Amino Acids, Peptides,
and Proteins
Dr Shahnaz Khaghani
Tehran University of Medical Sciences
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.
27- 2
Amino Acids : 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.
• Define the meaning of Isoelectric Point .
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
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.
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.
27- 6
Amino Acids
+
NH3

CO2–
+
–
H3NCH2CH2CO2

+
–
H3NCH2CH2CH2CO2

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- 7
Classification of Amino Acids
27- 8
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.
27- 9
H
+
H3N
C
O
C
O
–
R
The major differences among the side chains
concern:
Size and shape
Electronic characteristics
27- 10
General categories of -amino acids
nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains
27- 11
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
27- 12
1. Hydrophobic (non-polar) residues
Usually interior of proteins away from water.
Hydrocarbon: do not contain polar atoms.
27- 13
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]
27- 14
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
Glycine Gly [G] 0
Threonine Thr [T] 0.4
Alanine Ala [A] 0.5
Methionine Met [M]
1.3
Valine Val [V] 1.5
LeucineLeu [L] 1.8
Tyrosine Tyr [Y] 2.3
Isoleucine Ile [I] 2.5
Phenylalanine Phe [F] 2.5
Tryptophan Trp [W]3.4
27- 15
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
27- 16
20 Amino acids
Glycine (G)
Alanine (A)
Valine (V)
Isoleucine (I)
Leucine (L)
Proline (P)
Methionine (M)
Phenylalanine (F)
Tryptophan (W)
Asparagine (N)
Glutamine (Q)
Serine (S)
Threonine (T)
Tyrosine (Y)
Cysteine (C)
Lysine (K)
Arginine (R)
Histidine (H)
Asparatic acid (D) Glutamic acid (E)
White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic
27- 17
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.
27- 18
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.
27- 19
H
+
H3N
C
O
C
O
–
CH(CH3)2
Valine
(Val or V)
27- 20
H
+
H3N
C
O
C
O
–
CH2CH(CH3)2
Leucine
(Leu or L)
27- 21
H
+
H3N
C
O
C
O
–
CH3CHCH2CH3
Isoleucine
(Ile or I)
27- 22
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.
27- 23
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.
27- 24
H
+
H3N
C
CH2
O
C
O
–
Phenylalanine
(Phe or F)
The side chain in phenylalanine (a nonpolar
amino acid) is a benzyl group.
27- 25
H
+
H3N
C
O
C
O
–
Tryptophan
CH2
(Trp or W)
N
H
The side chain in
tryptophan (a nonpolar
amino acid) is larger
and more polarizable
than the benzyl group
of phenylalanine.
27- 26
H
+
H3N
C
O
C
O
–
CH2OH
Serine
(Ser or S)
The —CH2OH side chain in serine can be
involved in hydrogen bonding.
27- 27
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.
27- 28
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.
27- 29
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
27- 30
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.
27- 31
H
+
H3N
C
H2NCCH2CH2
O
C
O
–
Glutamine
(Gln or Q)
O
27- 32
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.
27- 33
H
+
H3N
–
C
OCCH2CH2
O
C
O
–
Glutamic Acid
(Glu or E)
O
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.
27- 35
H
Arginine
(Arg or R)
+
H3N
C
O
C
O
–
CH2CH2CH2NHCNH2
+ NH2
27- 36
H
Histidine
+
H3N
(His or H)
C
CH2
N
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
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
27- 39
R
H2N
CH
CO2H
All DNA encoded aa are 
CHO
All are chiral,
except Glycine
R=H
H
OH
CHO
HO
CH2OH
CH2OH
D-
All DNA
encoded aa
are usually L-
H
LR
CHO
=
HO
CH2OH
H
(S) - Glyceraldehyde
(-) -
=
H2N
C
CO2H
H
(L) - Amino Acids
(-) 27- 40
Acid-Base Behavior of Amino
Acids
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.
27- 42
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
For aa with basic R-groups, we require higher
pHs, and
for aa with acidic R-groups, we require lower
pHs
27- 43
to reach the Isoelectric Point
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
i.e. 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 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
•• O ••
•• •–
O•
••
••
H2NCH2C
••
OH
••
27- 48
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
•• •–
O•
••
called a zwitterion or
dipolar ion
27- 49
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
••
OH
••
27- 50
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
•• O ••
+
H3NCH2C
••
typical
carboxylic
acid: pKa ~5
OH
••
27- 51
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
••
typical
carboxylic
acid: pKa ~5
OH
••
27- 52
Acid-Base Properties of Glycine
Therefore, the more stable neutral form of
glycine is the zwitterion.
•• O ••
+
H3NCH2C
•• •–
O•
••
•• O ••
+
H3NCH2C
••
typical
carboxylic
acid: pKa ~5
OH
••
27- 53
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
••
typical
carboxylic
acid: pKa ~5
OH
••
27- 54
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).
27- 55
Isoelectric Point pI
•• O ••
+
H3NCH2C
••
OH
••
pKa = 2.34
•• O ••
+
H3NCH2C
•• •–
O•
••
pKa = 9.60
•• O ••
••
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- 56
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.
27- 57
Amino Acids with Neutral Side Chains
H
Glycine
+
H3N
C
O
C
O
–
pKa1 = 2.34
pKa2 = 9.60
pI = 5.97
H
27- 58
Amino Acids with Neutral Side Chains
H
Alanine
+
H3N
C
O
C
O
–
pKa1 = 2.34
pKa2 = 9.69
pI = 6.00
CH3
27- 59
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
27- 60
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
27- 61
Amino Acids with Neutral Side Chains
H
Isoleucine
+
H3N
C
O
C
O
–
pKa1 = 2.36
pKa2 = 9.60
pI = 5.98
CH3CHCH2CH3
27- 62
Amino Acids with Neutral Side Chains
H
Methionine
+
H3N
C
O
C
O
–
pKa1 = 2.28
pKa2 = 9.21
pI = 5.74
CH3SCH2CH2
27- 63
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
27- 64
Amino Acids with Neutral Side Chains
H
Phenylalanine
+
H3N
C
CH2
O
C
O
–
pKa1 = 1.83
pKa2 = 9.13
pI = 5.48
27- 65
Amino Acids with Neutral Side Chains
H
Tryptophan
+
H3N
C
CH2
O
C
O
–
pKa1 = 2.83
pKa2 = 9.39
pI = 5.89
N
H
27- 66
Amino Acids with Neutral Side Chains
H
Asparagine
+
H3N
C
O
C
O
–
pKa1 = 2.02
pKa2 = 8.80
pI = 5.41
H2NCCH2
O
27- 67
Amino Acids with Neutral Side Chains
H
Glutamine
+
H3N
C
H2NCCH2CH2
O
C
O
–
pKa1 = 2.17
pKa2 = 9.13
pI = 5.65
O
27- 68
Amino Acids with Neutral Side Chains
H
Serine
+
H3N
C
O
C
O
–
pKa1 = 2.21
pKa2 = 9.15
pI = 5.68
CH2OH
27- 69
Amino Acids with Neutral Side Chains
H
Threonine
+
H3N
C
O
C
O
–
pKa1 = 2.09
pKa2 = 9.10
pI = 5.60
CH3CHOH
27- 70
Amino Acids with Neutral Side Chains
H
Tyrosine
+
H3N
C
CH2
OH
O
C
O
–
pKa1 = 2.20
pKa2 = 9.11
pI = 5.66
27- 71
Amino Acids with Neutral Side Chains
H
Cysteine
+
H3N
C
O
C
O
–
pKa1 = 1.96
pKa2 = 8.18
pI = 5.07
CH2SH
27- 72
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.
27- 73
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
27- 74
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.
27- 75
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
27- 76
Amino Acids with Ionizable Side Chains
H
Histidine
+
H3N
C
CH2
N
O
C
O
–
pKa1 =
pKa2 =
pKa3 =
pI =
1.82
6.00
9.17
7.59
NH
27- 77
Reactions of Amino Acids
27- 78
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
(89-92%)
27- 79
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
–
+
H3NCHCOCH2CH3
CH3
(90-95%)
27- 80
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–
O
RCH + CO2 + H2O +
N
O
O
27- 81
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
F
27- 82
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
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
–
NHCHCO
CH3
CH(CH3)2 CH2C6H5
NO2
O
O
O
–
NHCHCO
CH3
27- 83
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
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
O
–
NHCHCO
CH3
27- 84
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.
27- 85
Edman Degradation
The key reagent in the Edman degradation is
phenyl isothiocyanate.
N
C
S
27- 86
Edman Degradation
Phenyl isothiocyanate reacts with the amino
nitrogen of the N-terminal amino acid.
O
C6H5N
C
S
+
+ H3NCHC
R
NH
peptide
27- 87
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
O
C6H5N
C
S
+
+ H3NCHC
R
NH
peptide
27- 88
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.
27- 89
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
HCl
S
C6H5NH
C
C
N
CH
R
O
+
+
H3N
peptide
27- 90
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
CH
R
O
+
+
H3N
peptide
27- 91
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
CH
R
O
+
+
H3N
peptide
27- 92
Some Biochemical Reactions
of Amino Acids
27- 93
27- 94
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
+ NH3
27- 95
Decarboxylation
N
CH2CH2 NH2
N
H
–CO2, enzymes
N
–
CH2CHCO2
N
H
+ NH3
27- 96
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
27- 97
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
27- 98
Neurotransmitters
Dopamine is formed
by decarboxylation
of L-DOPA.
H
H2N
H
H
H
HO
OH
Dopamine
27- 99
Neurotransmitters
H
H2N
H
H
OH
HO
OH
Norepinephrine
27- 100
Neurotransmitters
H
CH3NH
H
H
OH
HO
OH
Epinephrine
27- 101
Peptides
27- 102
Objectives
•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.
27- 103
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.
27- 104
Peptide Bond Formation
27- 105
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
H2O
27- 106
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
27- 107
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
27- 108
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- 109
27- 110
Alanine and Glycine
H
+
H3N
C
CH3
H
O
C
–
O
+
H3N
C
O
C
–
O
H
27- 111
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.
27- 112
Alanylglycine
H
+
H3N
N-terminus
C
CH3
H
O
C
N
C
H
H
O
C
–
O
C-terminus
Ala—Gly
AG
27- 113
Alanylglycine and glycylalanine are
constitutional isomers
H
+
H3N
C
C
CH3
H
+
H3N
C
H
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- 114
Alanylglycine
H
+
H3N
C
CH3
H
O
C
N
C
H
H
O
C
–
O
The peptide bond is
characterized by a
planar geometry.
27- 115
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
27- 116
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
27- 117
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.
27- 118
Leucine Enkephalin
Tyr—Gly—Gly—Phe—Leu
YGGFL
27- 119
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.
27- 120
Oxytocin
S—S bond
An S—S bond between two cysteines is
often referred to as a disulfide bridge.
27- 121
Introduction to Peptide
Structure Determination
27- 122
Primary Structure
The primary structure is the amino acid
sequence plus any disulfide links.
27- 123
Proteins are linear polymers of amino acids
R1
R2
NH3＋ C COO ＋ NH3＋ C COO ＋
ー
ー
H
H
A carboxylic acid
H 2O
condenses with an amino
group with the release of a
water
H 2O
R1
R2
R3
NH3＋ C CO NH C CO NH C CO
H
A
F
Peptide
bond
G
N
S
Peptide
bond
H
T
D
K
G
H
S
A
The amino acid
sequence is called as
primary structure
27- 124
Classical Strategy
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.
27- 125
Amino Acid Analysis
27- 126
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.
27- 127
Partial Hydrolysis of Proteins
27- 128
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.
27- 129
Partial Hydrolysis of Peptides and Proteins
The enzymes that catalyze the hydrolysis of
peptide bonds are called peptidases, proteases,
or proteolytic enzymes.
27- 130
Proteases
*
*
*
*
*
R2 Pro
R1 Pro
27- 131
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
27- 132
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
27- 133
Carboxypeptidase
Carboxypeptidase is selective for cleaving
the peptide bond to the C-terminal amino acid.
O
O
+
H3NCHC
R
protein
C
O
–
NHCHCO
R
27- 134
End Group Analysis
27- 135
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.
27- 136
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
F
27- 137
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
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
–
NHCHCO
CH3
CH(CH3)2 CH2C6H5
NO2
O
O
O
–
NHCHCO
CH3
27- 138
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
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
O
–
NHCHCO
CH3
27- 139
Insulin
27- 140
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.
27- 141
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
27- 142
The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
27- 143
The B Chain of Bovine Insulin
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
27- 144
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
27- 145
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
YTPKA
27- 146
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.
27- 147
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
GFFYTPK
YTPKA
27- 148
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
GFFYTPK
YTPKA
FVNQHLCGSHLVGALTLVCGERGFFYTPKA
27- 149
Insulin
The sequence of the A chain was determined
using the same strategy.
Establishing the disulfide links between cysteine
residues completed the primary structure.
27- 150
The Edman Degradation and
Automated Sequencing of
Peptides
27- 151
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.
27- 152
Edman Degradation
The key reagent in the Edman degradation is
phenyl isothiocyanate.
N
C
S
27- 153
Edman Degradation
Phenyl isothiocyanate reacts with the amino
nitrogen of the N-terminal amino acid.
O
C6H5N
C
S
+
+ H3NCHC
R
NH
peptide
27- 154
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
O
C6H5N
C
S
+
+ H3NCHC
R
NH
peptide
27- 155
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.
27- 156
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
HCl
S
C6H5NH
C
C
N
CH
R
O
+
+
H3N
peptide
27- 157
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
CH
R
O
+
+
H3N
peptide
27- 158
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
CH
R
O
+
+
H3N
peptide
27- 159
Secondary Structures
of Peptides and Proteins
27- 160
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
27- 161
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
27- 162
-helixes
Intra-chain
H-bonds
Secondary Structure
27- 163
 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.
27- 164
-strands
Inter-chain
H-bonds
Secondary Structure
27- 165
Two Types of -Pleated Sheets
27- 166
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.
27- 167
Tertiary Structure
of Peptides and Proteins
27- 168
Tertiary Structure
Specific compact
structure for one
entire polypeptide
Chain stabilizing
primarily through
weak bonds
27- 169
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
27- 170
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- 171
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.
27- 172
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- 173
Tertiary
structure
Hb monomer
(or myoglobin)
Quaternary
structure
Hb 22 tetramer
27- 174
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.
27- 175
Carboxypeptidase
Disulfide bond
Zn2+
Arg-145
N-terminus
C-terminus
tube model
ribbon model
27- 176
Myoglobin
C-terminus
Heme
N-terminus
Heme is the coenzyme that binds oxygen in myoglobin
(oxygen storage in muscles) and hemoglobin (oxygen
transport).
27- 177
Protein Quaternary Structure:
Hemoglobin
27- 178
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.
27- 179
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.
27- 180
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.
27- 181
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.
27- 182
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.
Denaturation agents
Heat: Break apart hydrogen bonds and disrupt hydrophobic attractions
between nonpolar side groups.
Acids/Bases: Break hydrogen bonds between polar R groups and
disrupt the ionic bonds (salt bridges).
Organic Compounds: Ethanol and isopropanol act as disinfectants by
forming their own hydrogen bonds with a protein and disrupting the
hydrophobic interactions.
Heavy metal ions: Heavy metal ions like Ag+, Pb2+ and Hg2+ React with
S-S bonds to form solids.
Agitation: Stretches chains until bonds break
27- 183
Different Classification of Proteins
On the basis of:
Shape:
Globular
Fibrilar
Homo or hetero
Function
27- 184
Immunoglobulin Structure
Heavy & Light
Chains
Disulfide bonds
Inter-chain
Intra-chain
Disulfide bond
Carbohydrate
CL
VL
CH2
CH1
VH
CH3
Hinge Region
27- 185
Hydroxylation of proline and lysine residues in collagen
Vitamin C is required for the maintenance of
normal connective tissue as well as for wound
healing since synthesis of connective tissue is the
first event in wound tissue remodeling.
27- 186
Globular Proteins
Myoglobin and hemoglobin are typical examples of
globular proteins.
Both are heme-containing proteins and each is involved
in oxygen metabolism.
27- 187
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..
27- 188
Myoglobin
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.
27- 189
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.
27- 190
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)
27- 191
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.
27- 192
Myoglobin
27- 193
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
27- 194
The Heme Group
-
-
CH2CH2COO
OOC CH2CH2
H3C
N
N
CH3
Fe(II)
H2C
N
CH
N
Pyrrole ring
CH3
CH3
CH CH2
27- 195
N of His F8 binds
to 5th coordination
site on heme iron
Oxygen binds to 6th
coordination site on
heme iron
27- 196
His E7 acts as a gate to favor oxygen
binding over carbon monoxide.
27- 197
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.
27- 198
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
27- 199
27- 200
Each chain is
in ribbon form.
The heme
groups are in
space filling
form
27- 201
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.
27- 202
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- 203
Oxygen Binding Curves-2
27- 204
Hemoglobin Equilibrium



O2
+
H ,CO
2,

BPG
T
R
(low affinity)
(high affinity)
27- 205
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
27- 206
Oxygen binding by hemoglobin
27- 207
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)
27- 208
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
27- 209
The Bohr Effect
27- 210
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
27- 211
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.
27- 212
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
27- 213
2,3-bisphosphoglycerate
(2,3-BPG) is a negative
allosteric effector of O2
binding to Hb - binds
tighter to deoxyHb
2,3-BPG
27- 214
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
=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- 215
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
Distal
His (E7)
27- 216
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)
27- 217
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)
27- 218
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)
27- 219
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.
27- 220
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)
27- 221
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
0
20
40
60
80
100
120
140
160
pO2 (mm Hg)
27- 222
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
27- 223
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)
27- 224
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)
27- 225
Effect of BPG


BPG

Side view (R)




Side view (T)
27- 226
Effect of BPG
- Lys His His
+
+
+
27- 227
Hemoglobin Equilibrium



O2
+
H ,CO
2,

BPG
T
R
(low affinity)
(high affinity)
27- 228
Hemoglobins in normal adults
α
β
α
γ
α
β
α
γ
α
δ
δ
α
HbA
HbF
HbA2
98%
~1%
<3.5%
27- 229
27- 230
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,
both expressed
27- 231
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
27- 232
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
hemoglobin
27- 233
Inherited Hemoglobin disorder
Definition: An inherited mutation of the
globin genes leading to a qualitative or
quantitative abnormality of globin
synthesis
27- 234
Inherited Hemoglobin
Abnormalities
Hemoglobinopathies - structurally abnormal
hemoglobin chains
Thalassemias - one or more of the normal
hemoglobin chains are synthesized at a
markedly reduced rate
27- 235
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
27- 236
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.
27- 237
Beta thalassemia
Impaired production of beta chain
beta thalassemia minor – heterozygous (or trait)
beta thalassemia major - homozygous
27- 238
Beta thalassemia - heterozygous (minor or trait)
Target cell
Oval cell
27- 239
Beta thalassemia major
27- 240
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.
27- 241
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
27- 242
27- 243
Alpha thalassemia
/

Normal
/
-
Mild microcytosis
/-
Mild microcytosis
-/- -
Hemoglobin H disease
- -/- -
Hemoglobin Barts – Hydrops Fetalis
27- 244
Structural hemoglobinopathy
(qualitative)
Amino acid substitution in the globin
chain e.g. sickle hemoglobin (HbS)
27- 245
Sickle cell hemoglobin
Glu
Glu
-
-
Glu






HbS
(heterozygous)
Sickle cell trait
HbS
(homozygous)
Sickle cell disease
-
HbA1
27- 246
27- 247
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
DEOXY-STATE
27- 248
Sickle Cell Anemia – blood film
Sickle
Cells
Erythroblasts
HowellJolly Body
27- 249
Fibres of Sickle Hemoglobin
27- 250
Fibres of Sickle
Hemoglobin –
cross section
27- 251
Hemoglobin S
Valine is exposed in deoxy-Hemoglobin
27- 252
Polymerization of HbS



  
  
  
  
  


27- 253