This document discusses the structure and function of haemoglobin and the transport of gases in the blood. It provides a history of discoveries about haemoglobin dating back to the 17th century. Key points covered include the tetrameric structure of haemoglobin, with each subunit binding one heme group and iron atom. Haemoglobin is able to efficiently transport oxygen and carbon dioxide via changes in its quaternary structure and binding of effectors like hydrogen ions, carbon dioxide and 2,3-BPG. The sigmoidal oxygen dissociation curve illustrates haemoglobin's ability to load and unload oxygen in the lungs and tissues respectively. Factors like pH, temperature and organic phosphates influence the curve.

1. BY
EBOT WALTER OJONG
Ph.D. CHEMICAL PATHOLOGY
HAEMOGLOBIN AND
GAS TRANSPORT

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HAEMOGLOBIN

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HISTORY OF HAEMOGLOBIN
 Marcello Malpighi described the RBCs in 1665.
 Felix Hoppe Seyler in 1862 isolated pure hemoglobin.
 Christian Bohr in 1904 discovered that hemoglobin is the
transporter of oxygen.
 In 1912 Kuster established the structure of hemoglobin.
 Hans Fischer synthesized heme in laboratory in 1920
(Nobel prize, 1930).
 Perutz (Nobel prize, 1962) studied the three dimensional
structure of hemoglobin.

4. HISTORY OF HAEMOGLOBIN
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STRUCTURE OF HAEMOGLOBIN
 i. Normal level of Hemoglobin (Hb) in blood in males is
14-16 g/dl and in females, 13-15 g/ dl.
 Hb is globular in shape.
 The adult Hb (HbA) has 2 alpha chains and 2 beta chains.
 Molecular weight of HbA is 67,000 Daltons (66,684 to be
exact).
 Hb F (fetal Hb) is made up of 2 alpha and 2 gamma
chains.

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STRUCTURE OF HAEMOGLOBIN
 Hb A2 has 2 alpha and 2 delta chains.
 Normal adult blood contains 97% HbA, about 2% HbA 2
and about 1% HbF.
 Alpha chain gene is on chromosome 16 while the beta,
gamma and delta chains are on chromosome 11.
 Each alpha chain has 141 amino acids. The beta, gamma
and delta chains have 146 amino acids.

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STRUCTURE OF HAEMOGLOBIN
 There are 36 histidine residues in Hb molecule; these are
important in buffering action.
 The 58th residue in alpha chain is called distal histidine,
because it is far away from the iron atom.
 The 87th residue in alpha chain is called proximal
histidine, because it lies near to the iron atom
 The alpha and beta subunits are connected by relatively weak non-
covalent bonds like van der Waals forces, hydrogen bonds and
electrostatic forces.

8. ATTACHMENT OF HEME WITH GLOBIN
 There are 4 heme residues per Hb molecule, one for each
subunit in Hb.
 The 4 heme groups account for about 4% of the whole
mass of Hb. The heme is located in a hydrophobic cleft of
globin chain.
 The iron atom of heme occupies the central position of
the porphyrin ring.
 The reduced state is called ferrous (Fe++) and the
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ATTACHMENT OF HEME WITH GLOBIN
 The ferrous iron has 6 valencies and ferric has 5
valencies.
 In hemoglobin, iron remains in the ferrous state.
 Iron carries oxygen: The iron is linked to the pyrrole
nitrogen by 4 coordinate valiancy bonds and a fifth one to
the imidazole nitrogen of the proximal histidine
 In oxy-Hb, the 6th valency of iron binds the O2

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ATTACHMENT OF HEME WITH GLOBIN
 The oxygen atom directly binds to Fe, and forms a
hydrogen bond with an imidazole nitrogen of the distal
histidine.
 In deoxy-Hb, a water molecule is present between the
iron and distal histidine.
 As the porphyrin molecule is in resonance, central iron
atom is linked by coordinate bond.
 The distal histidine lies on the side of the heme ring

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TRANSPORT OF OXYGEN BY HAEMOGLOBIN
 Hemoglobin has all the requirements of an ideal
respiratory pigment (Barcroft):
a. It can transport large quantities of oxygen
b. It has great solubility
c.It can take up and release oxygen at appropriate partial
pressures
d. It is a powerful buffer.

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OXYGEN DISSOCIATION CURVE
 The ability of hemoglobin to load and unload oxygen at
physiological pO2 (partial pressure of oxygen) is shown by the
oxygen dissociation curve (ODC) .
 Hemoglobin is oxygenated and not oxidized.
 At the oxygen tension in the pulmonary alveoli, the Hb is 97%
saturated with oxygen. Normal blood with 15 gm/dl of Hb can carry
1.34 x 15 = 20 ml of O2 /dl of blood.
 In the tissue capillaries, where the pO2 is only 40 mm of Hg,
theoretically, Hb saturation is 75%. Thus under NTP conditions,
blood can release only 22% .

13. OXYGEN DISSOCIATION CURVE
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14. OXYGEN DISSOCIATION CURVE
 But actually in tissue capillaries, where pO2 is 40 mm of
Hg, the Hb is about 60% saturated.
 So physiologically, 40% of oxygen is released .
 The pO2 in inspired air is 158 mm Hg; in alveolar air 100
mm Hg; in the blood in lungs, pO2 is 90 mm Hg; and in
capillary bed, it is 40 mm Hg. In tissues, oxygen is liberated
from hemoglobin. In lung capillaries, oxygen is taken up by
the hemoglobin. The following factors will affect the
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15. OXYGEN DISSOCIATION CURVE
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16. FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
1. Heme-heme Interaction and Cooperativity
i.The sigmoid shape of the oxygen dissociation curve (ODC) is due to the allosteric
effect, or cooperativity. The equilibrium of Hb with oxygen is expressed by the Hill
equation (AV Hill, Nobel prize, 1922).
ii.The binding of oxygen to one heme residue increases the affinity of remaining
heme residues for oxygen (homotropic interaction). This is called positive
cooperativity (CURVE B).
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17. FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
1. Heme-heme Interaction and Cooperativity
iii. Thus each successive addition of O2, increases the affinity of Hb to oxygen
synergistically.
iv.Similarly, binding of 2,3-BPG at a site other than the oxygen binding site, lowers
the affinity for oxygen (heterotropic interaction).
v. The quaternary structure of oxy-Hb is described as R (relaxed) form; and that of
deoxyHb is T (tight) form.
vi.When oxygenation occurs the salt bonds are broken successively. Thus, on
oxygenation, the hemoglobin molecule can form two similar dimers.
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FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
2. Effect of pH and pCO2
i.When the pCO2 is elevated, the H+ concentration increases and pH
falls. In the tissues, the pCO2 is high and pH is low due to the
formation of metabolic acids like lactate. Then the affinity of
hemoglobin for O2 is decreased (the ODC is shifted to the right) and
so, more O2 is released to the tissues (R → T change takes place)
(Curve C).
ii.In the lungs, the opposite reaction is found, where the pCO2 is low,
pH is high and pO2 is significantly elevated. More O2 binds to
hemoglobin and the ODC is shifted to the left. Moreover, T → R
change is seen.

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FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
3. The Bohr Effect
i.The influence of pH and pCO2 to facilitate oxygenation of Hb in the
lungs and deoxygenation at the tissues is known as the Bohr effect
(1904).
ii. Binding of CO2 forces the release of O2.
iii.When the pCO2 is high, CO2 diffuses into the red blood cells. The
carbonic anhydrase in the red cells favors the formation of carbonic
acid (H2CO3).
iv.When carbonic acid ionizes, the intracellular pH falls. The affinity of
Hb for O2 is decreased and O2 is unloaded to the tissues.

20. FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
2. The Bohr Effect
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FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
4. The Chloride Shift
i. When CO2 is taken up, the HCO3
¯ concentration within the cell
increases. This would diffuse out into the plasma. Simultaneously,
chloride ions from the plasma would enter in the cell to establish
electrical neutrality. This is called chloride shift or Hamburger
effect. Thus on venous side, RBCs are slightly bulged due to the
higher chloride ion concentration.
Ii When the blood reaches the lungs, the reverse reaction takes place.
The deoxyhemoglobin liberates protons. These would combine with
HCO3
– to form H2CO3 which is dissociated to CO2 and H2O by the
carbonic anhydrase. The CO 2 is expelled. As HCO – binds H+, more
3
HCO3
– from plasma enters the cell and Cl– gets out (reversal of chloride
shift).

22. FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
4. The Chloride Shift
a
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23. FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
4. The Chloride Shift
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FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
5. Effect of Temperature
The term p50 means, the pO2 at which Hb is half saturated
(50%) with O2. The p50 of normal Hb at 37oC is 26 mm Hg.
Elevation of temperature from 20 to 37oC causes 88%
increase in p50. Metabolic demand is low when there is
relative hypothermia. Shift in ODC to left at low temperature
results in release of less O2 to the tissues. On the other
hand, under febrile conditions, the increased needs of O2
are met by a shift in ODC to right (Curve D).

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FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
6. Effect of 2,3-BPG
i. Normally the 2,3-bisphosphoglycerate level is 15 + 1.5 mg /g Hb.
The 2,3-BPG concentration is higher in young children compared
to the elderly.
ii. The 2,3-BPG is produced from 1,3-BPG, an intermediate of glycolytic
pathway .
iii.The 2,3-BPG, preferentially binds to deoxy-Hb and stabilizes the T
conformation. When the T form reverts to the R conformation, the
2,3- BPG is ejected. During oxygenation, BPG is released.
iv. The high oxygen affinity of fetal blood (HbF) is due to the inability of
gamma chains to bind 2,3-BPG.

26. FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
6. Effect of 2,3-BPG
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FACTORS WHICH AFFECT THE OXYGEN DISSOCIATION CURVE
Adaptation to High Altitude
1. Increase in the number of RBCs
2. Increase in concentration of Hb inside RBCs
3. Increase in BPG.

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TRANSPORT OF CARBON DIOXIDE
At rest, about 200 ml of CO2 is produced per minute in tissues. The CO2
is carried by the following 3 ways.
1: Dissolved Form
About 10% of CO2 is transported as dissolved form.
– +
CO2 + H2O → H2CO3 → HCO3 + H
The hydrogen ions thus generated, are buffered by
the buffer systems of plasma.
2: Isohydric Transport of Carbon Dioxide
i.Isohydric transport constitutes about 75% of CO2. It means that there
is minimum change in pH during the transport. The H+ ions are
buffered by the deoxy-Hb and this is called the Haldane effect.
ii.In tissues: Inside tissues, pCO2 is high and carbonic acid is formed. It
ionizes to H+ and HCO3
– inside the RBCs.

29. TRANSPORT OF CARBON DIOXIDE
The H+ ions are buffered by deoxy-Hb and the HCO3
– diffuses out into
the plasma. In order to maintain ionic equilibrium, chloride ions are
taken into RBC. Thus the CO2 is transported from tissues to lungs, as
plasma bicarbonate, without significant lowering of pH. The H+ are
bound by N-terminal NH2 groups and also by the imidazole groups of
histidine residues.
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30. TRANSPORT OF CARBON DIOXIDE
iii. Oxy-Hb is More Negatively Charged Than Deoxy-Hb:
The iso-electric point of oxyhemoglobin is 6.6, while
that of deoxy-Hb is 6.8. Thus oxy- Hb is more
negatively charged than deoxy Hb. The reaction in
tissues may be written as
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31. TRANSPORT OF CARBON DIOXIDE
Therefore some cation is required to remove the extra
negative charge of Oxy-Hb. So H+ are trapped. Hemoglobin
binds 1 proton for every 2 oxygen molecules released. Or, 1
millimol of deoxy Hb can take up 0.6 mEq of H+, produced
from 0.6 mmol of carbonic acid.
iv. In the lungs: In lung capillaries, where the pO2 is high,
oxygenation of hemoglobin occurs. When 4 molecules of O2
are bound and one molecule of hemoglobin is fully
oxygenated, hydrogen ions are released.
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32. TRANSPORT OF CARBON DIOXIDE
v.The protons released in the RBC combine with HCO3– forming
H2CO3 which would dissociate to CO2, that is expelled through
pulmonary capillaries.
vi. As the HCO3
– level inside the erythrocyte falls, more and more
HCO – gets into the RBC, and chloride diffuses out (reversal of chloride
3
shift).
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TRANSPORT OF CARBON DIOXIDE
3: Carriage as Carbamino-Hemoglobin
The rest 15% of CO2 is carried as carbaminohemoglobin,
without much change in pH. A fraction of CO2 that enters
into the red cell is bound to Hb as a carbamino complex.
R–NH2 + CO2 --------- R–NH–COOH
The N-terminal amino group (valine) of each globin chain
forms carbamino complex with carbon dioxide. Deoxy-
hemoglobin binds CO2 in this manner more readily than oxy-
hemoglobin.

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FOETAL HAEMOGLOBIN
1. HbF has 2 alpha chains and 2 gamma chains. Gamma
chain has 146 amino acids.
2. The differences in physicochemical properties
compared with HbA are:
a. Increased solubility of deoxy HbF
b. Slower electrophoretic mobility for HbF
c. Increased resistance of HbF to alkali denaturation
d. HbF has decreased interaction with 2,3-BPG.
when

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FOETAL HAEMOGLOBIN
3.. The ODC of fetus and newborn are shifted to left. This
increase in O2 affinity is physiologically advantageous in
facilitating transplacental oxygen transport. The major
reason is the diminished binding of 2,3-BPG to HbF. When
pO2 is 20 mm Hg, the HbF is 50% saturated.
4.The synthesis of HbF starts by 7th week of gestation; it
becomes the predominant Hb by 28th week. At birth, 80%
of Hb is HbF. During the first 6 months of life, it decreases to
about 5% of total. There is a rapid postnatal rise in 2,3-BPG
content of RBC. However, HbF level may remain elevated in
children with anemia and beta thalassemia, as a
compensatory measure.

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FOETAL HAEMOGLOBIN
 In humans and other mammals, the
developing embryo and fetus express
different forms of hemoglobin than does the
mother.
 The oxygen affinities of fetal hemoglobin are
considerably greater than that of maternal
hemoglobin.
 This phenomenon fits with the fact that fetal
hemoglobin must be oxygenated in the
placenta, where the pO2 is lower than it is in
the lungs.

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FOETAL HAEMOGLOBIN
 The higher affinity (lower P 50) of fetal
hemoglobin is due to its lower affinity for
BPG. Because BPG binding and O2 binding
interfere with each other, reduced affinity
for the former means increased affinity for
the latter.
 Fetal hemoglobin is replaced by the mature
form in human infants by about six months
of age.