synthesis, structure & function
Ahmad Sh. Silmi
Msc Haematology, FIBMS
• The hemoglobin are red globular proteins which
have a molecular weight of about 64,500 and
comprise almost one third of the weight of a red
cell. Their primary function is the carriage of
oxygen from the lungs to the tissues.
• Over 500 different haemoglobin variants have
been described but all share the same basic
structure of four globin polypeptide chains each
with haem group. Functional haemoglobin
composed of two pairs of dissimilar globins.
Although haem & globin synthesis
separately within developing red cell
precursors their rate of synthesis are
carefully coordinated to ensure optimal
efficiency of haemoglobin assembly.
1st : Haem synthesis
The first step in
and glycin to
acid (δ ALA).
This reaction is
so occurs in the
• It’s catalyzed by the enzyme δ ALA synthetase.
• This step is a first-limiting step for the whole process of
• It is stimulated by the presence of globin chains and
inhibited by the presence of free haem groups.
• This represents an important control mechanism of the rate
of haem synthesis and it’s coordination with globin
• Several factors are required for this step, including vitamin
B6, free ferrous and copper ions.
• Synthesis of the enzyme δ ALA synthetase is inhibited by
the presence of free haem.
• This represents a further feedback mechanism for haem
Mitochondrial δ -aminolevulinic acid (ALA) is transported
to the cytoplasm, where ALA dehydratase (also called
porphobilinogen synthase) dimerizes 2 molecules of
ALA to produce the pyrrole ring compound
• The next step requires the synthesis of porphyrin
• The reactions involved are extremely complex
but can be summarized as the condensation of
four PBG molecules to form the asymmetric
cyclic uroporphyrinogen III (UPGIII).
• Synthesis of UPGIII requires the presence of two
enzymes (uroporphyrinogen I synthetase and
uroporphyrinogen III cosynthetase) and involves
the formation of several short-lived
• UPG III is converted to coproporphyrinogen III
(CPGIII) by decarboxylation of the acetate side
chains under the influence of the enzyme
• CPGIII enters the mitochondria where it converted to
protoporphyrinogen IX (PPG IX) by an unknown
mechanism. This reaction is catalyzed by the enzyme
PPG IX is further converted within the mitochondria to
• It only remains for the central ferrous ion to be inserted
to complete the synthesis of haem. This reaction is
catalyzed by the enzyme ferrochelatase and requires the
presence of reducing agents.
2nd : Globin synthesis
• Humans normally carry 8 functional globin
genes, arranged in two duplicate gene
• The β-like cluster on the short arm of
• The α-like cluster on the short arm of
• These genes code for 6 different types of
globin chains: α,β,γ,δ,ε,δ, globin.
Ontogeny of globin synthesis
Time Region Type of Globin Type of Hb
3 weeks of Yolk Sac δ&ε Hb Gawer1 δ ε)2
5 weeks of Yolk Sac γ&α Hb Portland(δ γ)2
Gestation Hb GawerII (αε)2
6-30 weeksof Liver & spleen α & γ & β Hb F (α γ)2
30 weeks of Liver δ Hb A2 (α δ(2
At Birth B.M ___ HbA(α β)2
Hb A Hb A2 Hb F
structure a2b2 a2d2 a2g2
Normal % 96-98 % 1.5-3.2 % 0.5-0.8 %
2nd : Globin synthesis
• Primary structure of globin
The primary structure of globin refers to the amino acid
sequence of the various chain types. Numbering
from the N-terminal end identifies the position of
individual amino acids. The identity and position of
these amino acids cannot be changed without
causing gross impairment to molecular function.
Secondary Structure of globin :
• The secondary structure of all globin chain types
comprises nine non-helical sections joined by
eight helical sections.
• The helical sections are identified by the letters
A-H while the non helical are identified by a pair
of letters corresponding to the adjacent helices
e.g. NA (N-terminal end to the start of A helix),
AB (joins the A helix to the B helix) etc.
Tertiary Structure of globin:
• The tertiary folding of each globin chain
forms an approximate sphere.
• The intra-molecular bonds which give rise
to the helical parts of the impart
considerable structure rigidity, causing
chain folding to occur in the non-helical
• Tertiary folding gives rise to at least 3 functionally
important characteristics of the hemoglobin molecule :
1- Polar or charged side chains tend to be directed to the
outside surface of the subunit and, conversely, non-polar
structures tend to be directed inwards. The effect of this is to
make the surface of the molecule hydrophilic and the interior
2- An open-toped cleft in the surface of the subunit known as
haem pocket is created. This hydrophobic cleft protects
the ferrous ion from oxidation.
3- The amino acids, which form the inter-subunit bonds
responsible for maintaining the quaternary structure, and
thus the function, of the haemoglobin molecule are
brought into the correct orientation to permit these bonds
Quaternary structure of Haemoglobin
The quaternary structure of haemoglobin has four
subunits arranged tetrahedrally. In adult
haemoglobin- (HbA), there are different contact
• α1β1 and α2 β 2 which confirms stability of
• α1 β2 and α2 β1 which confirms solubility
of the molecule.
• α1 α2 and β1 β2 which are weak bonds to
permit oxygenation and deoxygenation.
Functions of Haemoglobin
Oxygen delivery to the tissues
Reaction of Hb & oxygen
Oxygenation not oxidation
One Hb can bind to four O2 molecules
Less than .01 sec required for oxygenation
b chain move closer when oxygenated
When oxygenated 2,3-DPG is pushed out
b chains are pulled apart when O2 is unloaded, permitting
entry of 2,3-DPG resulting in lower affinity of O2
Oxy & deoxyhaemoglobin
Normal Hemoglobin Function
• When fully saturated, each gram of hemoglobin binds
1.34 ml of oxygen.
• The degree of saturation is related to the oxygen tension
(pO2), which normally ranges from 100 mm Hg in arterial
blood to about 35 mm Hg in veins.
• The relation between oxygen tension and hemoglobin
oxygen saturation is described by the oxygen-
dissociation curve of hemoglobin.
• The characteristics of this curve are related in part to
properties of hemoglobin itself and in part to the
environment within the erythrocyte, including pH,
temperature, ionic strength, and concentration of
phosphorylated compounds, especially 2,3-
Hb-oxygen dissociation curve
Normal Hemoglobin Function
• Oxygen affinity of hemoglobin is generally expressed in
terms of the oxygen tension at which 50% saturation
• When measured in whole erythrocytes, this value
averages 27.1 mm Hg in normal, nonsmoking males and
27.5 mm Hg in normal, nonsmoking females.
• When oxygen affinity is increased, the dissociation
curve is shifted Leftward, and the value is reduced.
• Conversely, with decreased oxygen affinity, the curve is
shifted to the right.
Hb-oxygen dissociation curve
The normal position of curve depends on
Concentration of 2,3-DPG
H+ ion concentration (pH)
CO2 in red blood cells
Structure of Hb
Hb-oxygen dissociation curve
Right shift (easy oxygen delivery)
Left shift (give up oxygen less readily)
• The change in oxygen affinity with pH is known
as the Bohr effect.
• Hemoglobin oxygen affinity is reduced as
the acidity increases.
• Since the tissues are relatively rich in carbon
dioxide, the pH is lower than in arterial blood;
therefore, the Bohr effect facilitates transfer of
• The Bohr effect is a manifestation of the acid-
base equilibrium of hemoglobin.
• This compound is synthesized from glycolytic intermediates by
means of a pathway known as the Rapoport-Luebering shunt.
• In the erythrocyte, 2-3-DPG constitutes the predominant
phosphorylated compound, accounting for about two thirds of the
red cell phosphorus.
• The proportion of 1,3-DPG pathway appears to be related largely to
cellular ADP and ATP levels; when ATP falls and ADP rises, a
greater proportion of 1,3-DPG is converted through the ATP-
• This mechanism serves to assure a supply of ATP to meet cellular
• In the deoxygenated state, hemoglobin A can bind 2,3-DPG in a
molar ratio of 1:1, a reaction leading to reduced oxygen affinity
and improved oxygen delivery to tissues.
• When oxygen is unloaded by the hemoglobin
molecule and 2,3 DPG is bound, the molecule
undergoes a conformational change becoming what is
known as the ""Tense" or "T" form.
• The resultant molecule has a lower affinity for
• As the partial pressure of oxygen increases, the 2,3,
DPG is expelled, and the hemoglobin resumes its
original state, known as the "relaxed" or "R" form, this
form having a higher oxygen affinity.
• These conformational changes are known as
• The increased oxygen affinity of fetal hemoglobin
appears to be related to its lessened ability to bind
• The increased oxygen affinity of stored blood is
accounted for by reduced levels of 2,3-DPG.
• Changes in 2,3-DPG levels play an important
role in adaptation to hypoxia. In a number of
situations associated with hypoxemia, 2,3-DPG
levels in red cells increase, oxygen affinity is
reduced, and delivery of oxygen to tissues is
• Such situations include abrupt exposure to high
altitude, anoxia due to pulmonary or cardiac
disease, blood loss, and anemia.
• Increased 2,3-DPG also plays a role in
adaptation to exercise. However, the compound
is not essential to life; an individual who lacked
the enzymes necessary for 2,3-DPG synthesis
was perfectly well except for mild polycythemia
• Transport of carbon dioxide by red
cells, unlike that of oxygen, does not
occur by direct binding to heme.
• In aqueous solutions, carbon dioxide
undergoes a pair of reactions:
1. CO2 + H2O H2CO3
2. H2CO3 H+ + HCO3
• Carbon dioxide diffuses freely into the red cell where the
presence of the enzyme carbonic anhydrase facilitates
• The H+ liberated in reaction 2 is accepted by deoxygenated
hemoglobin, a process facilitated by the Bohr effect.
• The bicarbonate formed in this sequence of reactions diffuses
freely across the red cell membrane and a portion is
exchanged with plasma Cl-, a phenomenon called the
"chloride shift." the bicarbonate is carried in plasma to the
lungs where ventilation keeps the pCO2 low, resulting in
reversal of the above reactions and excretion of CO2 in the
• About 70% of tissue carbon dioxide is processed in this way.
Of the remaining 30%, 5% is carried in simple solution and
25% is bound to the N-terminal amino groups of
deoxygenated hemoglobin, forming carbaminohemoglobin.
• In order to bind oxygen reversibly, the iron in the
heme moiety of hemoglobin must be maintained
in the reduced (ferrous) state despite exposure
to a variety of endogenous and exogenous
• The red cell maintains several metabolic
pathways to prevent the action of these oxidizing
agents and to reduce the hemoglobin iron if it
becomes oxidized. Under certain
circumstances, these mechanisms fail and
hemoglobin becomes nonfunctional.
• At times, hemolytic anemia supervenes as
well. These abnormalities are particularly
likely to occur
(1) if the red cell is exposed to certain oxidant drugs
(2) if the intrinsic protective mechanisms of the cell
are defective or
(3) if there are genetic abnormalities of the
hemoglobin molecule affecting globin stability or
the heme crevice.