to as a disulfide bridge.
Dr. Wolf's CHM 424
27- 118
Introduction to Peptide
Structure Determination
Dr. Wolf's CHM 424
27- 119
Primary Structure
The primary structure is the amino acid
sequence plus any disulfide links.
Dr. Wolf's CHM 424
27- 120
Classical Strategy (Sanger)
1. Determine what amino acids are present and
their molar ratios.
2. Cleave the peptide into smaller fragments,
and determine the amino acid composition of
these smaller fragments.
3. Identify the N-terminus and C-terminus in the
parent peptide and in each fragment.
4. Organize the information so that the
sequences of small fragments can be
overlapped to reveal the full sequence.
Dr. Wolf's CHM 424
27- 121
Amino Acid Analysis
Dr. Wolf's CHM 424
27- 122
Amino Acid Analysis
Acid-hydrolysis of the peptide (6 M HCl, 24 hr)
gives a mixture of amino acids.
The mixture is separated by ion-exchange
chromatography, which depends on the
differences in pI among the various amino
acids.
Amino acids are detected using ninhydrin.
Automated method; requires only 10-5 to 10-7 g
of peptide.
Dr. Wolf's CHM 424
27- 123
Partial Hydrolysis of Proteins
Dr. Wolf's CHM 424
27- 124
Partial Hydrolysis of Peptides and Proteins
Acid-hydrolysis of the peptide cleaves all of the
peptide bonds.
Cleaving some, but not all, of the peptide bonds
gives smaller fragments.
These smaller fragments are then separated
and the amino acids present in each fragment
determined.
Enzyme-catalyzed cleavage is the preferred
method for partial hydrolysis.
Dr. Wolf's CHM 424
27- 125
Partial Hydrolysis of Peptides and Proteins
The enzymes that catalyze the hydrolysis of
peptide bonds are called peptidases, proteases,
or proteolytic enzymes.
Dr. Wolf's CHM 424
27- 126
Trypsin
Trypsin is selective for cleaving the peptide bond
to the carboxyl group of lysine or arginine.
O
O
O
NHCHC
NHCHC
NHCHC
R
R'
R"
lysine or arginine
Dr. Wolf's CHM 424
27- 127
Chymotrypsin
Chymotrypsin is selective for cleaving the peptide
bond to the carboxyl group of amino acids with
an aromatic side chain.
O
O
O
NHCHC
NHCHC
NHCHC
R
R'
R"
phenylalanine, tyrosine, tryptophan
Dr. Wolf's CHM 424
27- 128
Carboxypeptidase
Carboxypeptidase is selective for cleaving
the peptide bond to the C-terminal amino acid.
O
O
+
H3NCHC
R
Dr. Wolf's CHM 424
protein
C
O
–
NHCHCO
R
27- 129
End Group Analysis
Dr. Wolf's CHM 424
27- 130
End Group Analysis
Amino sequence is ambiguous unless we know
whether to read it left-to-right or right-to-left.
We need to know what the N-terminal and Cterminal amino acids are.
The C-terminal amino acid can be determined
by carboxypeptidase-catalyzed hydrolysis.
Several chemical methods have been
developed for identifying the N-terminus. They
depend on the fact that the amino N at the
terminus is more nucleophilic than any of the
amide nitrogens.
Dr. Wolf's CHM 424
27- 131
Sanger's Method
The key reagent in Sanger's method for
identifying the N-terminus is 1-fluoro-2,4dinitrobenzene.
1-Fluoro-2,4-dinitrobenzene is very reactive
toward nucleophilic aromatic substitution
(Section 23.5).
NO2
O2N
Dr. Wolf's CHM 424
F
27- 132
Sanger's Method
1-Fluoro-2,4-dinitrobenzene reacts with the
amino nitrogen of the N-terminal amino acid.
NO2
O2N
O
O
F + H2NCHC
NHCHC
NHCH2C
O2N
Dr. Wolf's CHM 424
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
–
NHCHCO
CH3
CH(CH3)2 CH2C6H5
NO2
O
O
O
–
NHCHCO
CH3
27- 133
Sanger's Method
Acid hydrolysis cleaves all of the peptide bonds
leaving a mixture of amino acids, only one of
which (the N-terminus) bears a 2,4-DNP group.
NO2
O
O
O
O
+
+
+
NHCHCOH + H3NCHCO– + H3NCH2CO– + H3NCHCO–
O2N
CH(CH3)2
CH3
CH2C6H5
H3O+
NO2
O2N
Dr. Wolf's CHM 424
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
O
–
NHCHCO
CH3
27- 134
Insulin
Dr. Wolf's CHM 424
27- 135
Insulin
Insulin is a polypeptide with 51 amino acids.
It has two chains, called the A chain (21 amino
acids) and the B chain (30 amino acids).
The following describes how the amino acid
sequence of the B chain was determined.
Dr. Wolf's CHM 424
27- 136
The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Dr. Wolf's CHM 424
27- 137
The B Chain of Bovine Insulin
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Dr. Wolf's CHM 424
27- 138
The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Overlaps between the above peptide sequences were
found in four additional peptides:
SHLV
LVGA
ALT
TLVC
Dr. Wolf's CHM 424
27- 139
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
YTPKA
Dr. Wolf's CHM 424
27- 140
The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Overlaps between the above peptide sequences were
found in four additional peptides:
SHLV
LVGA
ALT
TLVC
Trypsin-catalyzed hydrolysis gave GFFYTPK which
completes the sequence.
Dr. Wolf's CHM 424
27- 141
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
GFFYTPK
YTPKA
Dr. Wolf's CHM 424
27- 142
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
GFFYTPK
YTPKA
FVNQHLCGSHLVGALTLVCGERGFFYTPKA
Dr. Wolf's CHM 424
27- 143
Insulin
The sequence of the A chain was determined
using the same strategy.
Establishing the disulfide links between cysteine
residues completed the primary structure.
Dr. Wolf's CHM 424
27- 144
Primary Structure of Bovine Insulin
N terminus
of A chain
S
S
C terminus
of A chain
15
5
E Q C
V
C S L Y Q L
I
F
E N
20
C
V
YC
A S
S
10
N
S
S
H
L
N Q
V
S
C
F
G S H L V G A L Y L V
5
C
15
G 20
10
N terminus
E
of B chain
G R
F
F
Y
K P T
A
C terminus
25
30
of B chain
Dr. Wolf's CHM 424
27- 145
The Edman Degradation and
Automated Sequencing of
Peptides
Dr. Wolf's CHM 424
27- 146
Edman Degradation
1. Method for determining N-terminal amino
acid.
2. Can be done sequentially one residue at a
time on the same sample. Usually one can
determine the first 20 or so amino acids from
the N-terminus by this method.
3. 10-10 g of sample is sufficient.
4. Has been automated.
Dr. Wolf's CHM 424
27- 147
Edman Degradation
The key reagent in the Edman degradation is
phenyl isothiocyanate.
N
Dr. Wolf's CHM 424
C
S
27- 148
Edman Degradation
Phenyl isothiocyanate reacts with the amino
nitrogen of the N-terminal amino acid.
O
C6H5N
Dr. Wolf's CHM 424
C
S
+
+ H3NCHC
R
NH
peptide
27- 149
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
O
C6H5N
Dr. Wolf's CHM 424
C
S
+
+ H3NCHC
R
NH
peptide
27- 150
Edman Degradation
S
O
C6H5NHCNHCHC
NH
peptide
R
The product is a phenylthiocarbamoyl (PTC)
derivative.
The PTC derivative is then treated with HCl in
an anhydrous solvent. The N-terminal amino
acid is cleaved from the remainder of the
peptide.
Dr. Wolf's CHM 424
27- 151
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
HCl
S
C6H5NH
C
C
N
Dr. Wolf's CHM 424
CH
R
O
+
+
H3N
peptide
27- 152
Edman Degradation
The product is a thiazolone. Under the
conditions of its formation, the thiazolone
rearranges to a phenylthiohydantoin (PTH)
derivative.
S
C6H5NH
C
C
N
Dr. Wolf's CHM 424
CH
R
O
+
+
H3N
peptide
27- 153
Edman Degradation
C6H5
S
N
C
C
The PTH derivative is
isolated and identified.
The remainder of the
peptide is subjected to
a second Edman
degradation.
O
CH
HN
R
S
C6H5NH
C
C
N
Dr. Wolf's CHM 424
CH
R
O
+
+
H3N
peptide
27- 154
The Strategy of Peptide Synthesis
Dr. Wolf's CHM 424
27- 155
General Considerations
Making peptide bonds between amino acids is
not difficult.
The challenge is connecting amino acids in the
correct sequence.
Random peptide bond formation in a mixture of
phenylalanine and glycine, for example, will give
four dipeptides.
Phe—Phe Gly—Gly Phe—Gly Gly—Phe
Dr. Wolf's CHM 424
27- 156
General Strategy
1. Limit the number of possibilities by
"protecting" the nitrogen of one amino acid
and the carboxyl group of the other.
N-Protected
phenylalanine
O
X
NHCHCOH
Dr. Wolf's CHM 424
CH2C6H5
C-Protected
glycine
O
H2NCH2C
Y
27- 157
General Strategy
2. Couple the two protected amino acids.
X
O
O
NHCHC
NHCH2C
Y
CH2C6H5
O
X
NHCHCOH
Dr. Wolf's CHM 424
CH2C6H5
O
H2NCH2C
Y
27- 158
General Strategy
3. Deprotect the amino group at the N-terminus
and the carboxyl group at the C-terminus.
X
O
O
NHCHC
NHCH2C
Y
CH2C6H5
O
+
H3NCHC
Dr. Wolf's CHM 424
O
–
NHCH2CO
CH2C6H5
Phe-Gly
27- 159
Amino Group Protection
Dr. Wolf's CHM 424
27- 160
Protect Amino Groups as Amides
Amino groups are normally protected by
converting them to amides.
Benzyloxycarbonyl (C6H5CH2O—) is a common
protecting group. It is abbreviated as Z.
Z-protection is carried out by treating an amino
acid with benzyloxycarbonyl chloride.
Dr. Wolf's CHM 424
27- 161
Protect Amino Groups as Amides
O
O
CH2OCCl
+
+
–
H3NCHCO
CH2C6H5
1. NaOH, H2O
2. H+
O
CH2OC
O
NHCHCOH
CH2C6H5
Dr. Wolf's CHM 424
(82-87%)
27- 162
Protect Amino Groups as Amides
O
CH2OC
O
NHCHCOH
CH2C6H5
is abbreviated as:
O
ZNHCHCOH
or Z-Phe
CH2C6H5
Dr. Wolf's CHM 424
27- 163
Removing Z-Protection
An advantage of the benzyloxycarbonyl
protecting group is that it is easily removed by:
a) hydrogenolysis
b) cleavage with HBr in acetic acid
Dr. Wolf's CHM 424
27- 164
Hydrogenolysis of Z-Protecting Group
O
CH2OC
O
NHCHCNHCH2CO2CH2CH3
CH2C6H5
H2, Pd
O
CH3
CO2
H2NCHCNHCH2CO2CH2CH3
CH2C6H5
Dr. Wolf's CHM 424
(100%)
27- 165
HBr Cleavage of Z-Protecting Group
O
CH2OC
O
NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
O
CH2Br
Dr. Wolf's CHM 424
CO2
+
H3NCHCNHCH2CO2CH2CH3
–
CH2C6H5 Br
(82%)
27- 166
The tert-Butoxycarbonyl Protecting Group
O
(CH3)3COC
O
NHCHCOH
CH2C6H5
is abbreviated as:
O
BocNHCHCOH
or Boc-Phe
CH2C6H5
Dr. Wolf's CHM 424
27- 167
HBr Cleavage of Boc-Protecting Group
O
(CH3)3COC
O
NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
O
H3C
C
H3C
Dr. Wolf's CHM 424
CH2
CO2
+
H3NCHCNHCH2CO2CH2CH3
–
CH2C6H5 Br
(86%)
27- 168
Carboxyl Group Protection
Dr. Wolf's CHM 424
27- 169
Protect Carboxyl Groups as Esters
Carboxyl groups are normally protected as
esters.
Deprotection of methyl and ethyl esters is
by hydrolysis in base.
Benzyl esters can be cleaved by
hydrogenolysis.
Dr. Wolf's CHM 424
27- 170
Hydrogenolysis of Benzyl Esters
O
O
C6H5CH2OC
O
NHCHCNHCH2COCH2C6H5
CH2C6H5
H2, Pd
O
C6H5CH3
Dr. Wolf's CHM 424
CO2
+
–
H3NCHCNHCH2CO
CH2C6H5
(87%)
CH3C6H5
27- 171
Peptide Bond Formation
Dr. Wolf's CHM 424
27- 172
aa are covalently linked by amide bonds
(Peptide Bonds)
The resulting molecules are called
Peptides & Proteins
R'
R'
N
C
O
R
N
C
R
O
Features of a Peptide Bond;
1. Usually inert
2. Planar to allow delocalisation
3. Restricted Rotation about the amide bond
4. Rotation of Groups (R and R’) attached to the amide
bond is relatively free
Dr. Wolf's CHM 424
27- 173
aa that are part of a peptide or 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
Dr. Wolf's CHM 424
27- 174
CH2OH
CH3
O
O
H3N
CH
H3 N
C
O
C
H3N
C
O
O
O
Serine
Alanine
Valine
- 2 H2O
CH3
O
H
N
H3N
C
C
O
CH2OH
O
N
H
C
O
Tripeptide : Ala . Ser. Val
Strong Acid Required to hydrolyse peptide bonds
Dr. Wolf's CHM 424
27- 175
Lys. Cys. Phe
Phe. Ser. Cys
1. RSH
2. 6 M HCl hydrolysis
Lys + 2 Cys
+ 2 Phe + Ser
Ph
Cysteine residues create
Disulfide Bridges
between chains
(CH2)4NH2
O
H
N
H2 N
C
C
OH
N
H
O
O
This does not reveal
Primary Structure
C
S
S
Ph
O
H
N
H2 N
C
OH
N
H
O
C
O
HO
Dr. Wolf's CHM 424
27- 176
Forming Peptide Bonds
The two major methods are:
1. coupling of suitably protected amino acids
using N,N'-dicyclohexylcarbodiimide (DCCI)
2. via an active ester of the N-terminal amino
acid.
Dr. Wolf's CHM 424
27- 177
DCCI-Promoted Coupling
O
O
ZNHCHCOH
+ H2NCH2COCH2CH3
CH2C6H5
DCCI, chloroform
O
ZNHCHC
O
NHCH2COCH2CH3
CH2C6H5
Dr. Wolf's CHM 424
(83%)
27- 178
Mechanism of DCCI-Promoted Coupling
O
+
ZNHCHCOH
C6H11N
C
NC6H11
CH2C6H5
H
C6H11N
O
C
C6H11N
Dr. Wolf's CHM 424
OCCHNHZ
CH2C6H5
27- 179
Mechanism of DCCI-Promoted Coupling
The species formed by addition of the Zprotected amino acid to DCCI is similar in
structure to an acid anhydride and acts as an
acylating agent.
Attack by the amine function of the carboxylprotected amino acid on the carbonyl group
leads to nucleophilic acyl substitution.
H
C6H11N
O
C
C6H11N
Dr. Wolf's CHM 424
OCCHNHZ
CH2C6H5
27- 180
Mechanism of DCCI-Promoted Coupling
O
H
C6H11N
C
O +
ZNHCHC
O
NHCH2COCH2CH3
CH2C6H5
C6H11NH
O
H2NCH2COCH2CH3
H
C6H11N
O
C
C6H11N
Dr. Wolf's CHM 424
OCCHNHZ
CH2C6H5
27- 181
The Active Ester Method
A p-nitrophenyl ester is an example of an "active
ester."
p-Nitrophenyl is a better leaving group than
methyl or ethyl, and p-nitrophenyl esters are
more reactive in nucleophilic acyl substitution.
Dr. Wolf's CHM 424
27- 182
The Active Ester Method
O
O
ZNHCHCO
NO2 +
H2NCH2COCH2CH3
CH2C6H5
chloroform
O
ZNHCHC
O
NHCH2COCH2CH3 + HO
CH2C6H5
Dr. Wolf's CHM 424
NO2
(78%)
27- 183
Solid-Phase Peptide Synthesis:
The Merrifield Method
Dr. Wolf's CHM 424
27- 184
Solid-Phase Peptide Synthesis
In solid-phase synthesis, the starting material is
bonded to an inert solid support.
Reactants are added in solution.
Reaction occurs at the interface between the
solid and the solution. Because the starting
material is bonded to the solid, any product from
the starting material remains bonded as well.
Purification involves simply washing the
byproducts from the solid support.
Dr. Wolf's CHM 424
27- 185
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
The solid support is a copolymer of styrene and
divinylbenzene. It is represented above as if it
were polystyrene. Cross-linking with
divinylbenzene simply provides a more rigid
polymer.
Dr. Wolf's CHM 424
27- 186
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
Treating the polymeric support with
chloromethyl methyl ether (ClCH2OCH3) and
SnCl4 places ClCH2 side chains on some of the
benzene rings.
Dr. Wolf's CHM 424
27- 187
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
CH2Cl
The side chain chloromethyl group is a benzylic
halide, reactive toward nucleophilic substitution
(SN2).
Dr. Wolf's CHM 424
27- 188
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
CH2Cl
The chloromethylated resin is treated with the Bocprotected C-terminal amino acid. Nucleophilic
substitution occurs, and the Boc-protected amino
acid is bound to the resin as an ester.
Dr. Wolf's CHM 424
27- 189
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
–
BocNHCHCO
CH
CH2
CH
CH2Cl
R
Dr. Wolf's CHM 424
27- 190
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
CH
CH2
CH
CH2
BocNHCHCO
Next, the Boc
protecting group is
removed with HCl.
Dr. Wolf's CHM 424
R
27- 191
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
CH
CH2
CH
CH2
H2NCHCO
DCCI-promoted
coupling adds the
second amino acid
Dr. Wolf's CHM 424
R
27- 192
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
BocNHCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
Remove the Boc
protecting group.
Dr. Wolf's CHM 424
27- 193
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
H2NCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
Add the next amino
acid and repeat.
Dr. Wolf's CHM 424
27- 194
The Merrifield Procedure
CH2
CH
CH2
O
CH
O
+
H3N peptide C NHCHC
R'
Dr. Wolf's CHM 424
CH2
CH
O
CH2
CH
CH2
NHCHCO
R
Remove the peptide
from the resin with
HBr in CF3CO2H
27- 195
The Merrifield Procedure
CH2
CH
CH2
CH
CH
CH2
CH
CH2Br
O
O
+
H3N peptide C NHCHC
R'
Dr. Wolf's CHM 424
CH2
O
–
NHCHCO
R
27- 196
The Merrifield Method
Merrifield automated his solid-phase method.
Synthesized a nonapeptide (bradykinin) in 1962
in 8 days in 68% yield.
Synthesized ribonuclease (124 amino acids) in
1969.
369 reactions; 11,391 steps
Nobel Prize in chemistry: 1984
Dr. Wolf's CHM 424
27- 197
Secondary Structures
of Peptides and Proteins
Dr. Wolf's CHM 424
27- 198
Levels of Protein Structure
Primary structure = the amino acid sequence
plus disulfide links
Secondary structure = conformational
relationship between nearest neighbor amino
acids
 helix
pleated  sheet
Dr. Wolf's CHM 424
27- 199
Levels of Protein Structure
The -helix and pleated  sheet are both
characterized by:
planar geometry of peptide bond
anti conformation of main chain
hydrogen bonds between N—H and O=C
Dr. Wolf's CHM 424
27- 200
-helixes
Intra-chain
H-bonds
Secondary Structure
Dr. Wolf's CHM 424
27- 201
-strands
Inter-chain
H-bonds
Secondary Structure
Dr. Wolf's CHM 424
27- 202
Pleated  Sheet
Shown is a  sheet of protein chains composed of
alternating glycine and alanine residues.
Adjacent chains are antiparallel.
Hydrogen bonds between chains.
van der Waals forces produce pleated effect.
Dr. Wolf's CHM 424
27- 203
Pleated  Sheet
 Sheet is most commonly seen with amino acids
having small side chains (glycine, alanine, serine).
80% of fibroin (main protein in silk) is repeating
sequence of —Gly—Ser—Gly—Ala—Gly—Ala—.
 Sheet is flexible, but resists stretching.
Dr. Wolf's CHM 424
27- 204
 Helix
Shown is an  helix of a protein
in which all of the amino acids
are L-alanine.
Helix is right-handed with 3.6
amino acids per turn.
Hydrogen bonds are within a
single chain.
Protein of muscle (myosin) and
wool (-keratin) contain large
regions of -helix. Chain can
be stretched.
Dr. Wolf's CHM 424
27- 205
Secondary Structure
The Development of Regular patterns of Hydrogen
Bonding, which result in distinct folding patterns
-helix
Dr. Wolf's CHM 424
-pleated sheets
27- 206
Tertiary Structure
of Peptides and Proteins
Dr. Wolf's CHM 424
27- 207
Tertiary Structure
Refers to overall shape (how the chain is folded)
Fibrous proteins (hair, tendons, wool) have
elongated shapes
Globular proteins are approximately spherical
most enzymes are globular proteins
an example is carboxypeptidase
Dr. Wolf's CHM 424
27- 208
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.
Dr. Wolf's CHM 424
27- 209
In globular proteins this tertiary structure or
macromolecular shape determines biological properties
Bays or pockets in proteins are called Active Sites
Enzymes are Stereospecific and possess Geometric Specificity
The range of compounds that an enzyme excepts varies
from a particular functional group to a specific compound
Emil Fischer formulated the lock-and-key mechanism for enzymes
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.
Dr. Wolf's CHM 424
27- 210
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
Dr. Wolf's CHM 424
27- 211
Tertiary
structure
Hb monomer
(or myoglobin)
Dr. Wolf's CHM 424
Quaternary
structure
Hb 22 tetramer
27- 212
Carboxypeptidase
Carboxypeptidase is an enzyme that catalyzes
the hydrolysis of proteins at their C-terminus.
It is a metalloenzyme containing Zn2+ at its
active site.
An amino acid with a positively charged side
chain (Arg-145) is near the active site.
Dr. Wolf's CHM 424
27- 213
Carboxypeptidase
Disulfide bond
Zn2+
Arg-145
N-terminus
C-terminus
tube model
Dr. Wolf's CHM 424
ribbon model
27- 214
What happens at the active site?
•• •
O•
+
H3N
peptide
C
O
NHCHC –
R
Dr. Wolf's CHM 424
O
H2N
+ C
Arg-145
H2N
27- 215
What happens at the active site?
•• •
O•
+
H3N
peptide
C
O
NHCHC –
R
O
H2N
+ C
Arg-145
H2N
The peptide or protein is bound at the active site
by electrostatic attraction between its negatively
charged carboxylate ion and arginine-145.
Dr. Wolf's CHM 424
27- 216
What happens at the active site?
•• •
O•
+
H3N
peptide
C
Zn2+
O
NHCHC –
R
O
H2N
+ C
Arg-145
H2N
Zn2+ acts as a Lewis acid toward the carbonyl
oxygen, increasing the positive character of the
carbonyl carbon.
Dr. Wolf's CHM 424
27- 217
What happens at the active site?
Zn2+
•• •
O•
+
H3N
peptide
C
O
NHCHC –
R
O
H2N
+ C
Arg-145
H2N
H
• O•
• •
H
Water attacks the carbonyl carbon. Nucleophilic
acyl substitution occurs.
Dr. Wolf's CHM 424
27- 218
What happens at the active site?
Zn2+
H2N
+ C
•• •
O•
+
H3N
peptide
C
•• –
O ••
••
H2N
O
+
H3NCHC –
R
Dr. Wolf's CHM 424
Arg-145
O
27- 219
Coenzymes
Dr. Wolf's CHM 424
27- 220
Coenzymes
The range of chemical reactions that amino acid
side chains can participate in is relatively
limited.
acid-base (transfer and accept protons)
nucleophilic acyl substitution
Many other biological processes, such as
oxidation-reduction, require coenzymes,
cofactors, or prosthetic groups in order to occur.
Dr. Wolf's CHM 424
27- 221
Coenzymes
NADH, coenzyme A and coenzyme B12 are
examples of coenzymes.
Heme is another example.
Dr. Wolf's CHM 424
27- 222
Heme
H 2C
CH
H 3C
CH3
N
N
CH
CH2
Fe
N
H 3C
HO2CCH2CH2
N
CH3
CH2CH2CO2H
Molecule surrounding iron is a
type of porphyrin.
Dr. Wolf's CHM 424
27- 223
Myoglobin
C-terminus
Heme
N-terminus
Heme is the coenzyme that binds oxygen in myoglobin
(oxygen storage in muscles) and hemoglobin (oxygen
transport).
Dr. Wolf's CHM 424
27- 224
Protein Quaternary Structure:
Hemoglobin
Dr. Wolf's CHM 424
27- 225
Protein Quaternary Structure
Some proteins are assemblies of two or more
chains. The way in which these chains are
organized is called the quaternary structure.
Hemoglobin, for example, consists of 4
subunits.
There are 2  chains (identical) and 2  chains
(also identical).
Each subunit contains one heme and each
protein is about the size of myoglobin.
Dr. Wolf's CHM 424
27- 226
Protein Structure
Primary structure is the amino acid sequence.
Secondary structure is how the amino acids in sequence fold up
locally. Examples are -helixes and -strands and loops.
Tertiary structure is the 3-dimensional folding of the secondary
structural elements and connecting loops in space.
Quaternary structure is the association of multiple subunits,
each with a tertiary structure and each a unique gene product.
Dr. Wolf's CHM 424
27- 227
Stabilization of Protein Structure
Electrostatic interactions involve the interaction of (+) and (-)
charged side groups.
Hydrogen bonds involve sharing of a hydrogen atom between
two eletronegative atoms (e.g., O, N).
Van der Waal’s forces are weak forces based on optimal
overlap of adjacent electronic orbitals. Can be repulsive.
Hydrophobic interactions are, by far, the most powerful force
stabilizing protein structure. Basis of force is entropy gain
realized by burying hydrophobic residues.
Dr. Wolf's CHM 424
27- 228
Cofactors
Cofactors are exogenous molecules that associate with
proteins to yield full activity. In the absence of cofactor,
protein is an apoprotein.
Co-enzymes are soluble and associate transiently with enzyme
during catalytic cycle. An example is vitamin K in activation of
blood clotting enzymes.
Prosthetic groups are covalently attached to the protein.
Examples are heme, in hemoglobin, and riboflavin, in
flavoproteins.
Dr. Wolf's CHM 424
27- 229
REVERSIBLE DENATURING
Oxidation
RS SR
RS H
Reduction
Prof. Linus Pauling
Dr. Frederick Sanger,
Prof. R. B. Merrifield
Nobel Prize for Chemistry Nobel Prize for Chemistry 1984
1958 and 1980
Automated Peptide Synthesis
Dr. Wolf's
CHM 424
Peptide
sequencing
27- 230
Globular Proteins
Globular proteins fold so as to "bury" the hydrophobic side
chains, minimizing their contact with water
Most polar residues face the outside of the protein and interact
with solvent
Most hydrophobic residues face the interior of the protein and
interact with each other
Packing of residues is close, but protein interiors contain some
empty space
The empty space is in the form of small cavities
Dr. Wolf's CHM 424
27- 231
Globular Proteins
Globular proteins fold so as to "bury" the hydrophobic side
chains, minimizing their contact with water
Most polar residues face the outside of the protein and interact
with solvent
Most hydrophobic residues face the interior of the protein and
interact with each other
Packing of residues is close, but protein interiors contain some
empty space
The empty space is in the form of small cavities
Dr. Wolf's CHM 424
27- 232
Objectives
Diagram and describe the effect of oxygen on the position of iron relative to the
heme plane.
Describe how cooperative binding of oxygen by hemoglobin improves its
effectiveness as an oxygen carrier.
Describe the relationship between Hb structure to the Bohr effect and explain its
physiological significance..
Discuss how carbon dioxide affects the affinity of Hb for oxygen and why this is
physiologically significant.
Explain the effect of bisphosphoglycerate (BPG) on the affinity of Hb for oxygen
and how this is related to altitude and HbF.
Explain how carbon monoxide (CO) binds to Hb and its affinity relative to that of
oxygen..
Describe the molecular basis of thalassemias and the aberrant Hb that are
produced in these diseases..
List three embryonic forms of Hb..
Dr. Wolf's CHM 424
27- 233
Myoglobin: 2o and 3o aspects
Myoglobin is a single peptide chain of 153 residues
arranged in eight -helical regions labeled A-H.
The heme cofactor is the oxygen binding site so it is
necessary for myoglobin’s function, oxygen storage in
mammalian muscle tissue.
His E7 and F8 are important for binding the heme group
within the protein and for stabilizing bound oxygen.
Dr. Wolf's CHM 424
27- 234
Myoglobin and Hemoglobin
Mb is monomer, Hb is a tetramer (22).
Hb subunits are structurally similar to Mb, with 8 helical regions, no -strands and no water.
Both contain heme prosthetic group
Both Mb and Hb contain proximal and distal
histidines.
Affinity of Mb for oxygen is high, affinity of Hb for
oxygen is low.
Dr. Wolf's CHM 424
27- 235
Myoglobin &Hemoglobin
Two related protein for O2 transportation.
Mb has one chain
Hb has four chains
Each chain has two parts: a globin ( protein) and
a heme ( non-protein)
Dr. Wolf's CHM 424
27- 236
Myoglobin
• 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
pocket.
Polar groups exposed to
solvent, Non-polar groups
buried.
Dr. Wolf's CHM 424
27- 237
Myoglobin:
Dr. Wolf's CHM 424
o
2
and
o
3
structure
27- 238
The 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
Dr. Wolf's CHM 424
27- 239
Binding of O2 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
Dr. Wolf's CHM 424
27- 240
The Heme Group
-
CH2CH2COO
OOC CH2CH2
H3C
N
-
N
CH3
Fe(II)
H2C
N
CH
CH3
CH3
Dr. Wolf's CHM 424
N
Pyrrole ring
CH CH2
27- 241
N of His F8 binds
to 5th coordination
site on heme iron
Oxygen binds to 6th
coordination site on
heme iron
Dr. Wolf's CHM 424
27- 242
His E7 acts as a gate to favor oxygen
binding over carbon monoxide.
Dr. Wolf's CHM 424
27- 243
Hemoglobin
A tetrameric protein
two -chains (141 AA)
two -chains (146 AA)
four heme cofactors, one in each chain
The  and  chains are homologous to myoglobin.
Oxygen binds to heme in hemoglobin with same
structure as in Mb but cooperatively: as one O2 is
bound, it becomes easier for the next to bind.
Dr. Wolf's CHM 424
27- 244
Hemoglobin
• 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
 11 (22)- 35 residues, 12 (21)- 19 residues
Dr. Wolf's CHM 424
27- 245
Dr. Wolf's CHM 424
27- 246
Each chain is in
ribbon form.
The heme
groups are in
space filling
form
Dr. Wolf's CHM 424
27- 247
Oxygen Binding Curves
Hemoglobin and myoglobin respond differently
to increase in O2 concentration.
Myoglobin shows normal saturation behavior
while hemoglobin shows cooperative behavior.
Each oxygen added to a heme of Hb makes
addition of the next one easier.
The myoglobin curve is hyperbolic.
The hemoglobin curve is sigmoidal.
Dr. Wolf's CHM 424
27- 248
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*
Dr. Wolf's CHM 424
27- 249
Oxygen Binding Curves-2
Dr. Wolf's CHM 424
27- 250
Hemoglobin Equilibrium



O2
+
H ,CO
2,

BPG
T
R
(low affinity)
(high affinity)
Dr. Wolf's CHM 424
27- 251
A Quaternary Structure
Change
One alpha-beta pair moves
relative to the other by 15
degrees upon oxygen binding
This large change is caused by
movement of Fe by only 0.039
nm when oxygen binds
Dr. Wolf's CHM 424
27- 252
Oxygen binding by hemoglobin
Dr. Wolf's CHM 424
27- 253
Allosteric 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)
Dr. Wolf's CHM 424
27- 254
The Bohr Effect
Competition between oxygen and H+
Discovered by Christian Bohr
Binding of protons diminishes oxygen binding
Binding of oxygen diminishes proton binding
Important physiological significance-O2 saturation
of Hb responds to pH
Dr. Wolf's CHM 424
27- 255
The Bohr Effect
Dr. Wolf's CHM 424
27- 256
Bohr Effect II
Carbon dioxide diminishes oxygen binding
CO2 produced in metabolically active tissue
(requires oxygen)
Hydration of CO2 in tissues and extremities
leads to proton production
CO2 + H2O  HCO3- + H+
These protons are taken up by Hb forcing more
oxygen to dissociate
The reverse occurs in the lungs
Dr. Wolf's CHM 424
27- 257
Carbon 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.
Dr. Wolf's CHM 424
27- 258
2,3-Bisphosphoglycerate
An Allosteric Effector of Hemoglobin
The sigmoid binding curve is only observed in the presence
of 2,3-BPG
Since 2,3-BPG binds at a site distant from the Fe where
oxygen binds, it is called an allosteric effector
Dr. Wolf's CHM 424
27- 259
2,3-bisphosphoglycerate
(2,3-BPG) is a negative
allosteric effector of O2
binding to Hb - binds
tighter to deoxyHb
2,3-BPG
Dr. Wolf's CHM 424
27- 260
Heme in hemoglobin
Proximal
His (F8)
Proximal
His (F8)
Proximal
His (F8)

=C
=N
=O
=C
=N
=O
Fe
Proximal
His (F8)
Distal
His (E7)
Heme prosthetic group
Dr. Wolf's CHM 424
=C
=N
=O
Fe
Distal
His (E7)
Distal
His (E7)
Proximal
His (F8)
Fe
=C
=N
=O
Fe

Distal
His (E7)

Fe
=C
=N
=O
Distal
His (E7)
Side view of Hb tetramer
27- 261
Binding of oxygen to heme iron
Ferrous is reduced
and +2 charge
Proximal
His (F8)
Ferric is oxidized and
+3 charge
Fe
-
e
=C
=N
=O
Dr. Wolf's CHM 424
Distal
His (E7)
27- 262
Effect of oxygen on heme iron
=C
=N
=O
FG1
C
N
F7
FG3
FG2
F6
Proximal
His (F8)
Fe
Plane of heme
D
istal
H
is(E
7)
Dr. Wolf's CHM 424
27- 263
Effect of oxygen on heme iron
=C
=N
=O
N
FG1
C
FG3
F7
FG2
F6
Proximal
His (F8)
Plane of heme
Fe
D
istal
H
is(E
7)
Dr. Wolf's CHM 424
27- 264
Effect of oxygen on heme iron
=C
=N
=O
FG1
FG1
CC
FG3FG3
N
N
F7
F7
FG2
FG2
F6F6
P
roximal
P
roximal
H
is
8)
H
is(F
(F
8)
Fe
Plane of heme
Fe
D
istal
H
is(E
7)
Dr. Wolf's CHM 424
27- 265
Cooperativity
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 second
four stamps.
To
To
pull
stamp,
To
pull
third
stamp,
you
To pull
fourth
stamp,
pull
first
stamp,
youonly
you
have
to
break
have
to
break
only
one
you
don’t
have
to
break
have
to break two edges.
one
edge.
edge.
any edges.
Dr. Wolf's CHM 424
27- 266
Cooperativity
100
BPGEffect
Mbalone
Hb
80
Hb +Hb
BPG
60
40
Sigmoid shape
indicates positive
cooperativity
20
pO2 vs p50=8
pO2 vs p50=26
0
0
20
40
60
80
100
120
140
160
pO2 (mm Hg)
Dr. Wolf's CHM 424
27- 267
Bohr Effect
Bohr Effect
100
7.4
7.6
80
7.0
7.2
60
O2 level in
arterial blood
40
20
0
O2 level in
venous blood
Dr. Wolf's CHM 424
0
20
40
60
80
100
120
140
160
pO2 (mm Hg)
27- 268
Hb structural families
Alpha family 1,2 - found in adult hemoglobins HbA1, HbA2.
z - found in embryonic hemoglobins Hb Gower 1 and
Hb Portland.
Beta family  - found in adult hemoglobin HbA1.
d - found in adult hemoglobin HbA2.
 - found in fetal hemoglobin HbF.
e - found in embryonic hemoglobin Hb Gower 1 and Hb
Gower 2
Dr. Wolf's CHM 424
27- 269
CO2 effect
CO2 Effect
100
pCO2 20 mm
80
pCO2 80 mm
60
40
20
pO2 vs p50=20
pO2 vs p50=40
0
0
20
40
60
80
100
120
140
160
pO2 (mm Hg)
Dr. Wolf's CHM 424
27- 270
Effect of BPG
BPG is responsible for
cooperativity.
BPGEffect
Hb alone
100
80
Hb + BPG
60
40
High altitude increases BPG,
pushing curve further to right
20
pO2 vs p50=8
pO2 vs p50=26
0
0
20
40
60
80
100
120
140
160
pO2 (mm Hg)
Dr. Wolf's CHM 424
27- 271
Effect of BPG


BPG

Side view (R)
Dr. Wolf's CHM 424




Side view (T)
27- 272
Effect of BPG
- Lys His His
+
+
+
Dr. Wolf's CHM 424
27- 273
Hemoglobin Equilibrium



O2
+
H ,CO
2,

BPG
T
R
(low affinity)
(high affinity)
Dr. Wolf's CHM 424
27- 274
Hemoglobins in normal adults
α
β
α
γ
α
β
α
γ
α
δ
δ
α
HbA
HbF
HbA2
98%
~1%
<3.5%
Dr. Wolf's CHM 424
27- 275
Dr. Wolf's CHM 424
27- 276
Globin gene clusters
 cluster, 16p 13.3
z
2


1
1,2= duplicate genes, both expressed
 cluster, 11p 15.5
e
G
A
d

G,=fetal genes, Gly and Ala at postion 136,
Dr. Wolf's CHM 424
both expressed
27- 277
Hb structural families
Alpha family 1,2 - found in adult hemoglobins HbA1, HbA2.
z - found in embryonic hemoglobins Hb Gower 1 and
Hb Portland.
 - (theta) newly discovered embryonic form.
Beta family  - found in adult hemoglobin HbA1.
d - found in adult hemoglobin HbA2.
 - found in fetal hemoglobin HbF.
e - found in embryonic hemoglobin Hb Gower 1 and Hb
Gower 2
Dr. Wolf's CHM 424
27- 278
FETAL AND NEONATAL ERYTHROPOIESIS
TABLE 1. Globin-chain development and composition
Developmental
stage
Hemoglobin
type
Globin-chain
composition
Embryo
Embryo
Embryo
Embryo to fetus
Fetus to adult
Adult
Adult
Gower 1
Gower 2
Portland
Fetal
A
A2
Fetal
Zeta2 , epsilon2a
Alpha2, epsilon2
Zeta2, gamma2
Alpha2, gamma2
Alpha2, beta2
Alpha2, delta2
Alpha2, gamma2b
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
Dr. Wolf's CHM 424
hemoglobin
27- 279
Inherited Hemoglobin disorder
Definition: An inherited mutation of the
globin genes leading to a qualitative or
quantitative abnormality of globin
synthesis
Dr. Wolf's CHM 424
27- 280
The Thalassemias
(quantitative)
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
Dr. Wolf's CHM 424
27- 281
Alpha Thalassemias
Rare, since  gene is duplicated (four genes per
diploid).
Usually more severe than beta thalassemia because
there is no substitute for  gene in adults.
Almost all  thalassemias are deletions
In  thalassemia major (0/00) - occurrence of HbH
(4) and Hb Bart’s (4).
BPG is ineffective in HbH & Hb Bart’s.
Dr. Wolf's CHM 424
27- 282
Beta thalassemia
Impaired production of beta chain
beta thalassemia minor – heterozygous (or trait)
beta thalassemia major - homozygous
Dr. Wolf's CHM 424
27- 283
Beta thalassemia - heterozygous (minor or trait)
Target cell
Oval cell
Dr. Wolf's CHM 424
27- 284
Beta thalassemia major
Dr. Wolf's CHM 424
27- 285
Beta Thalassemias
More common, since  gene is present in
only one copy per chromosome.
Less severe than  thalassemia, since d
chain can effectively substitute in adults.
The  chain can also persist into adulthood
(HPFH).
In d thal major (d0/d0) excess  chains do
not form soluble homotetramers.
Dr. Wolf's CHM 424
27- 286
Beta thalassemia major
No beta chain produced (no HbA)
Severe microcytic anemia occurs gradually in
the first year of life
Marrow expansion
Iron overload
Growth failure and death
Dr. Wolf's CHM 424
27- 287
Dr. Wolf's CHM 424
27- 288
Alpha thalassemia
/

Normal
/
-
Mild microcytosis
/-
Mild microcytosis
-/- -
Hemoglobin H disease
- -/- -
Hemoglobin Barts – Hydrops Fetalis
Dr. Wolf's CHM 424
27- 289
Structural hemoglobinopathy
(qualitative)
Amino acid substitution in the globin
chain e.g. sickle hemoglobin (HbS)
Dr. Wolf's CHM 424
27- 290
Sickle cell hemoglobin
Glu
Glu
-
-
Glu






HbS
(heterozygous)
Sickle cell trait
HbS
(homozygous)
Sickle cell disease
-
HbA1
Dr. Wolf's CHM 424
27- 291
Dr. Wolf's CHM 424
27- 292
Red Blood Cells from Sickle Cell Anemia
Deoxygenation of SS erythrocytes leads to intracellular
hemoglobin polymerization, loss of deformability and
changes in cell morphology.
OXY-STATE
Dr. Wolf's CHM 424
DEOXY-STATE
27- 293
Sickle Cell Anemia – blood film
Sickle
Cells
Erythroblasts
HowellJolly Body
Dr. Wolf's CHM 424
27- 294
Fibres of Sickle Hemoglobin
Dr. Wolf's CHM 424
27- 295
Fibres of Sickle
Hemoglobin – cross
section
Dr. Wolf's CHM 424
27- 296
Hemoglobin S
Valine is exposed in deoxy-Hemoglobin
Dr. Wolf's CHM 424
27- 297
Polymerization of HbS



  
  
  
  
  


Dr. Wolf's CHM 424
27- 298