Chromoproteins. Pathological and
physiological forms of hemoglobin
Hemoglobin
Hemoglobin is a conjugated protein, consisting of simple protein globin
and prosthetic group – hem. Hem is helatic complex of the porphyrin with an
iron atom in the center. Porphyrin is cyclic compound, which has 4 pyrrole groups, joined together by methane bridges.
There are different porphyrins that differ from each other by lateral groups of
the pyrrole rings. Heme forms part of mioglobin, cytochrom-b, cytochrome P-450,
catalases, peroxydases too (heme-containing proteins). Ion of Fe is combined
with four atoms of nitrogen of pyrrole rings (2 covalent bonds, 2
donor-acceptic bonds) and by coordination bond with atom of nitrogen of
histidine imidazole group in polypeptide chain.
Globin is protein consisting of four polypeptide chains (2a chains – each of them contains 141 amino acid residues, 2b chains containing of 146 amino acidic residues). Every chain is
connected only with one heme molecule. Four polypeptide subunits in space in
the form of tetrahedron in a compact packaged gives us globular molecule, where
subunits are connected with each other. This is a main form of hemoglobin of
adult person – hemoglobin A. Approximately 2 % of human hemoglobin is
hemoglobin A2, which contains d-chains
instead of β-chains (2a2d). Fetus has HbF (fetal),
consisting of 2a and
2g chains and forming 80 % of Hb.
At the last week of pregnancy and the first days after the birth HbF
gradually changes to HbA, and after 1st year content of Hb F is near
1,5 %. HbF has greater affinity for
oxygen than does HbA that is why fetus can pick up oxygen from the maternal
bloodstream.
In human blood nearly 300 variations of Hb are discovered, appeared as a
result of gene mutations, but majority of them don’t cause some disease. One of
most important anomalous hemoglobin is HbS. People – germ carriers of gene HbS
has sickle cell anaemia that is hemolytic by the mechanism of
development. HbS differs from HbA by the substitution of the one amino acid at
6th position of b
chain, where glutamate is substituted by valine. These amino acids are
different by the charge and hydrophobic interactions, the substitution lower
the solubility of HbS in deoxyform. Molecules of deoxyhemoglobin form the
threads, fibers and bunch of fibers, which cause the change of the erythrocytes
form. Sickle cells are less stable than normal ones and are destroying very
fast. The blood of homozygotic people has only HbS and the severe anemia are
developed, death comes in early childhood. The blood of the heterozygotic
people has HbS and HbA, so only the poor symptoms of disease appear. In such
individuals the process of development of malarial plasmodium delayed and they
do not ill to malaria or can easy cope with it. Gene HbS is common in malaria
regions.
The relation of some anomaly hemoglobins to the oxygen increases or
decreases which also can cause to hematolytic diseases. Besides these
hemoglobins diseases are hereditary ones as a result of dysfunction producing a and b
chains in equal quantities or the absolute absence of synthesis of one kind of
chain. These diseases are called talassemia. The result of misbalance a and b
chains is that superfluous chains sedimantats the level of hemoglobin and the
life period of erythrocytes decrease. Homozygotic form of talassemia is
resulted to death at the prenatal or neonatal periods.
Oxygen transport is one of the main blood functions. Only minor quantity
of the O2 is transported in soluble form, whereas bound with hemoglobin
quantity is greater in 70 times. One molecule of the hemoglobin consisting of
four hems can bind four molecules of the oxygen. Joining of oxygen doesn’t
change the iron valency, because it is providing by coordination bonds of iron
molecule. Hemoglobin bound with oxygen is
called oxyhemoglobin (oxygenated hemoglobin).
Carbonate is transported by the blood in soluble form - 6-7 %, bound
with hemoglobin (carbhemoglobin) – 3-10 %, and as hydrocarbonates – 80 %.
CO2+H2O«H2CO3®H++HCO3–
In carbhemoglobin molecule CO2 is connected with N-end
of each from 4 polypeptide chain. This compound is very unstable and can
dissociate in lungs capillaries with CO2 chipping off.
Carboxyhemoglobin is connection of hemoglobin with carbon monoxide CO.
The affinity of hemoglobin to CO is in 200 times higher than to O2.
Very small concentration of CO in air has toxic effect on the organism. When
one part of gem group is connected with CO and other one with O2, molecules of
hemoglobin give oxygen worse than hemoglobin in connection with 4 molecules of
oxygen. So in poisoning CO hypoxia is making not only by connection part of
hems with CO but also the shift of oxyhemoglobin dissociation.
The Fe2+ in hemoglobin is susceptible to oxidation to Fe3+
by superoxide and other oxidizing agents (amilnitrit, aniline, nitrobensol,
nitrates and nitrites, tiosulfats, fericianid), forming metHb, which cannot
transport oxygen. Only a very small amount of metHb is present in normal blood,
as the RBC possesses an effective system (the NADH-cytochrome b5 methemoglobin
reductase system) for reducing heme Fe3+ back to the Fe2+
state. This system consists of NADH (generated by glycolysis), a flavoprotein
named cytochrome b5 reductase (also known as metHb reductase), and
cytochrome b5. The Fe3+ of metHb is reduced back to the
Fe2+ state by the action of reduced cytochrome b5:
Hb-Fe3+ + Ñyt b5
red ® Hb-Fe2+ + Ñyt b5
ox
Reduced cytochrome b5 is then regenerated by the action of
cytochrome b5 reductase:
Ñyt b5 ox + NADH ® Ñyt b5 red + NAD
Haemoglobin or
hemoglobin (frequently abbreviated as Hb or Hgb) is the
iron-containing oxygen-transport metalloprotein in the red blood
cells of the blood in vertebrates and other animals; in mammals the protein
makes up about 97% of the red cell’s drycontent, and around 35% of the
total content including water. Hemoglobin transports oxygen from the lungs or
gills to the rest of the body, such as to the muscles, where it releases the
oxygen load. Hemoglobin also has a variety of other gas-transport and
effect-modulation duties, which vary from species to species, and which in
invertebrates may be quite diverse.
The name
hemoglobin is the concatenation of heme and globin, reflecting
the fact that each subunit of hemoglobin is a globular protein with an
embedded heme (or haem) group; each heme group
contains an iron atom, and this is responsible for the binding of oxygen. The
most common type of hemoglobin in mammals contains four such subunits, each
with one heme group.
Mutations in the
genes for the hemoglobin protein in humans result in a group of hereditary
diseases termed thehemoglobinopathies, the most common members of which are
sickle-cell disease and thalassemia. Historically in human medicine,
thehemoglobinopathy of sickle-cell disease was the first disease to be understood
in its mechanism of dysfunction, completely down to the molecular level.
However, not all of such mutations produce disease states, and are formally
recognized as hemoglobin variants (not diseases).
Hemoglobin (Hb) is
synthesized in a complex series of steps. The heme portion
is sythesized in both the the mitochondria
andcytosol of the immature red blood cell, while
the globin protein portions of the molecule
are sythesized by ribosomes in the cytosol[3].
Production of Hb continues in the cell throughout its early
development from the proerythroblast to the reticulocyte in
the bone marrow. At this point, the nucleus is lost in mammals, but not in
birds and many other species. Even after the loss of the nucleus in mammals,
however, residual ribosomal RNA allows further synthesis of Hb until
the reticulocyte loses its RNA soon after entering the vasculature
(this hemoglobin-synthetic RNA in fact gives the reticulocyte its
reticulated appearance and name).
The empirical
chemical formula of the most common human hemoglobin is
C2952H4664N812O832S8Fe4, but as noted above,hemoglobins vary widely across
species, and even (through common mutations) slightly among subgroups of
humans.
In humans, the
hemoglobin molecule is an assembly of four globular protein subunits. Each
subunit is composed of a protein chain tightly associated with a
non-protein heme group. Each protein chain arranges into a set of
alpha-helix structural segments connected together in a globin fold
arrangement, so called because this arrangement is the same folding motif used
in otherheme/globin proteins such as myoglobin. This folding pattern
contains a pocket which strongly binds the heme group.
A heme group
consists of an iron (Fe) atom held in a heterocyclic ring, known as
a porphyrin. The iron atom, which is the site of oxygen binding, bonds
with the four nitrogens in the center of the ring, which
all lie in one plane. The iron is also bound strongly to the globular
protein via the imidazole ring of a histidine residue below
the porphyrin ring. A sixth position can reversibly bind oxygen,
completing the octahedral group of six ligands. Oxygen binds in an
"end-on bent" geometry where one oxygen atom binds Fe and the other
protrudes at an angle. When oxygen is not bound, a very weakly bonded water
molecule fills the site, forming a distorted octahedron.
The iron atom may
either be in the Fe2+ or Fe3+ state,
but ferrihemoglobin (methemoglobin) (Fe3+) cannot bind oxygen. In
binding, oxygen temporarily oxidizes Fe to (Fe3+), so iron must exist in the +2
oxidation state in order to bind oxygen. The body reactivates hemoglobin found
in the inactive (Fe3+) state by reducing the iron center.
In adult humans,
the most common hemoglobin type is a tetramer (which contains 4 subunit proteins)
called hemoglobin A, consisting of two α and two β subunits non-covalently
bound, each made of 141 and 146 amino acid residues, respectively. This is
denoted as α2β2. The subunits are structurally similar and about the same size. Each
subunit has a molecular weight of about 17,000daltons, for a total molecular
weight of the tetramer of about 68,000 daltons. Hemoglobin A is the most
intensively studied of the hemoglobin molecules.
The four
polypeptide chains are bound to each other by salt bridges, hydrogen bonds, and
hydrophobic interactions. There are two kinds of contacts between the α and β chains: α1β1 and α1β2.
Oxyhemoglobin is
formed during respiration when oxygen binds to the heme component of
the protein hemoglobin in red blood cells. This process occurs in the pulmonary
capillaries adjacent to the alveoli of the lungs. The oxygen then travels
through the blood stream to be dropped off at cells where it is utilized in
aerobic glycolysis and in the production of ATP by the process of
oxidativephosphorylation. It doesn't however help to counteract a decrease in
blood pH. Ventilation, or breathing, may reverse this condition by
removal of carbon dioxide, thus causing a shift up in pH.[6]
Deoxyhemoglobin is
the form of hemoglobin without the bound oxygen. The absorption spectra
of oxyhemoglobin anddeoxyhemoglobin differ.
The oxyhemoglobine has significantly lower absorption of the 660 nm
wavelength than deoxyhemoglobin, while at 940 nm its absorption is
slightly higher. This difference is used for measurement of the amount of
oxygen in patient's blood by an instrument called pulse oximeter.
Iron's oxidation
state in oxyhemoglobin.
The oxidation
state of iron in hemoglobin is always +2. It does not change when oxygen binds
to the deoxy- form.
Assigning
oxygenated hemoglobin's oxidation state is difficult
because oxyhemoglobin is diamagnetic (no net unpaired electrons), but
the low-energy electron configurations in both oxygen and iron are paramagnetic.
Triplet oxygen, the lowest energy oxygen species, has two unpaired electrons
in antibonding π*
molecular orbitals. Iron(II) tends to be in a high-spin configuration
where unpaired electrons exist
in eg antibonding orbitals. Iron(III) has an odd number of
electrons and necessarily has unpaired electrons. All of these molecules are
paramagnetic (have unpaired electrons), not diamagnetic, so an unintuitive
distribution of electrons must exist to induce diamagnetism.
The three logical
possibilities are:
1) Low-spin Fe2+
binds to high-energy singlet oxygen. Both low-spin iron and singlet oxygen are
diamagnetic.
2) High-spin Fe3+
binds to .O2- (the superoxide ion) and antiferromagnetism oppositely
aligns the two unpaired electrons, giving diamagnetic properties.
3) Low-spin Fe4+
binds to O22-. Both are diamagnetic.
X-ray
photoelectron spectroscopy suggests that iron has an oxidation state of
approximately 3.2 and infrared stretching frequencies of the O-O bond
suggests a bond length fitting with superoxide. The correct oxidation
state of iron is thus the +3 state with oxygen in the -1 state. The
diamagnetism in this configuration arises from the unpaired electron on
superoxide aligningantiferromagnetically in the opposite direction from
the unpaired electron on iron. The second choice being correct is not
surprising because singlet oxygen and large separations of charge are both
unfavorably high-energy states. Iron's shift to a higher oxidation state
decreases the atom's size and allows it into the plane of the porphyrin ring,
pulling on the coordinated histidine residue and initiating
the allosteric changes seen in the globulins. The assignment of
oxidation state, however, is only a formalism so all three models may
contribute to some small degree.
Early postulates
by bioinorganic chemists claimed that possibility (1) (above) was correct and
that iron should exist in oxidation state II (indeed iron oxidation state III
as methemoglobin, when not accompanied by superoxide .O2- to
"hold" the oxidation electron, is incapable of binding O2). The iron
chemistry in this model was elegant, but the presence of singlet oxygen was
never explained. It was argued that the binding of an oxygen molecule placed
high-spin iron(II) in an octahedral field of strong-field ligands;
this change in field would increase the crystal field splitting energy, causing
iron's electrons to pair into the diamagnetic low-spin configuration.
Binding
of ligands
Binding and
release of ligands induces a conformational (structural) change in
hemoglobin. Here, the binding and release of oxygen illustrates the structural
differences between oxy- and deoxyhemoglobin, respectively. Only one of
the four heme groups is shown.
As discussed
above, when oxygen binds to the iron center it causes contraction of the iron
atom, and causes it to move back into the center of
the porphyrin ring plane (see moving diagram). At the same time,
the porphyrin ring plane itself is pushed away from the oxygen and
toward the imidizole side chain of the histidine residue
interacting at the other pole of the iron. The interaction here forces the ring
plane sideways toward the outside of the tetramer, and also induces a strain on
the protein helix containing thehistidine, as it moves nearer the iron. This
causes a tug on this peptide strand which tends to open up heme units
in the remainder of the molecule, so that there is more room for oxygen to bind
at their heme sites.
In
the tetrameric form of normal adult hemoglobin, the binding of oxygen
is thus a cooperative process. The binding affinity of hemoglobin for oxygen is
increased by the oxygen saturation of the molecule, with the
first oxygens bound influencing the shape of the binding sites for
the next oxygens, in a way favorable for binding. This positive cooperative
binding is achieved through stericconformational changes of the hemoglobin
protein complex as discussed above, i.e. when one subunit protein in hemoglobin
becomes oxygenated, this induces a conformational or structural
change in the whole complex, causing the other subunits to gain an increased
affinity for oxygen. As a consequence, the oxygen binding curve of hemoglobin
is sigmoidal, or S-shaped, as opposed to the normal hyperbolic curve
associated with noncooperative binding.
Hemoglobin's
oxygen-binding capacity is decreased in the presence of carbon monoxide because
both gases compete for the same binding sites on hemoglobin, carbon monoxide
binding preferentially in place of oxygen. Carbon dioxide occupies a different
binding site on the hemoglobin. Through the enzyme carbonic anhydrase,
carbon dioxide reacts with water to give carbonic acid, which decomposes into
bicarbonate and protons:
CO2 + H2O →
H2CO3 → HCO3- + H+
The sigmoidal shape
of hemoglobin's oxygen-dissociation curve results from cooperative binding of oxygen
to hemoglobin.
Hence blood with
high carbon dioxide levels is also lower in pH (more acidic). Hemoglobin can
bind protons and carbon dioxide which causes a conformational change in the
protein and facilitates the release of oxygen. Protons bind at various places
along the protein, and carbon dioxide binds at the alpha-amino group
forming carbamate. Conversely, when the carbon dioxide levels in the blood
decrease (i.e., in the lung capillaries), carbon dioxide and protons are
released from hemoglobin, increasing the oxygen affinity of the protein. This
control of hemoglobin's affinity for oxygen by the binding and release of
carbon dioxide and acid, is known as the Bohr effect.
The binding of
oxygen is affected by molecules such as carbon monoxide (CO) (for example from
tobacco smoking, cars and furnaces). CO competes with oxygen at
the heme binding site. Hemoglobin binding affinity for CO is 200
times greater than its affinity for oxygen, meaning that small amounts of
CO dramatically reduces hemoglobin's ability to transport oxygen. When
hemoglobin combines with CO, it forms a very bright red compound
called carboxyhemoglobin. When inspired air contains CO levels as low as
0.02%, headache and nausea occur; if the CO concentration is increased to 0.1%,
unconsciousness will follow. In heavy smokers, up to 20% of the oxygen-active
sites can be blocked by CO.
In similar
fashion, hemoglobin also has competitive binding affinity for cyanide (CN-),
sulfur monoxide (SO), nitrogen dioxide (NO2), and sulfide (S2-), including
hydrogen sulfide (H2S). All of these bind to iron in heme without
changing its oxidation state, but they nevertheless inhibit oxygen-binding,
causing grave toxicity.
The iron atom in
the heme group must be in the Fe2+ oxidation state to support oxygen
and other gases' binding and transport. Oxidation to Fe3+ state converts
hemoglobin into hemiglobin or methemoglobin (pronounced
"MET-hemoglobin"), which cannot bind oxygen. Hemoglobin in normal red
blood cells is protected by a reduction system to keep this from happening.
Nitrogen dioxide and nitrous oxide are capable of converting a small fraction
of hemoglobin to methemoglobin, however this is not usually of medical
importance (nitrogen dioxide is poisonous by other mechanisms, and nitrous
oxide is routinely used in surgical anesthesia in most people without
undue methemoglobin buildup).
In people
acclimated to high altitudes, the concentration of 2,3-bisphosphoglycerate
(2,3-BPG) in the blood is increased, which allows these individuals to deliver
a larger amount of oxygen to tissues under conditions of lower oxygen tension.
This phenomenon, where molecule Y affects the binding of molecule X to a
transport molecule Z, is called
a heterotropic allosteric effect.
A
variant hemoglobin, called fetal hemoglobin (HbF, α2γ2), is found
in the developing fetus, and binds oxygen with greater affinity than adult
hemoglobin. This means that the oxygen binding curve for fetal hemoglobin is
left-shifted (i.e., a higher percentage of hemoglobin has oxygen bound to it at
lower oxygen tension), in comparison to that of adult hemoglobin. As a result,
fetal blood in the placenta is able to take oxygen from maternal blood.
Hemoglobin also
carries nitric oxide in the globin part of the molecule. This
improves oxygen delivery in the periphery and contributes to the control of
respiration. NO binds reversibly to a specific cystein residue
in globin; the binding depends on the state (R or T) of the hemoglobin.
The resulting S-nitrosylated hemoglobin influences various NO-related
activities such as the control of vascular resistance, blood pressure and
respiration. NO is released not in the cytoplasm of erythrocytes but is
transported by an anion exchanger called AE1 out of them.
Degradation of
hemoglobin in vertebrate animals
When red cells
reach the end of their life due to aging or defects, they are broken down, the
hemoglobin molecule is broken up and the iron gets recycled. When
the porphyrin ring is broken up, the fragments are normally secreted
in the bile by the liver. This process also produces one molecule of carbon
monoxide for every molecule of heme degraded; this is one of the few
natural sources of carbon monoxide production in the human body, and is
responsible for the normal blood levels of carbon monoxide even in people
breathing pure air. The other major final product of heme degradation
is bilirubin. Increased levels of this chemical are detected in the
blood if red cells are being destroyed more rapidly than usual. Improperly
degraded hemoglobin protein or hemoglobin that has been released from the blood
cells too rapidly can clog small blood vessels, especially the delicate blood
filtering vessels of the kidneys, causing kidney damage
Role in disease
Decrease of hemoglobin,
with or without an absolute decrease of red blood cells, leads to symptoms of
anemia. Anemia has many different causes, although iron deficiency and its
resultant iron deficiency anemia are the most common causes in the Western
world. As absence of iron decreases heme synthesis, red blood cells
in iron deficiency anemia are hypochromic (lacking the red hemoglobin
pigment) and microcytic (smaller than normal).
Other anemias are rarer. In hemolysis (accelerated
breakdown of red blood cells), associated jaundice is caused by the hemoglobin
metabolite bilirubin, and the circulating hemoglobin can cause renal
failure.
Some mutations in
the globin chain are associated with the hemoglobinopathies,
such as sickle-cell disease and thalassemia. Other mutations, as discussed
at the beginning of the article, are benign and are referred to merely as
hemoglobin variants.
There is a group
of genetic disorders, known as the porphyrias that are characterized
by errors in metabolic pathways of hemesynthesis. King George III of the
United Kingdom was probably the most famous porphyria sufferer.
To a small extent,
hemoglobin A slowly combines with glucose at a certain location in the
molecule. The resulting molecule is often referred to as Hb A1c. As
the concentration of glucose in the blood increases, the percentage
of Hb A that turns into Hb A1cincreases. In diabetics whose
glucose usually runs high, the percent Hb A1c also runs high.
Because of the slow rate of Hb A combination with glucose,
the Hb A1c percentage is representative of glucose level in the
blood averaged over a longer time (the half-life of red blood cells, which is
typically 50-55 days).
Diagnostic use
Hemoglobin levels
are amongst the most commonly performed blood tests, usually as part of a full
blood count or complete blood count. Results are reported in g/L, g/dL or
mol/L. For conversion, 1 g/dL is 0,621 mmol/L. If the total
hemoglobin concentration in the blood falls below a set point, this is called
anemia. Normal values for hemoglobin levels are:
• Women: 12.1
to 15.1 g/dl
• Men: 13.8
to 17.2 g/dl
• Children:
11 to 16 g/dl
• Pregnant
women: 11 to 12 g/dl
Anemias are
further subclassified by the size of the red blood cells, which are
the cells which contain hemoglobin in vertebrates. They can be classified
as microcytic (small sized red blood
cells), normocytic (normal sized red blood cells),
or macrocytic (large sized red blood cells).
The hemaglobin is the typical test used for blood donation. A
comparison with the hematocrit can be made by multiplying
the hemaglobin by three. For example, if the hemaglobin is
measured at 17, that compares with a hematocrit of .51.
Glucose levels in
blood can vary widely each hour, so one or only a few samples from a patient
analyzed for glucose may not be representative of glucose control in the long
run. For this reason a blood sample may be analyzed
for Hb A1c level, which is more representative of glucose
control averaged over a longer time period (determined by the half-life of the
individual's red blood cells, which is typically 50-55 days). People
whose Hb A1c runs 6.0% or less show good longer-term glucose
control. Hb A1c values which are more than 7.0% are elevated.
This test is especially useful for diabetics.