Hemoglobin and myoglobin are the oxygentransporting
proteins in vertebrates. Hemoglobin
is found in red blood cells, myoglobin in
muscle. Hemoglobin arose from myoglobin
during the course of evolution. Hemoglobin has
four oxygen-binding sites, myoglobin has one.
Their genes and their three-dimensional protein
structures are completely known in atomic
detail. Different types of hemoglobin that are
optimally adapted to prenatal and postnatal life
have evolved in mammals from an ancestral
gene.
Sunday, April 12, 2009
Types of hemoglobin
Hemoglobin consists of four subunits (globin
chains), two pairs of identical polypeptides,
each polypeptide being attached to a heme
group with an oxygen-binding site. The different
kinds of hemoglobins, which are characteristic
of different stages of development
(embryonic, fetal, and postnatal), differ in the
composition of their chains: the hemoglobin of
adults (HbA) contains two ! and two " chains
(!2"2). A small proportion of adult hemoglobin
has two ! and two # chains (HbA2: !2#2).
Hemoglobin formed during the fetal period
(HbF) contains two ! and two $ chains (!2$2). In
the embryonic stage, % chains are joined to & or $
chains (Hb Gower 1: %2&2, and Hb Portland:
%2$2); two ! and two & chains form Hb Gower 2
(!2&2).
chains), two pairs of identical polypeptides,
each polypeptide being attached to a heme
group with an oxygen-binding site. The different
kinds of hemoglobins, which are characteristic
of different stages of development
(embryonic, fetal, and postnatal), differ in the
composition of their chains: the hemoglobin of
adults (HbA) contains two ! and two " chains
(!2"2). A small proportion of adult hemoglobin
has two ! and two # chains (HbA2: !2#2).
Hemoglobin formed during the fetal period
(HbF) contains two ! and two $ chains (!2$2). In
the embryonic stage, % chains are joined to & or $
chains (Hb Gower 1: %2&2, and Hb Portland:
%2$2); two ! and two & chains form Hb Gower 2
(!2&2).
Hemoglobins in thalassemia
The thalassemias are a group of genetically determined
disorders of hemoglobin synthesis.
Thalassemia occurs due to the absence or reduced
synthesis of a globin chain, which results
in unstable hemoglobin. It affects either the "
chain ("-thalassemia) or the ! chain (!-thalassemia).
Since the ! chain is a component of both
fetal and adult hemoglobins, the !-thalassemias
(!-thal) are especially severe. Hemoglobins
with four identical globin chains are
completely unstable and incompatible with life
(HbH with four " chains, Hb Bart’s with four $
chains).
disorders of hemoglobin synthesis.
Thalassemia occurs due to the absence or reduced
synthesis of a globin chain, which results
in unstable hemoglobin. It affects either the "
chain ("-thalassemia) or the ! chain (!-thalassemia).
Since the ! chain is a component of both
fetal and adult hemoglobins, the !-thalassemias
(!-thal) are especially severe. Hemoglobins
with four identical globin chains are
completely unstable and incompatible with life
(HbH with four " chains, Hb Bart’s with four $
chains).
Evolution of hemoglobin
Since hemoglobin has four polypeptide chains,
it is a much more efficient oxygen carrier than
the single-chained myoglobin molecule.
Furthermore, the existence of different globin
chains confers a selective advantage in evolution
because their slight functional differences
result in optimal adaptation to the differences
in oxygen concentration before and after birth.
The genes for individual hemoglobin chains
arose from myoglobin by a series of gene duplications
during evolution. The evolutionary age
of the individual Hb chains can be estimated
from their differences in relation to the rate of
mutation. When mammals began to evolve
about 100million years ago, the genes for ! and
" chainswere present, whereas "-like chains (&,
$, #) evolved later.
it is a much more efficient oxygen carrier than
the single-chained myoglobin molecule.
Furthermore, the existence of different globin
chains confers a selective advantage in evolution
because their slight functional differences
result in optimal adaptation to the differences
in oxygen concentration before and after birth.
The genes for individual hemoglobin chains
arose from myoglobin by a series of gene duplications
during evolution. The evolutionary age
of the individual Hb chains can be estimated
from their differences in relation to the rate of
mutation. When mammals began to evolve
about 100million years ago, the genes for ! and
" chainswere present, whereas "-like chains (&,
$, #) evolved later.
Globin formation in ontogeny
Different types of globin chains are formed at
different developmental stages: embryonic
hemoglobin during the early embryonic period
(to about the 12thweek), fetal hemoglobin from
about the 12th week until birth, and adult
hemoglobin thereafter. They differ in oxygenbinding
affinity. Thus, oxygen delivery is optimized
for different phases of development.
The site of synthesis also differs. During the
fetal phase, globin chains are synthesized
mainly in the liver, whereas after birth they are
synthesized in red blood cell precursors in the
bone marrow.
different developmental stages: embryonic
hemoglobin during the early embryonic period
(to about the 12thweek), fetal hemoglobin from
about the 12th week until birth, and adult
hemoglobin thereafter. They differ in oxygenbinding
affinity. Thus, oxygen delivery is optimized
for different phases of development.
The site of synthesis also differs. During the
fetal phase, globin chains are synthesized
mainly in the liver, whereas after birth they are
synthesized in red blood cell precursors in the
bone marrow.
Hemoglobin Genes
Each of the globin polypeptide chains is coded
for by a gene. In humans and in other mammals,
the !-like genes (!, ", #) are located together in
a cluster on one chromosome, while the $ genes
are located on another. They are arranged in the
order of their activation during ontogeny.
for by a gene. In humans and in other mammals,
the !-like genes (!, ", #) are located together in
a cluster on one chromosome, while the $ genes
are located on another. They are arranged in the
order of their activation during ontogeny.
The !-globin and "-globin genes
The !-globin-like genes (%, "G, "A, #, !) of man
are located on the short arm of chromosome 11
in region 1, band 5.5 (11p15.5). They span about
60000 base pairs (bp), or 60 kb (kilobases), of
DNA.
There are two " genes, "A and "G, which differ
only in codon 136. Codon 136 of "A is alanine,
and of "G, glycine. A pseudogene (&!1) is located
between the A" gene and the # gene. It is
similar to the ! gene, but has been permanently
altered by deletion and an internal stop codon,
so that it cannot code for a functional polypeptide.
A region that jointly regulates these genes
is located upstream (in the 5' direction) from
the ! genes (LCR, long-range control region).
In humans, two $-globin genes are located on
the short arm of chromosome 16 (16p13.11 to
16p13.33) on a DNA segment of about 30 kb. A '
gene, which is active only during the embryonic
period, lies in the 5' direction. Three pseudogenes:
&',&$2, and&$1 are located in between.
A further gene, (, with unknown function, has
been identified in this region.
are located on the short arm of chromosome 11
in region 1, band 5.5 (11p15.5). They span about
60000 base pairs (bp), or 60 kb (kilobases), of
DNA.
There are two " genes, "A and "G, which differ
only in codon 136. Codon 136 of "A is alanine,
and of "G, glycine. A pseudogene (&!1) is located
between the A" gene and the # gene. It is
similar to the ! gene, but has been permanently
altered by deletion and an internal stop codon,
so that it cannot code for a functional polypeptide.
A region that jointly regulates these genes
is located upstream (in the 5' direction) from
the ! genes (LCR, long-range control region).
In humans, two $-globin genes are located on
the short arm of chromosome 16 (16p13.11 to
16p13.33) on a DNA segment of about 30 kb. A '
gene, which is active only during the embryonic
period, lies in the 5' direction. Three pseudogenes:
&',&$2, and&$1 are located in between.
A further gene, (, with unknown function, has
been identified in this region.
Structure of the "-globin and !-globin genes
As a result of their origin froma common ancestral
gene, all globin genes have a similar structure.
Their coding sequences are arranged in
three exons. Each globin transcription unit includes
nontranslated sequences at the 5' and
the 3' ends (see section on the structure of
eukaryotic genes, p. 50). The lengths of the !-
globin and $-globin exons are similar (e.g., exon
1 of the ! gene has 30 codons; exon 1 of the $
gene has 31 codons), whereas the lengths of the
introns differ.
gene, all globin genes have a similar structure.
Their coding sequences are arranged in
three exons. Each globin transcription unit includes
nontranslated sequences at the 5' and
the 3' ends (see section on the structure of
eukaryotic genes, p. 50). The lengths of the !-
globin and $-globin exons are similar (e.g., exon
1 of the ! gene has 30 codons; exon 1 of the $
gene has 31 codons), whereas the lengths of the
introns differ.
Tertiary structure of the !-globin chain
The three-dimensional structures ofmyoglobin
and of the hemoglobin $ and ! chains are very
similar, although their amino acid sequences
correspond in only 24 of 141 positions. The !
chain, with 146 amino acids, is somewhat
longer than the $ chain, with 141 amino acids.
The structural similarity is functionally significant:
The oxygen-binding region lies inside the
molecule, where it is protected, and oxygen uptake
from the aqueous surroundings is reversible.
and of the hemoglobin $ and ! chains are very
similar, although their amino acid sequences
correspond in only 24 of 141 positions. The !
chain, with 146 amino acids, is somewhat
longer than the $ chain, with 141 amino acids.
The structural similarity is functionally significant:
The oxygen-binding region lies inside the
molecule, where it is protected, and oxygen uptake
from the aqueous surroundings is reversible.
Domains of the ! chain
Three functional and structural domains can be
distinguished in all globin chains. They correspond
to the three exons of the gene. Two
domains, consisting of amino acids 1–30 and
105–146 (coded for by exons 1 and 3), are located
on the outside. They are mainly formed of
hydrophilic amino acids. A third domain, lying
inside the molecule (coded for by exon 2), contains
the oxygen-binding site and consists
mainly of nonpolar hydrophobic amino acids.
The amino acid sequences of the hemoglobins
of more than 60 investigated species are identical
in nine positions. These invariant positions
are especially important for the function of the
molecule. Changes (mutations) in the invariant
positions affect function so severely that they
are not tolerated.
distinguished in all globin chains. They correspond
to the three exons of the gene. Two
domains, consisting of amino acids 1–30 and
105–146 (coded for by exons 1 and 3), are located
on the outside. They are mainly formed of
hydrophilic amino acids. A third domain, lying
inside the molecule (coded for by exon 2), contains
the oxygen-binding site and consists
mainly of nonpolar hydrophobic amino acids.
The amino acid sequences of the hemoglobins
of more than 60 investigated species are identical
in nine positions. These invariant positions
are especially important for the function of the
molecule. Changes (mutations) in the invariant
positions affect function so severely that they
are not tolerated.
Sickle Cell Anemia
Sickle cell anemia is a severe progressive disease
resulting from homozygosity for a mutation
in the !-globin gene. It is especially
frequent in Africa and in the black population of
North America. With a frequency of 1 in 500, it
is an important cause of morbidity and mortality
in these regions. The disease is transmitted
by autosomal recessive inheritance.
resulting from homozygosity for a mutation
in the !-globin gene. It is especially
frequent in Africa and in the black population of
North America. With a frequency of 1 in 500, it
is an important cause of morbidity and mortality
in these regions. The disease is transmitted
by autosomal recessive inheritance.
Sickle cells: erythrocytes deformed by hemoglobin S
In a normal blood smear under the light microscope
(1), erythrocytes (red blood cells) appear
as regular round disks of about 7 μm diameter.
Since a normal red blood cell is nonnucleated
and biconcave, the center appears paler than
the periphery. The erythrocytes of affected persons
are deformed and resemble sickles (2).
However, even the non-sickle-shaped red cells
display unusual sizes and shapes. In the course
of the disease, acute crises called sickle crises
(3) occur, during which sickle-like cells are
greatly increased and completely dominate the
blood picture. Heterozygotes show occasional
sickle cells but do not suffer from sickle crises,
and at the most have only very mild signs and
symptoms.
(1), erythrocytes (red blood cells) appear
as regular round disks of about 7 μm diameter.
Since a normal red blood cell is nonnucleated
and biconcave, the center appears paler than
the periphery. The erythrocytes of affected persons
are deformed and resemble sickles (2).
However, even the non-sickle-shaped red cells
display unusual sizes and shapes. In the course
of the disease, acute crises called sickle crises
(3) occur, during which sickle-like cells are
greatly increased and completely dominate the
blood picture. Heterozygotes show occasional
sickle cells but do not suffer from sickle crises,
and at the most have only very mild signs and
symptoms.
Result of a mutation: sickle cell anemia
All manifestations of sickle cell anemia are due
to the substitution of a single nucleotide base in
the !-globin gene. The sickle cell mutation is
the transversion of the second nucleotide base
of codon 6, adenine (A), to thymine (T). This
changes the codon GAG, for glutamic acid, to
GTG, the codon for valine. During the 1950s,
Vernon M. Ingram determined the amino acid
sequence of hemoglobin and found that the
only difference between sickle cell hemoglobin
(HbS) and normal adult hemoglobin (HbA) was
this exchange in the ! chain. This has far-reaching
pathophysiological consequences and explains
all manifestations of the disease. Sickle
cell hemoglobin (HbS) is less soluble than normal
hemoglobin and does not allow normal
erythrocyte distortion. It crystallizes in the
deoxy state and forms small rods. Thus, the
erythrocytes become firm and deform into
sickle cells. Unlike normal erythrocytes, sickle
cells are unable to pass through small arteries
and capillaries. These become clogged and
cause local oxygen deficiency in the tissues,
followed by infection. As a rule, learning disability
due to frequent illness occurs. Defective
erythrocytes are destroyed (hemolysis).
Chronic anemia and its numerous sequelae
such as heart failure, liver damage, and infection
are the result.
to the substitution of a single nucleotide base in
the !-globin gene. The sickle cell mutation is
the transversion of the second nucleotide base
of codon 6, adenine (A), to thymine (T). This
changes the codon GAG, for glutamic acid, to
GTG, the codon for valine. During the 1950s,
Vernon M. Ingram determined the amino acid
sequence of hemoglobin and found that the
only difference between sickle cell hemoglobin
(HbS) and normal adult hemoglobin (HbA) was
this exchange in the ! chain. This has far-reaching
pathophysiological consequences and explains
all manifestations of the disease. Sickle
cell hemoglobin (HbS) is less soluble than normal
hemoglobin and does not allow normal
erythrocyte distortion. It crystallizes in the
deoxy state and forms small rods. Thus, the
erythrocytes become firm and deform into
sickle cells. Unlike normal erythrocytes, sickle
cells are unable to pass through small arteries
and capillaries. These become clogged and
cause local oxygen deficiency in the tissues,
followed by infection. As a rule, learning disability
due to frequent illness occurs. Defective
erythrocytes are destroyed (hemolysis).
Chronic anemia and its numerous sequelae
such as heart failure, liver damage, and infection
are the result.
Selective advantage for HbS heterozygotes in areas of malaria
Heterozygotes for the sickle cell mutation are
relatively resistant to malarial infection. Erythrocytes
of heterozygotes for the sickle cell mutation
are a less favorable environment for the
malaria parasite than those of normal homozygotes.
Thus, heterozygotes develop malaria in
a much milder form or not at all. However, this
protection is at the expense of the affected homozygotes
(HbS/HbS): although they do not
contract malaria, they suffer from the severe
hemoglobin disorder. The protection against
malaria conferred by sickle cell heterozygosity
is an advantage in regions where malaria is
common. With reduced morbidity and mortality,
heterozygotes have a higher probability of
survival and of being able to reproduce (selective
advantage). This explains the high
frequency of the sickle cell gene observed there
(see p. 168). The sickle cell mutation has arisen
independently in at least four or five different
malaria-infested regions and has subsequently
spread out in the respective populations. Sickle
cell anemia is the best example in humans of a
selective advantage in heterozygotes for a mutant
allele that leads to severe illness in the homozygous
state.
relatively resistant to malarial infection. Erythrocytes
of heterozygotes for the sickle cell mutation
are a less favorable environment for the
malaria parasite than those of normal homozygotes.
Thus, heterozygotes develop malaria in
a much milder form or not at all. However, this
protection is at the expense of the affected homozygotes
(HbS/HbS): although they do not
contract malaria, they suffer from the severe
hemoglobin disorder. The protection against
malaria conferred by sickle cell heterozygosity
is an advantage in regions where malaria is
common. With reduced morbidity and mortality,
heterozygotes have a higher probability of
survival and of being able to reproduce (selective
advantage). This explains the high
frequency of the sickle cell gene observed there
(see p. 168). The sickle cell mutation has arisen
independently in at least four or five different
malaria-infested regions and has subsequently
spread out in the respective populations. Sickle
cell anemia is the best example in humans of a
selective advantage in heterozygotes for a mutant
allele that leads to severe illness in the homozygous
state.
Genetics and Medicine Mutations in Globin Genes
All types of mutations have been demonstrated
in the globin genes. The most frequent are point
mutations in a single codon. The functional consequences
vary, depending on the electrical
charge and size of the substituted amino acid
and its position in the polypeptide. If one of the
hydrophilic amino acids at the surface is replaced
by a hydrophobic amino acid (e.g., valine
for glutamic acid in the sickle cell mutation),
profound physicochemical changes will result.
Mutations may decrease the elasticity of the
molecule, alter its oxygen affinity, or cause instability.
in the globin genes. The most frequent are point
mutations in a single codon. The functional consequences
vary, depending on the electrical
charge and size of the substituted amino acid
and its position in the polypeptide. If one of the
hydrophilic amino acids at the surface is replaced
by a hydrophobic amino acid (e.g., valine
for glutamic acid in the sickle cell mutation),
profound physicochemical changes will result.
Mutations may decrease the elasticity of the
molecule, alter its oxygen affinity, or cause instability.
Point mutations of the !-globin gene
Over 300 point mutations in the !-globin gene
and over 100 in one of the "-globin genes have
been documented. Two clinically important
mutations affect codon 6: the sickle cell mutation,
6 Glu ! Val (sickle cell hemoglobin, HbS,
resulting in the incorporation of valine instead
of glutamic acid) and 6 Glu ! Lys (hemoglobin
C, HbC, incorporating lysine instead of glutamic
acid in codon 6). Compound heterozygoteswith
the HbS mutation on one chromosome and the
HbC on the other (HbSC) are not rare. The
marked methemoglobin formation in Hb Zürich
and Hb Saskatoon results fromsubstitutions for
histidine (His) in codon 63, which alter the oxygen-
binding region of the hemoglobin
molecule.
and over 100 in one of the "-globin genes have
been documented. Two clinically important
mutations affect codon 6: the sickle cell mutation,
6 Glu ! Val (sickle cell hemoglobin, HbS,
resulting in the incorporation of valine instead
of glutamic acid) and 6 Glu ! Lys (hemoglobin
C, HbC, incorporating lysine instead of glutamic
acid in codon 6). Compound heterozygoteswith
the HbS mutation on one chromosome and the
HbC on the other (HbSC) are not rare. The
marked methemoglobin formation in Hb Zürich
and Hb Saskatoon results fromsubstitutions for
histidine (His) in codon 63, which alter the oxygen-
binding region of the hemoglobin
molecule.
Deletion due to unequal crossing-over within a gene
Marked sequence homology of certain regions
of the globin genes may lead to nonhomologous
pairing and unequal crossing-over during
meiosis, e.g., in the regions of codons 90–94 of
one DNA strand and codons 95–98 of the other.
This explains the deletion of codons 91–95 in
hemoglobin Gun Hill.
of the globin genes may lead to nonhomologous
pairing and unequal crossing-over during
meiosis, e.g., in the regions of codons 90–94 of
one DNA strand and codons 95–98 of the other.
This explains the deletion of codons 91–95 in
hemoglobin Gun Hill.
Unequal crossing-over between similar genes
The sequence homology of the !-globin-like
genes (explained by their common evolution)
may lead to unequal crossover between regions
of the two #-globin genes (#A and #G), the $-
globin gene, or the !-globin gene. The best
known example is partial deletion of the $
genes (explained by their common evolution)
may lead to unequal crossover between regions
of the two #-globin genes (#A and #G), the $-
globin gene, or the !-globin gene. The best
known example is partial deletion of the $
Unstable hemoglobin due to chain elongation
If one of the globin chains is too long, it will
destabilize the tetrameric hemoglobin
molecule. Hemoglobin Cranston (HbCr) (1)
arises from the insertion of two nucleotide
bases (adenine and guanine) into positions 1
and 2 of codon 145 (tyrosine) of the ! chain,
which leads to a shift of the reading frame. This
changes the normal stop codon UAA into AGU,
the RNA codon for threonine (Thr). As a result,
the normally nontranslated sequences that follow
the stop codon are now translated, and a
polypeptide is formed that is 11 amino acids too
long, extending to position 157.With hemoglobin
Constant Spring (2), the " chain is
lengthened by mutation of the stop codon UAA
to CAA, which codes for glutamine (Gln). The
sequences that normally follow the stop codon
now become translated, and a peptide that is 31
amino acids too long is formed. A number of
other chain-elongation mutations due to similar
mechanisms, such as with hemoglobin
Ikaria
destabilize the tetrameric hemoglobin
molecule. Hemoglobin Cranston (HbCr) (1)
arises from the insertion of two nucleotide
bases (adenine and guanine) into positions 1
and 2 of codon 145 (tyrosine) of the ! chain,
which leads to a shift of the reading frame. This
changes the normal stop codon UAA into AGU,
the RNA codon for threonine (Thr). As a result,
the normally nontranslated sequences that follow
the stop codon are now translated, and a
polypeptide is formed that is 11 amino acids too
long, extending to position 157.With hemoglobin
Constant Spring (2), the " chain is
lengthened by mutation of the stop codon UAA
to CAA, which codes for glutamine (Gln). The
sequences that normally follow the stop codon
now become translated, and a peptide that is 31
amino acids too long is formed. A number of
other chain-elongation mutations due to similar
mechanisms, such as with hemoglobin
Ikaria
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