HAEMOGLOBIN

Dr. Shazia Nisar

 Hemoglobin or haemoglobin
(Hb or Hgb)
• iron-containing oxygen-transport metalloprotein
• Found in the red blood cells of all vertebrates as
well as the tissues of some invertebrates.
• Hemoglobin in the blood carries oxygen from the
respiratory organs (lungs or gills) to the rest of the
body (i.e. the tissues) where it releases the
oxygen to burn nutrients to provide energy to
power the functions of the organism in the
process called metabolism.
• In mammals, the protein makes up about 96% of
the red blood cells' dry content (by weight), and
around 35% of the total content (including water).
• It has an oxygen-binding capacity of 1.34 mL
O2 per gram of hemoglobin, which increases the
total blood oxygen capacity seventy-fold
compared to dissolved oxygen in blood.
• The mammalian hemoglobin molecule can bind
up to four oxygen molecules.
• transports other gases (such as CO2 , CO)
• carbaminohemoglobin, in which CO2 is bound to
the globin protein.
• The molecule also carries the important
regulatory molecule nitric oxide bound to a
globin protein thiol group, releasing it at the
same time as oxygen.
• Hemoglobin is also found outside red blood
cells. Other cells that contain hemoglobin
include the A9 dopaminergic neurons in
the substantia nigra, macrophages,
alveolar cells and mesangial cells in the
kidney. In these tissues, hemoglobin has a
non-oxygen-carrying function as an
antioxidant and a regulator of iron metabolism
.
• Hemoglobin and hemoglobin-like molecules are
also found in many invertebrates, fungi, and
plants.
• In these organisms, hemoglobins may carry
oxygen, or they may act to transport and regulate
other things such as carbon dioxide, nitric oxide,
hydrogen sulfide and sulfide.
• A variant of the molecule, called leghemoglobin,
is used to scavenge oxygen away from anaerobic
systems, such as the nitrogen-fixing nodules of
leguminous plants, before the oxygen can poison
the system.
 Synthesis

Hemoglobin (Hb) is synthesized in a complex

series of steps.
• The heme part is synthesized in a series of
steps in the mitochondria and the cytosol of
immature red blood cells,
• While the globin protein parts are synthesized
by ribosomes in the cytosol.
Structure of human hemoglobin. The proteins α and β subunits are in
red and blue, and the iron-containing heme groups in green.
• The three-dimensional structure of
hemoglobin was solved using X-ray
crystallography in 1959 by Max Perutz.
• The structure of hemoglobin is very similar to
the single polypeptide chain in myoglobin
despite the fact that their amino acid
sequences differ at 83% of the residues.
• 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
16,000 daltons, for a total molecular weight of the tetramer
of about 64,000 daltons (64,458 g/mol).
• In human infants, the hemoglobin molecule is made up of 2
α chains and 2 γ chains. The gamma chains are gradually
replaced by β chains as the infant grows.
• The four polypeptide chains are bound to each other by
salt bridges, hydrogen bonds, and the hydrophobic effect.
• It has a quaternary structure characteristic of
many multi-subunit globular proteins.
• Most of the amino acids in hemoglobin form
alpha helices, connected by short non-helical
segments.
• Hydrogen bonds stabilize the helical sections
inside this protein, causing attractions within the
molecule, folding each polypeptide chain into a
specific shape.
• Hemoglobin's quaternary structure comes from
its four subunits in roughly a tetrahedral
arrangement.
• This folding pattern contains a pocket that
strongly binds the heme group.
• A heme group consists of an iron (Fe) ion
(charged atom) held in a heterocyclic ring,
known as a porphyrin.
• This porphyrin ring consists of
four pyrrole molecules cyclically linked
together (by methine bridges) with the iron
ion bound in the center.
Heme b group Heme a group
• The iron ion, which is the site of oxygen binding,
coordinates with the four nitrogens in the center of
the ring, which all lie in one plane.
• The iron is bound strongly (covalently) to the
globular protein via the imidazole ring of
F8 histidine residue (also known as the proximal
histidine) below the porphyrin ring.
• A sixth position can reversibly bind oxygen by a
coordinate covalent bond, 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 ion may be either in the Fe2+ or in the
Fe3+ state, but ferrihemoglobin (methemoglobin)
(Fe3+) cannot bind oxygen.
• In binding, oxygen temporarily and reversibly
oxidizes (Fe2+) to (Fe3+) while oxygen temporarily
turns into superoxide, thus iron must exist in the
+2 oxidation state to bind oxygen.
• If superoxide ion associated to Fe3+ is protonated,
the hemoglobin iron will remain oxidized and
incapable of binding oxygen. In such cases, the
enzyme methemoglobin reductase will be able to
eventually reactivate methemoglobin by reducing
the iron center.
 Oxygen saturation
• hemoglobin saturated with oxygen molecules
(oxyhemoglobin), or
• desaturated with oxygen molecules
(deoxyhemoglobin).
Oxyhemoglobin
• is formed during physiological respiration
• 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 as a terminal
electron acceptor in the production of ATP by the
process of oxidative phosphorylation.
 T and R States of Hb
• Hemoglobin exists in two forms, a taut (tense)
form (T) and a relaxed form (R).
• The T state has a less of an affinity for oxygen than
the R state.
• The T-state of hemoglobin is the more "Tense" of
the two; this is the deoxy form of hemoglobin
(deoxyhemoglobin).
• The R-state of hemoglobin is more "Relaxed" and
is the fully oxygenated form (oxyhemoglobin).
• The conformation of hemoglobin also
changes as the oxygen binds to the iron,
raising both the iron and the histidine
residue bound to it. The oxygen binding
changes the position of the iron ion by
approximately 0.4 A.
• Before oxygenation the iron ion lies
slightly outside the plane of the
porphyrin upon oxygenation it moves
into the plane of the heme.
 Factors affecting T & R states
• low pH, high CO2 and high BPG (2,3-
Bisphosphoglycerate) at the level of the tissues favor
the taut form, which has low oxygen affinity and
releases oxygen in the tissues.
• a high pH, low CO2, or low 2,3 BPG favors the relaxed
form, which can better bind oxygen.
• The partial pressure of the system also affects
O2 affinity where, at high partial pressures of oxygen
(such as those present in the alveoli), the relaxed (high
affinity, R) state is favored.
• Inversely, at low partial pressures (respiring tissues),
the (low affinity, T) tense state is favored.
• Additionally, the binding of oxygen to the Iron-
II heme pulls the iron into the plane of the
porphyrin ring, causing a slight conformational
shift. The shift encourages oxygen to bind to
the three remaining hemes within hemoglobin
(thus, oxygen binding is cooperative).
 Deoxygenated hemoglobin

• Deoxygenated hemoglobin is the form of hemoglobin

without the bound oxygen.
• The absorption spectra of oxyhemoglobin and
deoxyhemoglobin differ.
• The oxyhemoglobin 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.
• This difference also accounts for the presentation of
cyanosis, the blue to purplish color that tissues
develop during hypoxia.
 Cooperativity

• A schematic visual model of oxygen-binding process, showing all four monomers

and hemes, and protein chains only as diagramatic coils, to facilitate visualization into
the molecule. Oxygen is not shown in this model, but, for each of the iron atoms, it
binds to the iron (red sphere) in the flat heme. For example, in the upper-left of the four
hemes shown, oxygen binds at the left of the iron atom shown in the upper-left of
diagram. This causes the iron atom to move backward into the heme that holds it (the
iron moves upward as it binds oxygen, in this illustration), tugging the histidine residue
(modeled as a red pentagon on the right of the iron) closer, as it does. This, in turn, pulls
on the protein chain holding the histidine.
• 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.
• When oxygen binds to the iron complex, it causes
the iron atom to move back toward the center of
the plane of the porphyrin ring.
• At the same time, the imidazole side-chain of the
histidine residue interacting at the other pole of
the iron is pulled toward the porphyrin ring.
• This interaction forces the plane of the ring sideways
toward the outside of the tetramer, and also induces
a strain in the protein helix containing the histidine
as it moves nearer to the iron atom.
• This positive cooperative binding is achieved
through steric conformational changes of the
hemoglobin protein complex.
• When one subunit protein in hemoglobin becomes
oxygenated, a conformational or structural change in
the whole complex is initiated, 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.
 Oxygen Binding Curve

• An oxygen-binding curve is a plot that shows fractional saturation versus

the concentration of oxygen.
• Fractional saturation indicates the presence of binding sites that have
oxygen.
• Fractional saturation can range from zero (all sites are empty) to one (all
sites are filled). The concentration of oxygen is determined by partial
pressure.
• Hemoglobin's oxygen affinity is relatively weak compared to myoglobin's
affinity for oxygen.
• Hemoglobin's oxygen-binding curve forms in the shape of a sigmoidal
curve. This is due to the cooperativity of the hemoglobin.
• As hemoglobin travels from the lungs to the tissues, the pH value of it's
surroundings decrease, and the amount of CO2 that it reacts with
increases.
• Both these changes causes the hemoglobin to lose it's affinity for oxygen,
therefore making it drop the oxygen into the tissues. This causes the
sigmoidal curve for hemoglobin in the oxygen-binding curve and proves
it's cooperativity.
• The first image shows hemoglobin's and
myoglobin's comparative oxygen binding affinity.
• Hemoglobin's sigmoidal curve is shown and how it
starts out with a little less affinity than myoglobin,
but comparable affinity to oxygen in the lungs.
• As the pressure drops and the myoglobin and
hemoglobin move towards the tissues, myoglobin
still attains it's high affinity for oxygen, while
hemoglobin, because of it's cooperativity,
suddenly loses it's affinity, therefore making it the
better transporter of oxygen than myoglobin.
 Equilibrium Binding Model
• A curve describing the fraction of hemoglobin molecules
bound to oxygen as a function of the oxygen concentration is
calculated assuming the sort of simple equilibrium binding
model as follows
Release of Oxygen in Tissues from RBCs
 Binding for ligands other than oxygen
• The other hemoglobin ligands include
competitive inhibitors such as carbon monoxide (CO)
and
• allosteric ligands such as carbon dioxide (CO2) and
nitric oxide (NO).
• The carbon dioxide is bound to amino groups of the
globin proteins as carbaminohemoglobin, and is
thought to account for about 10% of carbon dioxide
transport in mammals.
• Nitric oxide is bound to specific thiol groups in the
globin protein to form an S-nitrosothiol, which
dissociates into free nitric oxide and thiol again, as
the hemoglobin releases oxygen from its heme site.
 Competitive

• The binding of oxygen is affected by molecules

such as carbon monoxide (CO) (for example, from
tobacco smoking, car exhaust, and incomplete
combustion in furnaces).
• CO competes with oxygen at the heme binding
site. Hemoglobin binding affinity for CO is 250
times greater than its affinity for oxygen, meaning
that small amounts of CO dramatically reduce
hemoglobin's ability to transport oxygen.
• Since carbon monoxide is a colorless, odorless
and tasteless gas, and poses a potentially fatal
threat, detectors have become commercially
available to warn of dangerous levels in
residences.
• When hemoglobin combines with CO, it forms
a very bright red compound
called carboxyhemoglobin, which may cause
the skin of CO poisoning victims to appear
pink in death, instead of white or blue. 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.
 Impact of CO poisoning on body systems

• Neurologic: central nervous system depression,

causing headache, dizziness in mild cases and coma,
seizure in severe cases.
• Cardiac: decreased myocardial functions,
vasodilatation, and decreased oxygen delivery
causing chest pains, low blood pressure, fast heart
rates.
• Metabolic: hyperventilation in mild cases, metabolic
acidosis in severe cases.
• Pulmonary: pulmonary edema occurs in 10-30% of
acute cases.
• Multiple organ failure: happens at high level of CO
poisoning.
 Other competitive ligands
• cyanide (CN−), sulfur monoxide (SO),
nitric oxide (NO), 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.
 Allosteric affecters of hemoglobin

• Allosteric regulation is the process by which the

behavior of proteins is controlled by other
molecules; the molecules that perform this
regulation are known as allosteric regulators. This
process involves the binding of an allosteric
regulator molecule to the protein in question; the
result is a distinct effect on the protein's function.
Allosteric regulators that increase or supplement
a given protein's function are known as allosteric
activators. Those that decrease or interrupt a
given protein's function are known as allosteric
inhibitors.
 Carbon Dioxide
• Carbon dioxide occupies a different binding site
on the hemoglobin. Carbon dioxide is more
readily dissolved in deoxygenated blood,
facilitating its removal from the body after the
oxygen has been released to tissues undergoing
metabolism. This increased affinity for carbon
dioxide by the venous blood is known as the
Haldane effect. 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+
• 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 on the protein,
while carbon dioxide binds at the α-amino
group. Carbon dioxide binds to hemoglobin and
forms carbaminohemoglobin. This decrease in
hemoglobin's affinity for oxygen by the binding of
carbon dioxide and acid is known as the
Bohr effect (shifts the O2-saturation curve to
the right).
• 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.
• A reduction in the total binding capacity of
hemoglobin to oxygen (i.e. shifting the curve
down, not just to the right) due to reduced pH is
called the root effect. This is seen in bony fish.
• The pH, or proton concentration of a given
solution, is another allosteric regulator of
hemoglobin. Interestingly enough, pH can act as
both an allosteric activator and inhibitor,
depending on the direction of pH change.
• 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.
 Degradation in vertebrate animals
• When red cells reach the end of their life
due to aging or defects, they are broken
down in spleen.
• The hemoglobin molecule is broken up,
and the iron gets recycled.
• This process also produces one molecule
of carbon monoxide for every molecule
of heme degraded.
• Heme degradation is one of the few natural
sources of carbon monoxide 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 (yellow pigment
secreted as result of porphyrin ring
degradation.
• 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.
• Iron is removed from heme and
recovered for later use, it is stored as
hemosiderin or ferritin in tissues and
transported in plasma by beta globulins
as transferins.
 Role in Disease

• The Students are required to

prepare and submit an
Assignment regarding this
topic
 Other oxygen-binding proteins
• Myoglobin
• Found in the muscle tissue of many
vertebrates, including humans.
• It gives muscle tissue a distinct red or dark
gray color.
• Very similar to hemoglobin in structure and
sequence, but is not a tetramer; instead, it is a
monomer that lacks cooperative binding. It is
used to store oxygen rather than transport it.
Hemocyanin
• The second most common oxygen-
transporting protein found in nature.
• It is found in the blood of many
arthropods and mollusks.
• Uses copper prosthetic groups
instead of iron heme groups and is
blue in color when oxygenated.
• Hemerythrin
• Some marine invertebrates and a few species
of annelid use this iron-containing non-heme
protein to carry oxygen in their blood.
• Appears pink/violet when oxygenated, clear
when not.
• Chlorocruorin
• Found in many annelids,
• the heme group is significantly different in
structure.
• Appears green when deoxygenated and red
when oxygenated.
Vanabins
• Also known as vanadium chromagens, they are found
in the blood of sea squirts. There were once
hypothesized to use the rare metal vanadium as an
oxygen binding prosthetic group. However, although
they do contain vanadium by preference, they
apparently bind little oxygen, and thus have some
other function, which has not been elucidated .
• They may act as toxins.
Erythrocruorin
• Found in many annelids, including earthworms, it is a
giant free-floating blood protein containing many
dozens—possibly hundreds—of iron- and heme-
bearing protein subunits bound together into a single
protein complex with a molecular mass greater than
3.5 million daltons.
Pinnaglobin
• Only seen in the mollusc Pinna squamosa. Brown
manganese-based porphyrin protein.
Leghemoglobin
• In leguminous plants, such as alfalfa or soybeans, the
nitrogen fixing bacteria in the roots are protected from
oxygen by this iron heme containing oxygen-binding
protein. The specific enzyme protected is nitrogenase,
which is unable to reduce nitrogen gas in the presence
of free oxygen.
Coboglobin
• A synthetic cobalt-based porphyrin. Coboprotein
would appear colorless when oxygenated, but yellow
when in veins.