Hemoglobin

Dr. Srilatha Bashetti

Department of Biochemistry
 Heme proteins
Heme proteins are group of specialized proteins
which contain heme as tightly bound prosthetic
group
 Hemoglobin (oxygen transport)
 Myoglobin (oxygen transport)
 Cytochromes (electron transport)
 Catalase (H2O2 utilization)
Myoglobin
• Monomeric protein of red muscle

•stores O2 as a reserve against oxygen

deprivation
•A globular soluble protein
• 153 residues (16 kDa)
•Mole. Weight –17000
•Contains a heme prosthetic group
•functions as reservoir of oxygen
• Myoglobin consists of single polypeptide chain that
is structurally similar to each individual subunit
polypeptide chains of hemoglobin molecule.

• α-helical content :

• 80% of it’s polypeptide is folded in to 8 stretches of α

- helices (A,B,C,…..H)----- terminates as proline ,
whose 5 membered ring can not be accommodated in
α-helices (first protein crystal structure)

• ---by β-bends and loops stabilized by hydrogen

bonds and ionic bonds
•
• Typical of globular proteins , the
surface of myoglobin is polar,
the interior contains non polar
residues such as Leu, Val, Phe
and Met
• The heme group of Myoglobin sits in
crevice in the molecule ,which is lined by
non-polar amino acids .

• The exceptions are HisE7 and His F8,the

seventh & eight residues in helices E & F,
which lie close to heme iron ,where they
functions in O2 binding
HAEMOGLOBIN
 By Dr..Noorjahan
Structure of Hemoglobin
a2 Heme

b1

b2

a1

Inter-subunit contacts
 HAEMOGLOBIN
 Hemoglobin is found in RBC
 Hemoglobin A is major Hb in adults
 HEMOGLOBIN is a conjugated
protein
 heme ---non-protein
 Globin ----apo-protein
 Tetramer
Heme is iron containing compound
Belongs to the class of protoporphyrins

1 2
 A a
N
H
8 3
N
7D N B4
H
N
 b
6
C
5

 Made up of 4 pyrrole rings

 1 2
 a
N
H
8 3
N
7 N 4
H
N
 b
6 5
 Linked by 4 methene (=CH-) bridges
 STRUCTURE OF HAEM

Porphyrins found in nature are compounds in which side chains are substituted for the
eight hydrogen atoms at carbons numbered 1 through 8 in the pyrrole rings.
Protoporphyrin IX, found in heme, has 4 methyl, 2 vinyl
and 2 propionic acid side chains on the tetrapyrrole
ring.
Heme
 One per subunit
 Has porphyrin ring and iron atom,Carries O2
 M V O=O

A
N
M M
D N Fe2+ N B
P N V

C
N

NH CH2
P M Heme
 Heme
o Is a complex protoporphyrin IX
and Ferrous iron (Fe 2+)
o The iron atom held in the centre
of the heme molecule by bonds to
the 4 nitrogens of the porphyrin
ring .
o The heme Fe 2+ can form two
addittional bonds ,one on each
side of the planar porphyrin ring .
 Heme
o In myoglobin &hemoglobin ,one
of these positions is coordinated
to the side chain of Histidine
residue of the globulin molecule
,where as other position is
available to bind with oxygen
(oxygenated form)
GLOBIN  Multi-subunit protein
 2 a and 2 b subunits (
2, 2 , 2 as per type of
hemoglobin)
 a-chain contains 141 aa
 b-chain contains 146 aa
 4 subunits,4 haem held
together in a definite
conformation –quarternary
structure
 Stabilized non-hydrogen
bonds ,salt bridges and van
der waal forces
 Globin chains of hemoglobin forms a protective hydrophobic
pocket for binding of heme that protects the reduced form of
iron (Fe+2) of heme from oxidizing to ferric form (Fe+3)
from aqueous medium and permits reversible binding of
oxygen with Fe+2 of heme
 Bipyramidal

2 open positions on each

side of the heme plane

ferrous +2 ferric +3
heme hemin

Hb metHb
Heme ferriHb .

Mb metMb
 FUNCTIONs OF HEMOGLOBIN
Hemoglobin is found exclusively in RBC
Transport of O2 from lungs to the
tissues.(carries 4 molecules of O2 )
Transport of CO2 and H+ from tissues
to lungs.
Acts as an intracellular buffer and is
thus involved in acid base balance.
 The Hemoglobin tetramer –composed of 2 identical dimers
 The polypeptide chains in each dimer held together tightly,
primarily by hydrophobic interactions (also forms
inter chain by hydrophobic bonds )
 Ionic and hydrogen bonds also occur between the members of
the dimers .
 As a result of the weaker interactions in between dimers they
occupy different relative positions in deoxy Hemoglobin as
compared to oxy Hemoglobin
 T form : deoxy form --T form – taut
form
 Low oxygen affinity form
 The two ab dimers interact through a
network of ionic & hydrogen bonds .
 R form : relaxed form --R form
 High oxygen affinity form
 The polypeptides have more freedom of
movement
Formation of Oxy Hb
 O2 first binds to α chain as β chains are blocked by valine
residues
 Binding of O2 breaks salt bridges which leads to

 Widening of Heme pockets

 Movt. of Subunits
 Movt. of Fe+2 atom
 As a result tense T structure changes to R form
The ‘T’ – ‘R’ State Transition
• Binding of O2 causes a series of shifts in all subunits
• Change in heme structure upon binding O2
• Since His F8 is covalently attached, all of F helix shifts
• Reorganization of helix alters tertiary structure, which
in turn alters the quaternary structure- 4 chains behave
as a single cooperative structural unit
 Changes in packing of hydrophobic side chain
 Changes in pairing of charged side chains
The change in conformation of Hemoglobin from the T to
the R state increases O2 affinity at ALL sites
 Forms of Hb
T form R form
 Oxy Hb relaxed form.
Deoxy Hb tense/taut
 High affinity
Has low O2 affinity  Binding of O2 destabilizes
H & ionic bonds limit some of the H & ionic
the movements of bonds b/w αβ dimers
monomers. (subunits move a
little freely).
Iron moves towards the
plane of Heme

Rotation increases the affinity of O2 to heme

Binding of O2 to the Heme
Changes the Whole Structure
of Hemoglobin
R state T state

Shifts at the b chains

ab interfaces further apart
Myoglobin can bind to 1
molecule of O2
Hemoglobin can bind to 4
molecules of O2
Oxygen dissociation curve (odc)
 A plot of degree of saturation
measured at different partial
pressures of oxygen (pO2 ) is called
ODC curve

 The partial pressure of O2 needed to achieve half –

saturation of the binding sites (P 50) is approximately
100 mm Hg for Myoglobin,26 mm for hemoglobin
The ability of Hb to
load or unload oxygen
at physiological pO2
is shown by ODC
Transport of O2 to the tissues
 In lungs – high pO2,Hb gets fully
saturated (loaded with O2)
 At tissue level – low pO2, oxyHb releases
O2 (unloads).
 Mediated by binding O2 to myoglobin
(immediate reservoir and supplier of O2)
 (lungs)

Oxygen dissociation curves of Hb and

Mb.
Note the sigmoidal shape of the
hemoglobin dissociation curve and the
hyperbolic shape for myoglobin.

Affinity for O2
Cyt Oxidase > Mb > Hb
Oxygen dissociation curve for
myoglobin (Mb)---hyperbolic
shape

Hemoglobin (Hb)------sigmoidal in
shape- indicating that the subunits
cooperate in binding oxygen .
heme-heme interactions and cooperativity

 Sigmoid shape of ODC is due to Allosteric effect or

cooperativity
 The equilibrium of Hb with oxygen is expressed by the
Hill equation
Hill
Plot
 Effect of temperature
 p50- --the pO2 at which Hb is half saturated
with O2 (normal 26 mm of Hg )
 Elevation of temp from 20 to300 ---
88%increase in p50
 Hypothermia -----shift of ODC to left
 Hyperthermia -----shift of ODC to right
 Cooperative binding of
O2 by the four subunits of
Hb means that the binding
of oxygen molecule at one
heme group increases the
oxygen affinity of the
remaining heme groups
in the same Hb
(homotropic effect)
 Co-Operative binding of Hb
 Binding of first O2 enhances the binding of other molecules
 Binding of 2,3BPG at a site other than
oxygen binding site, lowers the affinity
for oxygen (heterotropic effect)
 pH and pCO2
 In Tissues :
pCO2 high, pH low (metabolic acids like lactate)

affinity of Hb for O2 is decreased(ODC shift to right )

O2 is released(R---T)
 pH and pCO2
 In lungs :
pCO2 low, pH high, pO2 is significantly elevated

affinity of Hb for O2 is increased(ODC shift to left )

O2 binds to Hb(T---R)
LINKAGE BETWEEN
pH, CO2 BINDING, & O2 BINDING
 The influence pH, CO2 to
facilitate oxygenation of Hb in
the lungs and deoxygenation at
the tissues is known as
BOHR’SEFFECT.
Bohr’s effect
Mechanism of Bohr effect
 Terminal a.a βHis 146 is largely responsible
for the Bohr effect.
 Imidazole ring of His (146) is in close
proximity with the Aspartate (94) of the
same β-chain.
 H+ binds (Bohr Protons) to the His and this
binding is promoted by the –ve charge on
the Aspartate.
a- chain : N-terminal a.a
residues, His 122 nd

β – chain : C- terminal carboxy

gp residues, His 146 , Asp
th

(94 )
th
 Role of Cl¯ in O2 transport :

 Cl¯ is bound more tightly to deoxy Hb than to oxy

Hb, facilitates the release of O2.
 HCO¯3 is freely permeable and equilibrates with the
surround plasma.

 To maintain neutrality Cl¯ enter the erythrocytes

and bind with deoxy Hb.

 Conc of Cl¯ is greater in venous blood than in

arterial blood.
Chloride shift or Hamburger effect
 Effect of 2,3- BPG
 Reduces the affinity of O2 to Hb
 HbO2 + 2,3 BPG Hb-2,3BPG +O2
 Binding site
 Pocket formed by the β- globin chain at central cavity
 Forms ionic bond with His-2 , His-143, lys-82
 Oxygenation prolapse this pouch.
 Without it Hb can deliver only 8% of O2
Normal level 15 mg/g Hb.

The high O2 affinity of

fetal Hb is due to the
inability of gamma chains
to bind 2,3 BPG
Effect of 2,3-
BPG on the
oxygen
affinity of
hemoglobin
 In chronic tissue deprivation levels increase
 Individuals in high altitude
 COPD
 Anemia
 Cardiac failure
 Increased 2,3-BPG 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-BPG synthesis was perfectly well except for
mild polycythemia
 2,3-BPG levels decrease in blood stored for
transfusion
 It is prevented by adding Inosine
 Dissolved form
 Isohydric transport
 As carbamino Hb
Carbon Dioxide
(CO2)
 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+ + HCO-3
 (CO2)
 Carbon dioxide diffuses freely into the red cell where the
presence of the enzyme carbonic anhydrase facilitates
reaction 1.

 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 expired air.
 About 70% of tissue carbon dioxide is processed in
this way.

 Of the remaining 30%:

5% is carried in simple solution
25% is bound to the N- terminal amino (Valine)
groups of deoxygenated hemoglobin, forming
carbaminohemoglobin.

R-NH2 + CO2 R-NH-COOH

 Dissolved form---10%
 Isohydric transport---75%
 Minimum change in pH during
transport
 H+ ions are buffered the deoxy
Hb----Explained by HALDANE
effect
Deoxygenation of the blood
increases its ability to carry
carbon dioxide
From Tissues to Lungs Lungs
Hb-oxygen dissociation curve
 Right shift (easy oxygen delivery)

 High 2,3-BPG
 High H+
 High CO2
 HbS
 Cl-

 Left shift (give up oxygen less readily)

 Low 2,3-BPG
 HbF
Ontogeny of globin synthesis
Time Region Type of Globin Type of Hb
Gene

3 weeks of Yolk Sac ζ&ε Hb Gawer1 ζ ε)2

Gestation )

5 weeks of Yolk Sac γ&α Hb Portland(ζ γ)2

Gestation Hb GawerII (αε)2

6-30 weeksof Liver & spleen α & γ & β Hb F (α γ)2

Gestation

30 weeks of Liver δ Hb A2 (α δ(2

Gestation

At Birth B.M ___ HbA(α β)2

Adult haemoblobin
Hb A Hb A2 Hb F

structure a2b2 a22 a22

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

Embryonic Hb Gower-I (ε2ζ2) 2nd to 3rd wk
Gower-II(a2ε2) 3rd to 7th wk
HbF (a2 γ2) 2nd to 3rd
trimester (0.5-0.8 %)

HbA2 (a2 δ 2) Minor (1.5-3.2 %)

HbA (a2β2) Adult Hb

(96-98 %)

HbA1C Glycated 4% and 5.6%

hemoglobin