Haemoglobin-Structure Synthesis and

Iron Metabolism

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 Haemoglobin and Haemoglobinisation

▪ Haemoglobin is an iron containing protein synthesised (

in the adult) by the erythroblast precursors in the bone
marrow that will give rise to the mature red blood cell.
▪ A haemoglobin molecule is composed of 2 dissimilar
pairs of polypeptide chains each of which encloses an
iron containing porphyrin ring , called haem.
▪ Haemoglobin has a molecular mass of 64-65.5 kDa.
▪ Haem is essential for oxygen transport, while globin
serves to protect haem from oxidation, renders the
molecule soluble and permits variation in oxygen
affinity.

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 Haemoglobin
▪ 3 phases of Haemoglobin formation. Embryonic, Foetal
and adult.
▪ In each phase we have a different types of
Haemoglobins formed which is specific for the stage of
life.
▪ The difference in the Haemoglobin is dependant upon
the different globin chains in the Haemoglobin
molecule
▪ 2 chromosomes are responsible for globin synthesis 16
and 11

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 Genes for chain synthesis
The genes for the globin chains occur in two
clusters
▪ Chromosome 11 = has the genes for ε, δ, γ and β
globin
▪ Chromosome 16 = has the genes for ζ and α globin

▪ The different globin chains are synthesised

independently and then combine with each other to
produce the different types of Haemoglobin
molecules.

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Definitions and Symbols of Globin Chains

▪ alpha α
▪ beta β
▪ gamma γ
▪ Delta δ
▪ epsilon ε
▪ zeta ζ

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 Different Types of Haemoglobin

▪ A
▪ A2
▪ F (foetal)
▪ Gower 1
▪ Gower 2
▪ Portland 1
▪ Portland 2

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 Production of Haemoglobin
▪ In the early embryo Haemoglobin is synthesised in the
yolk sac where specific embryonic Haemoglobin are
produced :
▪ By 5 weeks of gestation the zeta, and epsilon chains, are
already been synthesised in primitive erythroblasts in the
yolk sac, with the production of Gower 1, Gower 2 and
Portland 1 and Portland 2 Haemoglobins.
▪ From the 6th week onwards these same cells start to
synthesise alpha, beta, and gamma globin chains which
will be used to make other Haemoglobin molecules.
▪ The formation of Gower 2 Haemoglobin in the embryo is
essential for life.

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 Production of Haemoglobin

▪ Starting from about the 10th –12th week of gestation

haemoglobin is synthesis in the liver and spleen, with the
production of foetal haemoglobin and later a haemoglobin A
▪ Later in intrauterine life the bone marrow takes over as the
main site of haemoglobin synthesis and increasing amounts of
haemoglobin A are produced.
▪ In adult life the bone marrow erythroblasts synthesise
haemoglobin A and other minor haemoglobins.

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 Haemoglobins Present During Adult,
Foetal and Embryonic life

Haemoglobin Globin Present

A α2 β2 Major Haemoglobin in adult
life.
A2 α2 δ2 Minor Haemoglobin in adult
life, more minor in foetal and
neonatal period.
F α2 γ2 Major Haemoglobin in foetal
life, declining through neonatal
period, and a minor
Haemoglobin in adult life.

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 Haemoglobins Present During adult,
foetal and embryonic life
Haemoglobin Globin Present

Gower 1 ζ2 ε2 All are Significant

Gower 2 α2 ε2 Haemoglobins
during early
Portland 1 ζ2 γ2 Intrauterine
Life
Portland 2 β2 ζ2

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 Haemoglobin
▪ In the adult there are three types of Haemoglobin.
▪ Levels of Haemoglobin in a normal Healthy adult.
▪ Haemoglobin A : ~ 97 %
▪ Haemoglobin A2: 2.0-3.3 %
▪ Haemoglobin F : 0.2- 1.0 %

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 Iron Stores

▪ Iron in the body: Male 50 mg/kg, female 35 mg/kg.

▪ Haemoglobin contains most of the body’s iron (65% of
total) , where it is incorporated in the red blood precursor
cells in the bone marrow for use in erythropoiesis.
▪ Iron is also found in the following-
▪ 30% of total iron is stored as Ferritin and its insoluble form
Hemosiderin in the macrophages of the bone marrow, liver,
spleen and is available for Haem synthesis if necessary.
▪ Iron is found in Myoglobin in the muscles.
▪ Iron is found in Cytochrome enzymes in the cells
▪ Iron is also bound to the iron transport protein in the blood
transferrin

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 Diet Sources of Iron
▪ Red meat- especially liver.
▪ Animal products and fish
▪ Vegetables usually poor source of iron, with the exception
of the legumes.
▪ Many cereals are now fortified with iron.
▪ Neonates require iron supplements if they are not breast
fed.
▪ The average western diet contains 10-15mg of iron daily
from which 5-10% is normally absorbed. The proportion of
iron absorbed can be increased to 20-30% in iron
deficiency or pregnancy.

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 Haem and Non Haem Forms of Iron
▪ In a normal healthy individual daily absorption will
equate with daily losses of iron.
▪ Iron can be found in different forms.
▪ Iron can be found in Haem and non Haem forms in
Ferrous ( Fe++ ) and Ferric ( Fe+++ ) states.
▪ Iron in food in the form of Haem as is found in liver
and other forms of red meat or fish and is usually in
Fe++ ( Ferrous) state which is absorbed more rapidly
than the Fe+++ (Ferric) form .
▪ Haem iron is released is released from Haem by the
enzyme haemoxgenase 1.

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Haem and Non Haem Forms of Iron
▪ Non Haem iron is usually in the Ferric Fe+++
state is more likely to be found in vegetables and
cereals.
▪ Non Haem iron , is released from protein
complexes in foods by the action of enzymes and
acids in the gut.
▪ Ferric iron is converted to Ferrous iron to enable
easy absorption of iron into the blood system.
▪ Ferric iron conversion to the ferrous form is
facilitated by the by an enzyme ferroreductase in
the gut

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Haem structure
▪ Haem consists of a
protoporphyrin ring with an
iron atom at its Centre.
▪ The protoporphyrin ring
consists of four pyrrole
groups which are united by
methane bridges (=C-).
▪ The hydrogen atoms in the
pyrrole groups are replaced
by four methylene (CH3-),
two vinyl (-C=CH2) and two
propionic acid (-CH2-CH2-
COOH) groups.
 Absorption of Iron
▪ Free iron is absorbed by the intestinal epithelial cells
( enterocytes) via a specific cell membrane molecule
called dimetal transporter-1. (DMT I)
▪ Another protein Ferroportin on the surface of the
enterocytes and also found on Macrophages and
Hepatocytes has a role in controlling the export of iron
into the circulation.
▪ Hepcidin a liver protein regulates iron absorption into the
body , by controlling the level of Ferroportin in the cells .
Increased Hepcidin levels, lowers Ferroportin, and thus
reduces iron absorption from the gut or from iron stores
in the macrophages. Decreased production of Hepcidin in
with iron deficiency, results in an increase in Ferroportin
levels which allows more iron to enter the Portal system.
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 Iron Absorption
▪ Iron is transported over the brush border in the small
intestine and transported by the transferrin molecule to
the bone marrow for use in the formation of haemoglobin
or to be stored in the tissue macrophages as ferritin or
Haemosiderin. ( Stores of iron are found in all cells but
primarily the Liver, spleen and bone marrow)
▪ Iron is primarily absorbed in the duodenum and upper
jejunum, where the acidic conditions help the absorption
of iron.
▪ Absorption is helped by the presence of other reducing
substances such as hydrochloric acid and ascorbic acid.
▪ The body has the capacity to increase iron absorption in
the face of increase demands, i.e. Pregnancy, lactation,
growth spurts and iron deficiency.

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 Iron Metabolism
▪ Iron absorption is controlled by
1. The amount of iron in the diet
2. The availability and the form its in and how easy its
to absorb.
3. The bodies store and requirement for iron.
▪ Losses of body iron.
1. Menstruation ( large iron loss in females)
2. Gastrointestinal bleeds- blood in stools
3. Epithelial cells from skin, gut or urinary track

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 Iron Transport
▪ Once in the circulation free iron is picked up by carrier
proteins that are synthesised in the liver.
▪ These carrier proteins include albumin, lactoferrin and
transferrin.
▪ Transferrin is the most important iron carrier. It is 76-80 kDA
protein that can carry two atoms of ferric iron.
▪ Transferrin the iron transport protein delivers iron to the
tissues and cells that have transferrin receptors, especially
the erythroblasts in the bone marrow, which incorporate iron
into haem in the synthesis of the haemoglobin molecule.
Transferrin receptors are also present on the cells where
iron is stored.
▪ The transferrin is then reutilised pick up more free iron in
the body.

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 Iron Absorption Transport and Storage

12/02/2024 Michael Healy 21

 Iron Transport and Iron Cycle
▪ At the end of their life, red cells are broken down by the
macrophages of the RE system and the iron that is
released from haemoglobin enters the plasma and
provides most of the iron for the transport protein
transferrin to be reutilised in the synthesis of new
haemoglobin.
▪ The amount of iron being carried by transferrin is a useful
indicator of general iron status.
▪ Note: Only a small proportion of plasma iron comes from
dietary iron, which is absorbed through the duodenum
and jejunum. The main source of iron is from
Haemoglobin breakdown from effete red blood cells

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 Transferrin Receptors
▪ Transferrin receptors are receptors that are on the
lining of cells.
▪ They are present on the cells in the bone marrow
involved in erythropoiesis and are also present on the
cells where iron is stored.
▪ They bind to transferrin and play a major role in the
release of iron into the cell. The expression of surface
transferrin receptors is increased if cells have
inadequate iron. This occurs in iron deficiency when
ferritin ( storage form of iron ) is low. Some of the
receptors are shed into the plasma and can be
measured.

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 Haemoglobin

▪ Haemoglobin Synthesis begins in

proerythroblast ( early precursor of the red cell
found in the bone marrow). Haemoglobin
synthesis occurs at both the erythroblast and
reticulocyte stage, but not in the mature red
cell.
 65% at erythroblast stage
 35% at reticulocyte stage

▪ Haemoglobin Synthesis involves-

1. Haem synthesis
2. Globin synthesis

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 Synthesis of Haemoglobin

▪ Haem and globin produced at two

different sites in the cells.
▪ Haem primarily in mitochondria
▪ Globin in the ribosome's
▪ Haem and globin synthesis is well
synchronized.

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 Haem Synthesis
▪ Haem belongs to the class of pigments known as porphyrin.
▪ Haem is composed of 4 pyrrole rings linked by methene
bridges each bound to a central ferrous ion (Fe2+).
▪ Haem is synthesised in virtually all human tissues but the
more important sites is in the developing red cell
precursors, the erythroblasts.
▪ Enzymes involved in Haem synthesis include- Succinyl Co
Enzyme A transferase, aminolaevulininic acid synthase.
(ALAS):
▪ Haem Synthase also called Ferrocheletase is the enzyme
that catalyses the insertion of iron into the Haem molecule
in the mitochondria.

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Haem structure
▪ Haem consists of a
protoporphyrin ring with an
iron atom at its Centre.
▪ The protoporphyrin ring
consists of four pyrrole
groups which are united by
methane bridges (=C-).
▪ The hydrogen atoms in the
pyrrole groups are replaced
by four methylene (CH3-),
two vinyl (-C=CH2) and two
propionic acid (-CH2-CH2-
COOH) groups.
 Steps in Haem Synthesis
▪ 1st step involves combination of glyceine and
succinyl-Co A to produce δ- aminolaevulinic acid (δ-
ALA.) The catalysts for this reaction is δ-ALA synthase.
Several co factors are required δ-ALA synthesis and
these include Vitamin B6 derivative Pyridoxal
phosphate, the presence of free ferrous and copper
ions. This occurs in the mitochondria of cells.
▪ 2nd step is that two δ-ALA combine to form a Pyrrole
called Porphobilinogen (PBG), under the influence of
δ-ALA Dehydrogenase and glutathione. This and
subsequent reactions occur in the cytoplasm of the
cell.

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 Steps in Haem Synthesis
▪ 3rd step involves four PBG molecules combining to form a
Tetrapyrrolle, Uroporphyrinogen (UPG) in the
cytoplasm.
▪ 4th step is the conversion of UPG to
Coproporphyrinogen. ( CPG)
▪ 5th step is the movement of CPG from the cytoplasm into
the mitochondria and is converted to
Protoporphyrinogen IX which in turn is converted to
Protoporphyrin IX
▪ The final step involves a central ferrous ion to be
inserted to complete haem synthesis.
▪ Haem Synthase also called Ferrocheletase is the enzyme
that catalyses the insertion of iron into the Haem
molecule in the mitochondria
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Synthesis of Haemoglobin

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 Haemoglobin Structure
Synthesis and Function
▪ Haemoglobin is a protein consisting of 4 globulin chains
each containing a prosthetic haem group.
▪ The synthesis of the haem and the globin moieties
proceeds separately, but are well synchronised.
▪ Haem is inserted into a globin subunit followed by final
assembly of the whole molecule.
▪ The synthesis of Haem is co-ordinated with that of globin
synthesis and requires control of the entry of iron into the
cell.
▪ The formation of Haem increases globin synthesis and lack
of haem formation reduces globin synthesis.

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 Globin Synthesis
▪ The Globin part in the Haemoglobin molecule is made up of
4 protein chains.
▪ Each globin chain is made up of a series of amino acids.
▪ In the adult there are 2 alpha chains and 2 beta chains.
▪ The genes controlling the production of the alpha genes are
located on Chromosome 16.
▪ The genes controlling the production of the beta genes are
located on Chromosome 11.
▪ Other globin chains are also formed in the embryo and the
foetus.

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Globin Gene Clusters

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Globin Chain Switch

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 Haemoglobin Structure

▪ When discussing the structure of the

Haemoglobin molecule in addition to the
structure of Haem you have to make reference
to the –
▪ Primary structure of globin
▪ Secondary structure of globin
▪ Tertiary structure of globin
▪ Quaternary structure of Haemoglobin

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 Haemoglobin Structure
▪ Primary structure of globin- refers to the amino acid
sequence of each globin chain. The identity and
position of each amino acid in the chain is known.
▪ Secondary structure of all globin chain types comprise
of 9 non helical sections and 8 helical sections .
▪ Tertiary structure of globin chains refers to the folding
of each individual chain into an approximate three
dimensional sphere.
▪ Quaternary structure of Haemoglobin has 4 sub units.
Each sub unit has a globin chain with a haem group.
The haemoglobin molecule is a tetramer of the four
globin chains each with its own haem group in the
pocket.

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Alpha and Beta globin chains and Haem

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Quaternary structure of Haemoglobin

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 Functions of Haemoglobin
▪ Oxygen binding in the lungs and delivery to the tissues.
▪ Carbon dioxide transport from the tissues for excretion via
the lungs.
▪ Each Haemoglobin molecule can bind to four O2
molecules. Less than .01 sec required for oxygenation. b
chains move closer when Haemoglobin is oxygenated
▪ When Haemoglobin is oxygenated, the molecule 2,3-DPG
is pushed out of the Haemoglobin molecule.
▪ b chains are pulled apart when O2 is unloaded, permitting
entry of 2,3-DPG resulting in lower affinity of O2

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Oxy and Deoxyhaemoglobin

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 Oxygen-Haemoglobin
Dissociation Curve
▪ The ability of Haemoglobin to pick up and carry
oxygen is affected by different conditions and is
represented by the oxygen dissociation curve.
▪ The curve plots the % saturation of Haemoglobin
against the partial pressure of O2. (O2 carrying
capacity of Haemoglobin at different oxygen
pressures ( PO2)
▪ Haemoglobin Oxygen Dissociation Curve is
sigmoidal

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 Oxygen-Haemoglobin
Dissociation Curve
▪ P 50 (pressure of O2 at which Haemoglobin is 50%
saturated with O2) is normally 26.6mmHg.
▪ The position of curve depends on the concentration of
2,3-DPG, H+ ion concentration (pH) and the CO2 in red
blood cells.
▪ Also the type of Haemoglobin will effect the position
of the curve. For examples adult Haemoglobin and
foetal Haemoglobin will have different affinities for
oxygen and thus different oxygen association curves.
 Hb-Oxygen Dissociation Curve
▪ Right shift (easy for oxygen delivery to the
tissues)
▪ High 2,3-DPG
▪ High H+
▪ High CO2
▪ HbS

▪ Left shift (give up oxygen less readily to the

tissues )
▪ Low 2,3-DPG
▪ HbF

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Haemoglobin and Carbon Dioxide Transport

▪ Carbon dioxide is excreted via the lungs in exhaled

air. It is transported from the tissues to the lungs in
the blood in three forms-
▪ Approximately 78% is transported in the form of
bicarbonate ions ( HCO3-)
▪ Approximately 13% is transported bound to
Haemoglobin and other proteins in the blood.
▪ Approximately 9% is transported in solution in
plasma.

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Hb-Oxygen Dissociation Curve

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 Red Cell Ageing and
Haemoglobin Breakdown (Catabolism)

▪ As the red cell ages, the enzymes that are responsible

for producing the energy in the cell decrease in
activity.
▪ At the end of a normal lifespan, red cells are destroyed
within the RE system in the spleen, bone marrow and
liver.
▪ Free haemoglobin can be broken down within the
spleen, bone marrow or transported to the liver where
it is further downgraded.
▪ The iron released from the Haemoglobin can be
reutilised to form new Haemoglobin in primitive red
cells in the bone marrow.
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 Haemoglobin Breakdown In Old Red Cells
haemoglobin

haem globin
iron protoporphyrin
Amino acids

CO Bilirubin
transferrin Expired air (free)
Liver
conjugation
erythroblast
Bilirubin glucuronides

Urobilin(ogen) Stercobilin(ogen)

Urine faeces