Hemoglobin

Haemoglobin structure
 Haemoglobin (Hb) is the most abundant
porphyrin – containing compound.

 It is a tetramer made up of four subunits.

 Each subunit contains a heme group and a

polypeptide chain.

06/27/2025
 Haemoglobin structure

• Normal level of Hb in blood of males is 14 – 16

g/dl and in females, 13 – 15 g/dl.

• Normal adult blood contains 97% HbA, about 2%

HbA2 and about 1% HbF.

06/27/2025
 Hemoglobin is a O2 and CO2 transport protein
found in the RBCs

 Hemoglobin is an oligomeric protein made up

of 2 α β dimers, a total of 4 polypeptide
chains: α1 β1 α2 β2.

 Total Mr of hemoglobin is 64,500.

 The α (141 aa) and β (146 aa) subunits have <

50 % identity.
06/27/2025
• • The 3D- structures of α (141 aa) and β (146
aa) subunits of hemoglobin and the single
polypeptide of myoglobin are very similar; all
three are members of the globin family.

• • Each subunit has a haem-binding pocket

06/27/2025
• The polypeptide chains are of five types viz. α,
β, γ, δ and ε

• The α chain is made up of 141 amino acids.

• The β , γ, δ and ε chains are made up of 146

amino acid residues each.

06/27/2025
 • Normal adult haemoglobin (HbA) is made up
of four haem groups, two α chains and two β
chains, and is represented as α2 β2.

• A small amount of HbA2 is also found in

adults which is α2 δ2.

• Foetal haemoglobin (HbF) is α2 γ2

• Embryonic haemoglobin is α2 ε2

06/27/2025
 Adult haemoblobin
Hb A Hb A2 Hb F

structure a 2b 2 a 2d 2 a 2g 2

Normal % 96-98 % 1.5-3.2 % 0.5-0.8 %

Synthesis of globin
 Synthesis of globin
• Various types of globin combines with haem
to from different haemoglobin

• Eight functional globin chains, arranged in two

clusters the
• b- cluster (b, g, d and e globin genes) on the short arm
of chromosome 11
• a- cluster (a and z globin genes) on the short arm of
chromosome 16
Globin gene clusters
 Synthesis of globin
Globin synthesis, starts at 3rd week of gestation
• Embryonic
Haemoglobin Gower I ( z2e2)
Haemoglobin Portland ( z2g2)
Haemoglobin Gower II (a2e2)
• Fetal : HbF (a2g2), HbA (a2b2)
• Adult : HbA, HbA2 ( a2d2), HbF.
 Normal hemoglobins

Embryonic
Hemoglobins Fetal hemoglobin Adult hemoglobin

• Hemoglobin A-
alpha (2), beta (2)
• Gower 1- zeta (2), • ~95%
epsilon (2) • Hemoglobin F- • Hemoglobin A2-
• Gower 2- alpha alpha(2), delta(2)
alpha(2), gamma
(2), epsilon (2) (2)
• ~1.5-3.7%
• Portland-Zeta (2), • Hemoglobin F-
gamma (2) alpha(2), gamma
(2)
• <2 %
Globin chain switch
• The histidine residues linked to iron are
present at positions 58 and 87 in α chains and
at positions 63 and 92 in other chains.

• The bond between iron and the distal

histidine residue (His87 or His92) is unstable.

06/27/2025
• The distal iron-histidine bond is broken when
haemoglobin is exposed to high oxygen
tension

• This results in the formation of an iron-oxygen

bond

• The binding of oxygen to haemoglobin changes the

conformation of haemoglobin

06/27/2025
• Two conformations have been described, T
(taut) and R (relaxed).

• Deoxygenated Hb exists in T form which is stabilised by

2,3-bisphosphoglycerate (2,3-BPG) which is formed from
1, 3-BPG (an intermediate in glycolytic pathway) when
there is a deficiency of oxygen in the tissues.

06/27/2025
• T and R states of Hemoglobin
• Hemoglobin exists in two major conformational
states: Relaxed (R ) and Taut or Tense (T)

• R state has a higher affinity for O2.

• In the absence of O2, T state is more stable; when O2

binds, R state is more stable, so hemoglobin
undergoes a conformational change to the R state.

• The structural change involves readjustment of

interactions between subunits
06/27/2025
• There is a central cavity in the haemoglobin molecule
surrounded by the four polypeptide chains.

• 2,3-BPG enters this cavity and cross links the two β

chains.

• When oxygen tension increases, 2,3-BPG is displaced

and the T form changes into R form.

• During this transition, one pair of α and β subunits

rotates by 15° relative to the other pair.

06/27/2025
 • Each subunit of haemoglobin can bind one
oxygen molecule.

• Since there are four subunits in a molecule of

haemoglobin, one molecule can bind four
oxygen molecules.

06/27/2025
 Oxygen transport
• The amount of O2 bound to hemoglobin and
released to tissues depends on PO2 and PCO2,
but also the affinity of hemoglobin for O2.
• Oxyhemoglobin: hemoglobin with oxygen
• Deoxyhemoglobin: hemoglobin without
oxygen
• Oxygen affinity is the ease with which
hemoglobin binds and releases oxygen.
 Oxygen Affinity
• Determines the proportion of O2 released to
the tissues or loaded onto the cell at a given
oxygen pressure.
• Increases in oxygen affinity means hemoglobin
has an increased affinity for O2, so it binds
more. However, it does not want to give it up.
• Decreases in oxygen affinity, cause O2 to be
released.
 Oxygen dissociation curve

The ability of Hb to load and unload oxygen at

physiological pO2 is shown by oxygen
dissociation curve (ODC)

06/27/2025
 Oxygen Dissociation Curve
• Right-Shift
– Hgb has less attraction
for O2
– Hgb willing to release O2
to tissue
– Examples: anemia,
acidosis
– Even though there may
be less RBC’s, they act
more efficiently to
deliver O2 to target
 Oxygen Dissociation Curve
• Left shift
– Hgb has more attraction
for O2
– Hgb less willing to
release O2 to tissue
– Examples: presence of
abnormal Hgb’s, alkalosis
 A
Percentage
saturation

06/27/2025
pO2 in mm of Hg
 B
Percentage
saturation

06/27/2025
pO2 in mm of Hg
 C
Percentage
saturation

06/27/2025
pO2 in mm of Hg
 D
Percentage
saturation

06/27/2025
pO2 in mm of Hg
• A. Theoretical curve as per mass action.

• B. Sigmoid curve, due to heme-heme interaction (hill

effect).

• C. Further shift to right due to carbon dioxide (Bohr

effect) and BPG. This curve represents the pattern under
normal conditions.

• D. further shift to right when temp is increased to 420C.

06/27/2025
 • ii. 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 20
ml of oxygen /dl of blood.

• iii. In the tissue capillaries, where the pO2 is

only 40 mmHg, theoretically Hb saturation is
75%. Thus under STP conditions, blood can
release only 22%.

06/27/2025
 Factors affecting ODC
• 1. Heme-heme interaction and co-operativity:-
• A. the sigmoid shape of ODC – due to
allosteric effect, or co-operativity.
• equilibrium of Hb=O2
Hill equation (A V Hill, nobel prize,1922)

06/27/2025
 B. Positive co operativity

Hb HbO2 HbO4 HbO6 HbO8

Homotropic interaction

06/27/2025
 c. Each successive addition of O2, increase the
affinity of Hb to O2 synergistically.

• D. Similarly, binding of 2, 3 – BPG at a site other

than the oxygen binding site, lowers the affinity
for oxygen (heterotropic interaction).

06/27/2025
 Alteration of structure

Diagrammatic representation of subunit interaction in Hemoglobin

06/27/2025
• During oxygen uptake, the T form to the R form
with disruption of the salt bridges .
• The Hb subunits are moved relative to one
another.
• During oxygenation, the α1 - β2 interface shows
movement.
• The two subunits slip over each other.
• The quaternary structure of oxy Hb is described
as R form; and that of de-oxy Hb is T form.

06/27/2025
• When oxygenation occurs the salt bonds are
broken successively. Thus on oxygenation, the
Hb molecule can form two similar dimers.
(2x alpha)+(2x beta)→2x(alpha-beta)
(Deoxy-Hb) (oxy-Hb)

06/27/2025
06/27/2025
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 high, CO2 diffuses into the RBCs
CO2 + H2O → H2CO3 → H+ + HCO3-
Carbonic

Anhydrase

Iv. When carbonic acid is ionizes, the intracellular pH

falls. The affinity of Hb for oxygen is decreased and
oxygen is unloaded
06/27/2025
4. The chloride shift
When CO2 is taken up ----HCO3- ↑

Cl shift (in tissues) CO2

H2O + CO2

Carbonic anhydrase

H2CO3 HbO2
H+

N N
HCO3-

HHb +O2

HCO3- Cl-

O2-
To cells
Cl-

Chloride enters into RBC

06/27/2025
• When the blood reaches the lungs, reverse
reaction takes place

06/27/2025
4. The chloride shift
When O2 is taken up ----

Cl shift (in lungs) H2O + CO2

CO2 Air

Carbonic anhydrase

H2CO3 HbO2
H +

N N
HCO3-

HHb +O2

HCO3- Cl-

O2-
Air
Cl-

Chloride comes out of RBC

06/27/2025
5. Effect of temperature
• p50 = the pO2 at which Hb is half saturated
• p50 of normal Hb = 26 mmHg (at 37oC)
• Elevation of temp. causes 88 % increase in p50
• ODC shifts to left at low temp.
• Under febrile conditions , increased needs of
oxygen met by a shift in ODC to right.

06/27/2025
6. Effect of 2,3-BPG
• Normal 2,3-BPG level=15 ± 1.5 mg/g Hb.
• 2,3-BPG == high in children
• 2,3-BPG is produced from 1,3-BPG, an
intermediate of glycolytic pathway.
• 2,3-BPG, preferentially binds to deoxyHb and
stabilizes T form
• When T form reverts to R, 2, 3-BPG ejected
• During oxygenation, BPG released
06/27/2025
 Carbon Dioxide Transport
• Three mechanisms of transport
– Dissolution in the plasma
– Formation of bicarbonic acid
– Binding to carbaminohemoglobin
 Nonfunctional hemoglobins
• What do they do?
– Hypoxia
• Inadequate amount of O2 in the blood
– Cyanosis
• Presence of > 5 g/dl deoxyhemoglobin in blood
• Patient appears blue
 Nonfunctional hemoglobins

– Carboxyhemoglobin
• Oxygen molecules bound to heme are replaced by carbon
monoxide.
• Slightly increased levels of carboxyhemoglobin are present in
heavy smokers and as a result of environmental pollution.
• Can revert to oxyhemoglobin.
– Methemoglobin
• Iron in the hemoglobin molecule is in the ferric (Fe3) state instead
of the ferrous (Fe2) state. Incapable of combining with oxygen.
• Can occur as a result of strong oxidative drugs or to an enzyme
deficiency (more discussion to follow).
• Can revert to oxyhemoglobin
– Sulfhemoglobin
• Hemoglobin molecule contains sulfur.
• Caused by certain sulfur-containing drugs or chronic constipation.
• Cannot revert to oxyhemoglobin and may cause death.