Download Hemoglobin

Hemoglobin; structure
and function
Mahmoud A. Alfaqih BDS PhD
Jordan University of Science and
Technology (JUST)
Heme-proteins




Heme-proteins are a group of specialized
proteins that contains heme (prosthetic
group).
Examples of hemeproteins:
I- Myoglobin and hemoglobin
II- Other Hemeproteins
 1- Cytochromes
 2- Peroxidases
 3- catalase
MYOGLOBIN AND
HEMOGLOBIN

Both proteins are formed of heme
attached to a globin chain
 Myoglobin is formed of one heme
attached to one polypeptide chain,
while
 Hemoglobin is formed of four heme
groups attached to four polypeptide
chains i.e. one heme per one chain.
Heme

A cyclic tetrapyrrole consisting of four molecules of pyrrole
linked by methylene bridges

This planar network of conjugated double bonds absorbs visible
light and colors heme deep red

The substituents at the -positions of heme are methyl (M), vinyl
(V), and propionate (Pr) groups arranged in the order M, V, M,
V, M, Pr, Pr, M

One atom of ferrous iron (Fe2+) resides at the center of the
planar tetrapyrrole

Oxidation of the Fe2+ of myoglobin or hemoglobin to Fe3+
destroys their biologic activity
Heme is a complex of protoporphyrin IX
and ferrous iron (Fe2+).
M
V
N
M
M
Fe++
N
N
V
P
N
P
M
Heme
Ferrous Protoporphyrin IX
M = methyl
V = Vinyl
P = Propionyl
Attachment of Heme to The
Polypeptide Chain




Ferrous of heme can form up to 6 bonds as
follows:
1- Four bonds are formed between Fe2+ and
the four nitrogens of the porphyrin ring.
2- The fifth bond is for histidine (His F8) of
the globin chain.
3- The sixth bond is for oxygen which lies
between Fe++ and another histidine termed
distal histidine (E7)
A-Myoglobin

Location: In skeletal and cardiac muscles

Function: a storage form of oxygen in muscles,
enhances transport of oxygen inside muscle cells

Structure : single polypeptide chain, similar to
individual subunit polypeptides of hemoglobin
Oxygen stored in red muscle myoglobin is released
during O2 deprivation (eg, severe exercise) for use in
muscle mitochondria for aerobic synthesis of ATP
Helical content of Myoglobin

Single polypeptide chain, 80% helical, forms 8
helices named starting from the N-terminal A
through H

Soluble protein with polar outer surface and nonpolar inner groups except for the two histidines that
function in oxygen binding i.e. histidines F8 and E7.
Hemoglobin


Location: Exclusively in red blood cells
Function:
 Transports oxygen from lungs to
every tissue in the body
 Removes CO2 from tissues
 Also Hb / oxy-Hb system acts as
buffer in RBCs
Note
Myoglobin & the β Subunits of Hemoglobin
Share Almost Identical Secondary and
Tertiary Structures
Structure Of Adult Hemoglobin
1- HbA

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HbA is formed of four polypeptide chains
Two hetero-dimers, dimer is formed of one
alpha (α) and one beta (β) chain
Tetrameric structure (αβ)2 i.e. (α1 β1 – α2 β2)
Each subunit is similar in structure to
Myoglobin
Continued


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The α & β chains are tightly held
together by hydrophobic interactions
The two dimers are less tightly held
together by hydrogen & ionic bonds
Allows movement of the 2 dimers
relative to each other.
Movement occurs during oxygenation
& deoxygenation
The “ T “ form ( Hb ):
OR [ Taut ( tense ) Form ]

Low-oxygen affinity form of hemoglobin

Network of ionic bonds between the two
dimers, constrains movement

Dimers are difficult to move relative to each
other
The “ R “ Form ( HbO2 ):
OR [ Relaxed Form ]
 Binding of oxygen disrupts ionic and
hydrogen bonds between two dimers
 Dimers are free to move relative to each
other
 It has a higher affinity to oxygen.
Binding Of oxygen to myoglobin and
hemoglobin

Myoglobin can bind only one molecule of oxygen
(O2), because it contains only one heme group

Hemoglobin can bind four oxygen molecules

The degree of saturation (Y) can vary between zero
(all sites are empty) and 100% (all sites are full)
Oxygen dissociation curve



A plot of Y measured at
different partial pressures
of oxygen (pO2)
Myoglobin has a higher
oxygen affinity at all pO2
values
(P50) is 1 mm Hg for
myoglobin and 26 mm
Hg for hemoglobin.
Oxygen dissociation curve of
Myoglobin

It has a hyperbolic shape

Oxygenated (MbO2) and deoxygenated (Mb) myoglobin
exist in a simple equilibrium:
Oxygen dissociation curve of
Hemoglobin

Sigmoidal in shape

Subunits cooperate in binding oxygen

Binding of an oxygen molecule at one heme group
increases the affinity of the remaining groups to O2

This is referred to as heme-heme interaction
Hemoglobin binds oxygen with
increasing affinity

It is more difficult for the first
oxygen molecule to bind to
hemoglobin, subsequent binding of
oxygen occurs with higher affinity

Affinity of hemoglobin for the last
oxygen bound is approximately 300
times greater than its affinity for the
first oxygen bound
Allosteric effects
Interaction at one site on the hemoglobin
molecule affects the binding of oxygen to heme
groups at other locations on the molecule
 Allosteric effectors:
1. pO2
2. pH
3. pCO2
4. 2,3 - bisphosphoglycerate

Bohr effect

Affinity of hemoglobin for oxygen decreases
when the pH is lowered (when the partial
pressure of CO2 increases)

Oxygen dissociation curve shifts to the right

Stabilization of the T state of Hemoglobin
Bohr effect
Source of Protons

The concentration of CO2 and H+ is higher around
metabolically active tissues compared to lung alveoli

CO2 is converted by carbonic anhydrase to carbonic
acid:

Carbonic acid spontaneously loses a proton:

Lungs have higher pH compared to tissues
Source of protons…..cont.

Hemoglobin carries CO2 as carbamates formed with the amino
terminal nitrogens of the polypeptide chains

Formation of carbamates gets rid of the positive charge of
terminal nitrogen and allows the gain of a net negative charge

The above reaction also causes the release of protons
Source of protons

The net negative charge gained following carbon
dioxide binding favors salt bond formation between the
α and β chains

This change stabilizes T state and facilitates delivery of
oxygen
What happens at the lung side?

In the lungs, the process reverses.

O2 binds to deoxyhemoglobin. Protons are released and
combine with bicarbonate to form carbonic acid.

Dehydration of H2CO3, catalyzed by carbonic anhydrase, forms
CO2, which is exhaled.

Binding of oxygen drives the exhalation of CO2
Mechanism of Bohr effect
Deoxy form of hemoglobin has a greater affinity
for protons than does oxyhemoglobin
 N-terminal α-amino groups and specific
histidine side chains have higher pKas in
deoxyhemoglobin than in oxyhemoglobin
 A decrease in pH causes them to become
protonated (charged) and form ionic bonds
 This effect stabilizes the deoxy form of
hemoglobin

Continued

An increase in protons shifts the equilibrium to
the right (favoring deoxyhemoglobin)

An increase in pO2 (or a decrease in protons)
shifts the equilibrium to the left
Tissue side
Lung side
Effect of 2,3-bisphosphoglycerate (BPG) on
oxygen affinity

It is the most abundant
organic phosphate in
the red blood cell

BPG is synthesized
from an intermediate of
the glycolytic pathway
Binding of 2,3-BPG to deoxyhemoglobin

BPG decreases the oxygen affinity of hemoglobin by
binding to deoxyhemoglobin but not to
oxyhemoglobin

This preferential binding stabilizes the taut
conformation of deoxyhemoglobin
Binding site of 2,3-BPG

BPG binds to a pocket,
formed by the two β-globin
chains, in the center of the
deoxyhemoglobin tetramer

This pocket contains several
positively charged amino
acids that form ionic bonds
with BPG
Effect of 2,3-BPG on oxygen dissociation curve

Hemoglobin from which 2,3BPG has been removed has a
high affinity for oxygen

The presence of 2,3-BPG
shifts the oxygen-dissociation
curve to the right
Response of 2,3-BPG levels to chronic hypoxia
or anemia


The concentration of 2,3-BPG increases in chronic
hypoxia, pulmonary emphysema, at high altitudes and
in chronic anemia
Elevated 2,3-BPG levels lower the oxygen affinity of
hemoglobin, permitting greater unloading of oxygen
to tissues
Adaptation to high altitude

Physiologic changes due to prolonged
exposure to high altitude include:
 Increase in the number of red cells
 Increase in the concentration of Hb
 Increase in the concentration of BPG
Role of 2,3-BPG in transfused blood

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Storing blood in acid-citrate-dextrose leads to a
decrease of 2,3-BPG in RBCs
Such blood fails to unload oxygen in the tissues
Hemoglobin deficient in 2,3-BPG acts as an oxygen
“trap”
Transfused RBCs can restore 2,3-BPG in 24 to 48
hours
This might represent a problem for the severely ill
patient
Solution
The decrease in 2,3-BPG can be prevented by
adding inosine (hypoxanthine-ribose)
 Inosine can enter RBC, its ribose moiety is
released and phosphorylated
 It then enters the hexose monophosphate
pathway to be converted to 2,3-BPG

Binding of CO2



Most of the CO2 is hydrated and transported as
bicarbonate ion
Some CO2 is carried as carbamate bound to α-amino
groups of hemoglobin (carbamino-hemoglobin)
The binding of CO2 stabilizes the T (taut) or deoxy
form of hemoglobin (lower affinity for oxygen)
Binding of CO

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CO binds tightly to the hemoglobin iron
When CO binds, hemoglobin shifts to the relaxed
conformation
This causes hemoglobin to bind oxygen with higher
affinity
This shifts curve to the left, and changes the normal
shape to a hyperbola
Hemoglobin is unable to release oxygen to the tissues
Effect of CO
Hemoglobin
Myoglobin
Location
In Red Blood Cells
In Cardiac & Skeletal
Muscles
No Of
Polypeptides
4 polypeptides
One polypeptide
No Of Heme /
molecule
4 Heme
One Heme
No Of O2 mol.
Can bind
4 O2 mol.
One O2 mol.
Structure
Quaternary
Tertiary
Affinity For O2
( At low pO2 )
Lower than Myoglobin
Higher than Hb
O2
Dissociation
Curve
Sigmoidal
Cooperative Binding Of
O2
Hyperbolic
Mb + O2
MbO2
Forms
2 forms R & T
One form
Allosteric
Effect
Affected
Not
Minor Hemoglobins


Hb A is just one member of
a family of proteins, the
hemoglobins
All hemoglobins are
tetramers of two α-globin
like chains and two β-globinlike chains
Fetal Hemoglobin (Hb F)



A tetramer consisting of two α chains identical
to those found in Hb A, plus two γ chains
γ chains are members of the β-globin gene
family
Hb F's binds only weakly to BPG, it has a
higher affinity for oxygen than Hb A
Hb F synthesis during development

Embryonic hemoglobin ( (ζ2ε2), is
synthesized in the first three months of
pregnancy

Hb F is the major hemoglobin in the
fetus and newborn

Hb F makes sixty percent of the total
hemoglobin during the last months of
fetal life

Hb A synthesis starts at about the eighth
month of pregnancy and gradually
replaces Hb F.
Hemoglobinpathies

1.
2.
A family of genetic disorders caused by
production of structurally abnormal
hemoglobin
synthesis of insufficient quantities of normal
hemoglobin
Hemoglobinpathies

1.
2.
3.
Hemoglobinpathies include the following
Sickle cell anemia (Hb S)
Hemoglobin C disease (Hb C)
Thalassemia syndromes
Sickle cell disease (Hemoglobin S disease)

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
A genetic disorder caused by a single nucleotide
alteration in the β-globin gene
Sickle cell disease is a homozygous recessive
disorder
It occurs in individuals who have inherited two
mutant genes
Heterozygotes have one normal and one sickle cell
gene
Heterozygotes have sickle cell trait and can have a
normal life span
Amino acid substitution in Hb S β
chains


A molecule of Hb S
contains two normal
α-glob in chains and
two mutant β-globin
chains
Glutamate at position
six is replaced with
valine
Effect of amino acid substitution




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The substitution forms a protrusion on the β-globin
chain of hemoglobin
Protrusion fits into a complementary site on the α chain
of another hemoglobin molecule
At low oxygen, deoxyhemoglobin S polymerizes into a
network of fibres that distort RBCs
sickled cells block the flow of blood in capillaries.
This leads to localized anoxia (oxygen deprivation) in
the tissueand eventually death (infarction) of tissue cells
Variables that increase sickling
1. Decrease in pO2
2. Increase in pCO2
3. Increase in BPG
concentration
4. Decrease in pH
Hemoglobin C disease




A hemoglobin variant that has a single amino acid
substitution in the sixth position of β-globin
A lysine is substituted for glutamate
Patients homozygous for hemoglobin C have a mild,
chronic hemolytic anemia
No specific therapy is required for these patients
Hemoglobin SC disease





Some β-globin chains have the sickle cell mutation
Other β-globin chains have Hemoglobin C mutation
Hemoglobin levels tend to be higher in Hb SC disease
than in sickle cell disease
Patients with Hb SC disease remain well, until they
suffer an infarctive crisis
This crisis may follow childbirth or surgery and may be
fatal
Hemoglobin electrophoresis
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Some β-globin chains have the sickle cell mutation
Other β-globin chains have Hemoglobin C mutation
Hemoglobin levels tend to be higher in Hb SC disease
than in sickle cell disease
Patients with Hb SC disease remain well, until they
suffer an infarctive crisis
This crisis may follow childbirth or surgery and may be
fatal
Thalassemias
Hereditary hemolytic diseases in which an imbalance
occurs in the synthesis of globin chains
 The synthesis of either the α- or the β-globin chain is
defective
 Thalassemias are classified into
A. β-Thalassemias
B. α-Thalassemias

β-Thalassemias





1.
2.
Synthesis of β-globin chains is decreased or absent (as a result
of point mutations)
α-Globin chain synthesis is normal. α-Globin tetramers form
in RBCs
α-Globin tetramers are not stable and precipitate causing the
premature death of cells
Accumulation of α2γ2 (Hb F) and γ4 (Hb Bart's) also occurs
There are two forms of β-Thalassemia
β-Thalassemia major
β-Thalassemia minor
β-Thalassemias

β-thalassemia trait (βthalassemia minor): only
one defective β-globin
gene

β-thalassemia major
(Cooley anemia) if both
genes are defective

Clinical course is different
between the two diseases
β-Thalassemias



β-thalassemia minor patients make some β
chains; do not require specific treatment
β-thalassemia major patients do not develop
symptoms until after the first year of life. Why?
β-thalassemia major patients require regular
transfusions of blood
α-Thalassemias



Synthesis of α-globin chains is decreased or
absent
Each individual has four copies of the α-globin
gene (two on each chromosome 16)
There are several levels of α-globin chain
defecencies

One of the four genes is defective, the
individual is a silent carrier of α-thalassemia
and no physical manifestations of the disease
occur

If two α-globin genes are defective, the
individual has α-thalassemia trait. Why?
If three α-globin genes are
defective, the individual has
hemoglobin H (Hb H) disease,
mild to moderate anemia

 If all four α-globin genes are
defective, hydrops fetalis and
fetal death result