CHAPTER

OXYGEN TRANSPORTERS:
HAEMOGLOBIN AND
17 MYOGLOBIN

Molecular oxygen is sparingly soluble in body fluids. This limits the amount that can be transported in physical solution to
less than 30 g per day, whereas the body requires approximately 500 g of molecular oxygen per day. An effective
oxygen transport is made possible by haemoglobin, the oxygen-binding protein, present in the erythrocytes. Because of
the oxygen-binding properties of this protein, blood can dissolve approximately 70 times more oxygen than the plasma
free of haemoglobin can do.
Just as haemoglobin is the oxygen-binding protein in erythrocytes, the myoglobin binds oxygen in muscle tissue. It per-
mits storage of oxygen in muscles. The stored oxygen is released when it is required, for example, during vigorous mus-
cular exercise. Binding of oxygen to haemoglobin and myoglobin is called oxygenation.
Protein  O2  Oxygenated protein
It is a non-enzymatic, reversible process. Therefore, oxygen, when available in plenty, binds to the oxygen-binding
proteins. Conversely, oxygen is released from these proteins when it is scarce. None of the constituent amino acids of these
proteins has any binding affinity for molecular oxygen. Therefore, the oxygen-binding proteins have to employ a non-protein
component to serve as the oxygen-binding site. This component is haem in haemoglobin as well as in myoglobin.
In this chapter, general characteristics and oxygen-binding properties of haemoglobin and myoglobin are described.
After going through this chapter, the student should be able to understand:
 General properties of haemoglobin, such as structure of haem, organization of various globin chains, positioning of
iron in the tetrapyrrole ring and mode of binding of oxygen; structural and functional peculiarities of various types of
haemoglobins.
 Behaviour of haemoglobin as an allosteric oxygen binder, sigmoid shape of the oxygen saturation curve of haemo-
globin, Bohr effect, and role of 2,3-BPG in oxygen off-loading.
 Aetiology and oxygen-binding peculiarities in various types of haemoglobinopathies.
 Oxygen-binding properties of myoglobin and its suitability to store oxygen in muscles.

I. Haemoglobin Upon oxygenation, the haemoglobin is converted to

oxyhaemoglobin. This is not a simple metal–gas interac-
Haemoglobin is a tetrameric metalloprotein consisting tion, but is a dynamic process subject to various allo-
of four polypeptide chains, each with its own haem. In steric controls. Binding of other ligands such as carbon
addition to carrying oxygen from lungs to the peripheral dioxide (and H) to haemoglobin is also regulated by
tissues, it carries carbon dioxide from actively metaboliz- allosteric mechanism. The allosteric mechanism is very
ing tissues to lungs. Haemoglobin possesses buffering well understood in case of haemoglobin, and discussed
property as well. in detail in this chapter.
 Oxygen Transporters: Haemoglobin and Myoglobin 351

A. Basic Structure bends and interhelical segments. -chain has 7, and -chain
has 8 helical segments, labelled as A (N-terminal) through
Humans have several types of haemoglobins. All types are H. They are organized into a tightly packed, nearly spher-
made up of two ␣ chains and two non-␣ chains (b, d or g) ical, globular tertiary structure, shown in Figure 17.1.
with a total molecular weight of approximately 65,000. Like most soluble proteins, haemoglobin has a rela-
These chains, collectively termed globin polypeptides, dif- tively hydrophilic surface and a hydrophobic interior.
fer in primary structures but have similar secondary and ter- Polar amino acids are located almost exclusively on the exte-
tiary structures. The differences in tertiary structure among rior surface of the globin polypeptides, and contribute to
them are critical to the functional properties of each. A non- their remarkably high solubility in cytoplasm of erythro-
covalently bound haem group with a molecular weight cytes (5.2 mmol/L). This permits building up of a high
of 616 is associated with each chain (Fig. 17.1). It should β1
be noted that all globin polypeptides are homologous
β2
proteins, having arisen from the same ancestral gene.

Globin chains: The globin chains present in the major adult

haemoglobin, called HbA, are  and , and its subunit
structure is 22. The minor adult haemoglobin (HbA2)
and fetal haemoglobin (HbF) also have two -chains but
instead of the -chains they have -chains and -chains
respectively (Fig. 17.2).
The -chain has 141 amino acids (MW 15,750) and
the -chain has 146 amino acids (MW 16,500). Like the
-chains, the other two non--chains ( and ) also have
146 amino acids and are structurally related.
The - and -chain differ in 10 and the - and -chains
α2
in 39 of their 146 amino acids. The - and -chains are not
as closely related: they are identical only in 64 of their
amino acids. α1

Haem group
Secondary and tertiary structures of globin polypeptides:
The secondary structure of all globin polypeptides consists Fig. 17.1. Structure of HbA molecule consisting of two -chains
largely of -helical segments and short stretches of various and two -chains, each with a non-covalently bound haem group.

Type of haemoglobin Structure Composition Importance

α2β2 97% of adult

Major adult (HbA) α α haemoglobin
β

α2δ2 2–3% of adult

Minor adult (HbA2) α α
haemoglobin
δ

γ
Major haemoglobin
α2γ2 in second and
Fetal (HbF) α α
third trimester
γ of pregnancy

Fig. 17.2. Subunit structure and importance of various haemoglobin types. A subunit consists of a polypeptide chain and its
haem group (  haem,  polypeptide chain).
352 Textbook of Medical Biochemistry

intracellular concentration of Hb within red cells, which  Fifth is an imidazole nitrogen of the so called proxi-
appear as “bags” filled with Hb. Amino acids having mal histidine.
both polar and non-polar side chains, such as tyrosine,  The sixth ligand is molecular oxygen bound between the
threonine and tryptophan, are oriented with their polar iron and the imidazole side chain of distal histidine.
functions toward the protein’s exterior. Hydrophobic
In deoxyhaemoglobin, the iron is coordinated with
amino acids are buried within the interior, where they stabi-
only five ligands since oxygen is not present.
lize the folding of the polypeptide and binding of the
iron-porphyrin prosthetic group. 
Haem group is the oxygen-binding site. It consists of a
 porphyrin ring system with ferrous iron fixed in the centre.
Polar amino acids, located towards the exterior, make
haemoglobin highly soluble, whereas the non-polar amino
Proximal and distal histidine: They are located on either
acids are buried in the interior and influence folding.
side of the haem prosthetic group. Side chains of both
these histidines are oriented perpendicular to the planar
The only exception to this general distribution of
haem group. The proximal histidine is bound to iron,
amino acid residues in globins are the two histidine resi-
and the distal histidine guards the entrance on the oppo-
dues, termed proximal- and distal-histidines, which are
site site (Fig. 17.3). Their presence ensures that the imme-
oriented perpendicular to and on either side of the pla-
diate environment of the haem iron is the same (i.e.
nar haem prosthetic group. They create a hydrophobic
hydrophobic).
pocket in which the haem-group resides.
Haem group: A haem group is associated with each glo- 
bin chain in the hydrophobic interior (tucked between Proximal and distal histidine, located on either side of
the E and the F helices). Haem consists of a porphyrin planar haem, create a suitable hydrophobic environment
ring system with various side chains: methyl, vinyl and for binding of oxygen to ferrous iron.
propionate (Chapter 16). A ferrous iron, fixed in the cen-
tre of the ring through complexation to the nitrogen The ferrous iron in haem: The haem iron occurs in the ferrous
atoms of the four pyrrole rings, is functionally the most state in haemoglobin (and myoglobin). During oxygen
important element (Fig. 17.3). binding, it is not oxidized to the ferric form; it becomes oxy-
The ferrous iron has an octahedral geometry and can genated but not oxidized. Oxidation to the ferric state converts
be attached to six ligands: the haemoglobin to methaemoglobin, which is function-
ally inactive. It can no longer carry oxygen. Oxidation to fer-
 Four of these ligands are the pyrrole nitrogen atoms
ric form imparts a net positive charge (haem is neutral),
of the porphyrin ring system.
therefore, a negative counter ion must be associated with
Apoprotein the molecule. If the counter ion is Cl then the compound
is haemin; if it is OH then the compound is haematin.

N Proximal 
histidine
Haemoglobin A is a tetrahedral complex of two identi-
N cal -globins and two identical -globins. The minor
adult haemoglobin and fetal haemoglobin also have
Fe -chains, but instead of -chains, they have -chains
N N
and -chains, respectively.
N N

B. Quaternary Structure
Haemoglobin is a tetramer of four subunits, each sub-
Oxygen
unit consisting of a polypeptide chain with a non-
N
Distal
covalently bound haem group. Packing of these subunits
histidine relative to one another is referred to as the quaternary
N
structure, as shown earlier in Figure 17.1.
The four polypeptide chains are visualized as compris-
Fig. 17.3. The haem iron lying at centre of the tetrapyrrole ring. ing two identical dimers, each dimer having an - and
 Oxygen Transporters: Haemoglobin and Myoglobin 353

-chain. The dimers are designated ()1 and ()2, Effectively, binding of oxygen with one of the haem groups
where the numbers refer to dimer 1 and dimer 2. Two of haemoglobin increases the oxygen affinities of the remain-
chains within a dimer are held together tightly by the ing haem groups. This effect is called positive cooperativity.
ionic bonds and the hydrophobic interactions, which It indicates a cooperative interaction between different
prevent their movement relative to each other. However, subunits of haemoglobin (Box 17.1) because the ligand-
the two dimers are linked with each other by weak polar binding information is transmitted from one subunit to
bonds, so that movement at the interface of these two another.
occurs more freely. These characteristics are shown by the sigmoid
(S-shaped) curve of oxygen dissociation of haemoglobin
(Fig. 17.4). This curve describes the fractional saturation of
C. Allosteric Effects the haem groups at various oxygen partial pressures. The
initial flat region reflects low oxygen affinity of the deox-
Oxygen-binding characteristics of haemoglobin change ygenated haemoglobin for the first oxygen. With increas-
by the following effectors: ing oxygen partial pressure, however, the first haem group
 pO2: The partial pressure of oxygen (pO2). gets oxygenated. This facilitates subsequent binding of
 pCO2: The partial pressure of carbon dioxide (pCO2). oxygen molecules. Therefore, the curve becomes steeper,
 pH of the surrounding medium. indicating that subsequent oxygen molecules are bound
 Presence of 2,3-bisphosphoglycerate. with much higher affinity. In fact, the fourth oxygen mole-
cule binds haemoglobin 300 times as tightly as the first; the
These effectors are collectively called allosteric (“other binding affinities of the second and the third oxygen
site”) modulators because their interaction at one site of molecules are intermediate between those of the first and
haemoglobin molecule influences the binding character- the last.
istics of some other site within the same molecule.

 100
The ligand-binding information is communicated within
the haemoglobin molecule from one subunit to another.
% Saturation

Such subunit interaction makes the haemoglobin well

adapted for integrating the transport of oxygen, car- 50
bon dioxide and H.
P50 = 26 torr c = 100 torr

Binding of Oxygen: Positive Cooperativity 0

Binding affinity of haemoglobin for oxygen changes with 0 20 40 60 80 100
the state of oxygenation. As the first oxygen molecule a = 20 torr b = 40 torr
binds with one of the haem groups, the binding affinity
Fig. 17.4. The sigmoid curve of oxygen dissociation of hae-
of the haemoglobin for the second oxygen molecule is
moglobin. The arrows indicate: a  pO2 in capillaries of work-
enhanced. As a result, the second oxygen molecule binds
ing muscles; b  pO2 in capillaries of resting muscles; c  pO2
much more easily. The same process is repeated for the in alveoli of lungs, P50 indicates 50% saturation of haemoglo-
third and fourth oxygen molecules. bin when pO2 equals 26 torr.

BOX 17.1
Hill Coefficient
The measure of cooperativity among ligand-binding sites is indicated by Hill coefficient(n), named after the scientist Archibald
Hill (Nobel Prize in 1922). It reflects the extent to which the interaction of oxygen with one subunit influences the interaction
of oxygen with other subunits. For fully cooperative interaction (meaning that binding at one site maximally enhances bind-
ing at other sites in the same molecule), value of Hill coefficient is equal to the number of sites. The theoretical maximum
for haemoglobin would, therefore, be four. In practice, however, lower values are observed: the normal value for adult
haemoglobin (n  2.7) indicates that there is a substantial functional subunit-subunit interaction. In the absence of coop-
erative ligand binding, even with multiple sites, the Hill coefficient would be one. This is what is observed for those haemo-
globin mutants that have lost cooperative ligand binding, as also for myoglobin.
354 Textbook of Medical Biochemistry

 Table 17.1. Per cent saturation of haemoglobin at different

Oxygen binding to a haem group in haemoglobin partial pressures of oxygen
increases the oxygen affinities of the remaining haem pO2 (torr) Per cent saturation of Hb
groups; this effect is positive cooperativity and it leads 100 (in alveoli) 96
to sigmoid oxygen-binding curve.
40 (in resting muscles) 64
20 (in working muscles) 20
The oxygen dissociation curve is characterized by P50
value, the oxygen pressure at which the haemoglobin is 10 (in vigorously exercising muscles) 10
50% saturated with oxygen. Its value is 26 torr. It should
be noted that in proteins that are not allosteric and do
not show coooperative oxygen-binding kinetics, have a sim- 100
pH 7.4 A
ple hyperbolic curve with low P50; this is what is observed B
with myoglobin (see Fig. 17.14).

% Saturation
Note: The term ligand refers to specific molecule (such pH 7.2
as, oxygen in case of haemoglobin), that is bound by a 50
protein. The word ligand comes from Latin, meaning
“bound entity”.
H+ releases O2
Significance of Positive Cooperativity
The cooperative binding of oxygen enhances the effi-
0 20 40 60
ciency of haemoglobin as an oxygen transporter. Without pO2 (torr)
positive cooperativity, an 81-fold increase of the pO2
would be required to raise the oxygen saturation from Fig. 17.5. Effect of pH on the oxygen affinity of haemoglo-
10% to 90%. In case of haemoglobin, however, about bin. The decreased affinity in response to fall in pH is reflected
fivefold increase is sufficient to do the same. This means by rightward shift of the oxygen dissociation curve.
that haemoglobin can rapidly bind oxygen in lungs
(where pO2 is high) and then readily liberate it in the tis-
sue capillaries (where pO2 is low).
Bohr Effect
The oxygen affinity of haemoglobin exhibits exquisite sensitiv-
This may be seen from the oxygen dissociation curve:
ity to pH; the affinity decreases as the pH becomes more acidic
 Haemoglobin is 96% saturated with oxygen in lungs (Fig. 17.5). This is known as the ‘Bohr effect’ after the
(pO2  100 torr). discoverer Christian Bohr, father of Neil Bohr, the atomic
 In resting muscle (pO2  40 torr) it is only about 64% physicist.
saturated (Fig. 17.4). Thus, it delivers 32% (96%
minus 64%) of its oxygen content to the resting muscle. HbO2  H  H.Hb  O2
 In exercising muscles, further fall in pO2 occurs Closely related to the Bohr effect is the ability of car-
because of rapid oxygen consumption. Since haemo- bon dioxide to alter the oxygen affinity of Hb. Like the
globin is only about 20% saturated at this partial pres- negative allosteric effect of H , increasing levels of CO2
sure (Table 17.1), further liberation of oxygen occurs. decrease affinity for oxygen. Thus, there exists an inverse
 Likewise, in the vigorously exercising muscles (pO2  10 relation of oxygen binding affinity of haemoglobin vis-á-vis
torr), where oxygen need is maximum, the saturated hae- binding of CO2 and H.
moglobin may release up to 90% of its oxygen content. Decreased affinity of haemoglobin for oxygen is
Note: Torr is a unit of pressure equal to that exerted by a reflected as rightward shift of the oxygen dissociation
column of mercury 1 mm high at O°C and standard curve. It represents oxygen-offloading.
gravity (1 mmHg). This unit is named after Evangelista Effect of CO2 on oxygen affinity is mediated via [H]
Torricelli, the inventor of mercury barometer. because in solution there exists the equilibria:

 CO2 + H2O
CA
H2CO3
CA
H+ + HCO−3
Haemoglobin binds oxygen efficiently in lung alveoli and
releases it with similar efficiency to peripheral cells. This These reactions are enhanced in the red blood cells
remarkable duality of function is achieved by cooperative through the action of carbonic anhydrase (CA). Thus, rise
interactions among the globin subunits of haemoglobin,
in pCO2 induces a decrease in pH, which then causes a
which is reflected by a sigmoid oxygen-dissociation curve.
loss of oxygen from oxyhaemoglobin.
 Oxygen Transporters: Haemoglobin and Myoglobin 355

Significance of Bohr Effect Range of O2 partial

pressures in tissues
Bohr effect changes the oxygen-binding characteristics of
100
haemoglobin according to the requirements of the tis-
No BPG
sue. Since an increase in pCO2 and a decrease in pH are (Hb)
Mb
both characteristics of actively metabolizing cells, these

% Saturation
cells promote the release of oxygen from oxyhaemoglo- Normal:
bin. This is desirable since the actively metabolizing tis- 5 mM BPG Altitude-adapted:
50
(Hb) 7.5 mM BPG (Hb)
sues require an adequate supply of oxygen.
A closer look at the curves A and B (Fig. 17.5) will
illustrate this point further. At tissue pO2  40 torr, hae-
moglobin will be saturated ⬃64% at physiologic pH
(curve A), and ⬃50% at acidic pH (curve B). In lungs the
0 10 20 30 40 50
haemoglobin is 96% saturated with oxygen. Thus, for
pO2 (torr)
curve A, 96  64%  32% of the oxygen bound to hae-
moglobin would be released to the tissues, whereas for Fig. 17.6. Effect of 2,3-BPG on the oxygen-binding affinity
curve B the rate of delivery is 96  50%  46%. Thus, a of haemoglobin (Hb). Oxygen binding by myoglobin (Mb) is
rightward shift of the curve provides a mechanism whereby also shown ( ).
additional oxygen can be supplied to tissues.

 Clinical Significance of 2,3-BPG

Oxygen-binding affinity of haemoglobin is reduced by low BPG has an indispensable physiological function. As
pH. This phenomenon, known as the Bohr effect, is impor- noted, arterial blood, haemoglobin is ⬃96% saturated,
tant for delivering adequate oxygen in metabolizing tissue. and in venous blood (pO2  ⬃40 torr) it is only 64%
saturated, and so unloads 32% (96–64%) of its bound
oxygen in passing through capillaries. In the absence of
Effect of 2,3-bisphosphoglycerate (2,3-BPG) BPG, little of this oxygen would be released, since hae-
This compound is an alternative intermediate in the glyco- moglobin’s oxygen affinity will be increased, thus shift-
lytic pathway, and serves as the third important modulator ing the oxygen dissociation curve significantly towards
of oxygen affinity (others are oxygen and carbon dioxide/ the left (Fig. 17.6). The erythrocyte concentration of BPG
protons). It is formed in the human erythrocytes from is responsive to various physiologic and pathologic con-
1,3-bisphosphoglycerate (1,3-BPG), where it is present at ditions: it increases when there is deprivation of oxygen,
a concentration of approximately 5 mM, roughly equimo- including lung diseases, severe anaemia and during adap-
lar with haemoglobin. Like H and CO2, 2,3-BPG is a nega- tation to high altitude. This enhances unloading of oxygen
tive allosteric effector of oxygen binding to haemoglobin: it causes in the tissues, thereby ensuring adequate oxygenation.
a marked increase in P50, thereby causing unloading of oxy- 2,3-BPG accounts for oxygen-binding peculiarity of
gen from oxyhaemoglobin. Indeed, if it were not for the the fetal haemoglobin, discussed later.
high erythrocyte concentration of 2,3-BPG, the oxygen sat-
uration curve of Hb would approach that of myoglobin. Role in Stored Blood
2,3-BPG plays an important role in blood that has been
Glucose 1,3-BPG 3 Phosphoglycerate
stored for transfusion. The concentration of 2,3-BPG shows
Mutase Phosphatase a drastic fall in the red blood cells from 2.2 mM to one-
2,3-BPG
tenth of this concentrations within a few days of storage.
In hypoxic states, production of 2,3-BPG is increased This causes increased affinity of Hb for oxygen, so that the
which enhances release of oxygen from oxyhaemoglobin transferred blood cannot effectively release O2 to periph-
(Fig. 17.6). eral tissues. This problem is solved by addition of inosine
(ribose  purine base). Haemoglobin releases a significant
Rapoport–Luebering Cycle amount of bound oxygen while passing through capillar-
It is a shunt pathway in RBCs, that bypasses the glycerophos- ies of metabolizing tissues. 2,3-BPG enhances this release
phate kinase reaction (step 6) of glycolysis. As diagrammed to the medium, which is readily transported into red cells
above, the 2,3-BPG is produced from 1,3-BPG, an interme- (2,3-BPG cannot move across cell membrane). Inside the
diate of glycolysis, which is hydrolyzed to 3-phosphoglyc- cell, ribose moiety of inosine is split off from purine base
erate. This is useful not only in oxygen-offloading, but also and the former is metabolized (via a section of the HMP
in dissipation of energy, as described earlier in Box 9.1. shunt and glycolysis) to produce 2,3-BPG.
356 Textbook of Medical Biochemistry

 Mechanism of Positive Cooperativity

Haemoglobin responds to body requirements and alters in Oxygenation
its binding properties accordingly. It can sense changes Binding of oxygen to haemoglobin causes rupture of
in the concentration of any of its four ligands—oxygen, some of the salt linkages, thus favouring the T to R shift.
carbon dioxide, H and 2,3-BPG. The message is trans- Since the binding affinity of the R form for oxygen is
mitted between different subunits through change in the higher than that of the T form, it accepts oxygen mole-
conformation of the molecule. cules readily. Thus, T  R transition explains why bind-
ing of the first oxygen enhances the binding of subsequent
Interactions between different ligands, such as BPG
oxygen molecules to the other haem groups in the hae-
and oxygen, are called heterotropic effects and interac-
moglobin molecule.
tions between identical ligands, as in the cooperativity of
oxygen binding, are called homotropic effects. 
Haemoglobin has two conformational states: the T state
D. Molecular Mechanism of (conformation of deoxyhaemoglobin) and the R state (con-
Allosteric Effects* formation of oxyhaemoglobin). Oxygen binding to a haem
group induces a T to R transition, which in turn induces con-
formational change in the entire haemoglobin molecule.
The allosteric effects depend on transmission of molecu-
lar signal from one subunit of the protein to another. The
mechanisms underlying these effects were elucidated How does oxygen binding cause rupture of salt
following the discovery that the subunits of haemoglo- linkages for T to R transition? Detailed information
bin can take on either one of the two conformations: about the molecular mechanisms responsible for this
the R (relaxed) and T (tense) form. The overall interac- has been elucidated by comparative X-ray analysis. In the
tions between the subunits in the T form are stronger deoxygenated state, the iron atom is about 0.6Å out of
than in the R form, as discussed below. the haem plane, due in part to stearic repulsion between
the proximal histidine and the nitrogen atoms of the
The Tense (T) Form prophyrin ring (Fig. 17.7). Oxygen binding induces cer-
The deoxyhaemoglobin conformation is called the tense (T) tain changes in the haem’s electronic state, which short-
or taut form, in which the subunits are held together by eight ens the Fe–porphyrin bonds by ⬃0.1 Å. Consequently,
salt bonds between polypeptides in addition to a number the iron atom moves into the centre of the haem plane,
of hydrogen bonds and other non-covalent interactions. where it can more tightly bind oxygen, and in doing so it
drags the covalently linked proximal histidine (F8 His)
The Relaxed (R) Form and its attached F helix. A subtle conformational change
It is a less constrained form produced by tearing away of is thereby induced, which strains other non-covalent
some of the salt linkages that stabilize the T form. Binding bonds elsewhere in the subunit causing some of them to
of oxygen to the T form causes rotation of an  pair rela- break. This is beginning of a series of reactions leading to
tive to the other  pair by 15 degrees. This brings about the quaternary structure transition from the deoxy-T
rupture of some of the salt linkages to produce the R form. form to oxy-R form.
The binding affinity of the R form for oxygen is 150 – 300 In defining transition from T to R form, a number of
times greater than that of the T form. Hence, the R form has models have been developed (Box 17.2).
greater tendency for getting oxygenated. Thus, any alloste-
ric modulator that favours formation of the R form (i.e. T to Mechanism of Bohr Effect
R shift) favours oxygen binding. Conversely, the allosteric The principal residues involved in Bohr effect are the
modulators favouring R to T shift increase oxygen liberation. N-terminal amino groups of the -chains, the C-terminal
O2 carboxy groups of -chains and the imidazole side chains
T R of His122. They interact with protons to acquire more
positive charge. The protonated, positively charged resi-
O2 dues then form electrostatic interactions with nega-
With this information, it is now possible to understand tively charged residues, thus converting the R forms into
the mechanism of oxygenation, the Bohr effect and to T form and thereby releasing oxygen.
appreciate role of protons at molecular level.
*This topic relates to higher level learning and it is meant only for post- H+
graduates. Undergraduates or MBBS students may refer to it for higher
learning. Oxy-R state Deoxy-T state O2-release
 Oxygen Transporters: Haemoglobin and Myoglobin 357

N
C N
C
CH Proximal
histidine HC CH
HC
N
N
Stearic
repulsion

Fe +O2
Plane of haem Fe

Fig. 17.7. Movement of iron atom into the plane of the haem upon oxygenation.

BOX 17.2
Sequential and Symmetrical Models
The sequential (Aldair-Koshland) model; proposed that as dexoyhaemoglobin (all T forms) bound the first molecule of
oxygen and changed its conformation to R form; the conformational change would exert an influence on a neighbouring
subunit to change its shape to the R form. The latter would then pick up O2 more easily than the first subunit that. The
sequential model thus proposed that each subunit sequentially responds to oxygen binding with a conformational change,
so that partially oxygenated haemoglobin molecules would consist of hybrids of the R and T forms.
The symmetrical (Monod) model on the other hand proposed that any given haemoglobin molecule was in either all
R or all T conformations and that all subunits switched together (concertedly, not one by one) as increasing quantities of
oxygen got bound to haemoglobin. Hybrid states are forbidden in this model and oxygen binding causes concerted tran-
sition between T and R forms. The majority of evidence, however, points toward the sequential model.

For every equivalent of oxygen released from haemo- to release its bound oxygen. The H produced in the
globin, about 0.3 equivalents of [H] is bound. The above reaction further promotes oxygen release through
bound H are called Bohr protons. They are released in the Bohr effect. Clearly, effects of Hⴙ and CO2 are often
the lungs, where pO2 is high and pCO2 is low, and the tightly associated, both promoting oxygen release from
T form changes back to R form. oxyhaemoglobin.
It may be re-emphasized that like the pH effect, the CO2
 effect ensures that oxygen is released in actively metabolizing
Binding of Bohr protons to certain amino acid residues
tissues, where it is most needed. On return to the lungs,
favours formation of new bonds, thus causing R  T tran-
blood is exposed to low pCO2 and by law of mass action
sition and off-loading of oxygen from oxy-haemoglobin.
the carbamination reaction is reversed and T to R conver-
sion occurs, therefore binding of oxygen is again favoured.
Effect of CO2: Lowering of oxygen-binding affinity of
haemoglobin by carbon dioxide is effected by the spon- High CO2
taneous covalent binding of CO2 with the N-terminal (in tissues)
Oxy-R state Deoxy-T state + O2
group (valine) of each of the globin polypeptides to give
carbamino complexes.
Low CO2 (lungs)
O2
R – NH2  CO2  RNHCOO  H
Deoxyhaemoglobin because of its higher pI (7.6) has
a greater affinity for CO2 than oxyhaemoglobin (pI  6.7). Mechanism of Oxygen Off-loading by BPG
When the CO2 concentration is high, as it is in the capillar- The oxygen unloading effect of 2,3-BPG is accounted
ies, the deoxy-T state is favoured, stimulating haemoglobin by the fact that it binds tightly to the deoxy-T state of
358 Textbook of Medical Biochemistry

haemoglobin but only weakly to the oxy-R state. The result Since the -chain differs from -chain in 39 of the
is stabilization of the T conformation, which has low residues, the physicochemical properties of the HbF iso-
oxygen-binding affinity. Moreover, the presence of 2,3-BPG form are different: its electrophoretic mobility is slower and
also lowers intracellular pH (6.95). This dual role leads the deoxy HbF has higher solubility than HbA. The most
to the observed effect of oxygen unloading. How does 2,3- striking difference between the two is: HbF has lower
BPG stabilize the deoxy-T state? The X-ray structure of a affinity for 2,3-BPG (because the residue 143 of the
BPG-deoxyhaemoglobin complex shows that BPG binds -chain, which is histidine in HbA but is replaced by ser-
in the central cavity formed among the four subunits of Hb. ine in HbF; the absence of this histidine eliminates a pair
The binding site for BPG is created at one end of this cav- of interactions that stabilize the BPG-deoxyhaemoglobin
ity by multiple positive charges, contributed by several complex). As a result, affinity of HbF for oxygen (P50  19
residues, e.g. 2nd histidine, 143rd histidine, 82nd lysine torr, vs 26 torr for HbA) is much higher and formation of
in -chain. BPG interacts electrostatically with these and the oxygenated R form is favoured. The oxygen dissocia-
the interaction in turn stabilizes the complex between tion curve in fetus consequently is shifted to left, a direct
the effector (BPG) and the Hb in its deoxy-T state. benefit of which is that there is a more efficient transpla-
cental transfer of oxygen from maternal HbA to fetal HbF.
HbF is elevated up to 15–20% in individuals with
 mutant adult HbS, such as sickle cell disease where it has
BPG decreases haemoglobin’s oxygen affinity by bind-
a compensatory effect. Children with anaemia and
ing to the deoxy-T state, and stabilizing it.
-thalassaemia also show such compensatory response.

B. Embryonic Haemoglobins
II. Haemoglobin Variants
The non--chains present in embryonic haemoglobins
are the embryonic chains, i.e. epsilon ( ) and zeta ( ).
A. Fetal Haemoglobin (HbF) Expression of these chains is limited to early embryonic
stage, from 3rd to 8th week of gestation (Fig. 17.8). The
Fetal haemoglobin differs from the adult haemoglobin
embryonic haemoglobin Gower-2 has two alpha and two
in having -polypeptide chain, a variant of the -chain
epsilon chains, and the haemoglobin Gower-1 comprises
(subunit composition 22; see Fig. 17.2). While it accounts
only embryonic chains.
for less than 1% total adult haemoglobin in adults, HbF
predominates in fetus during the second and third tri-
mesters of gestation. Its synthesis starts by seventh week of C. Minor Adult Haemoglobin (HbA2)
gestation and at birth it accounts for about 75% haemo-
globin. There is rapid post-natal decline and within It is a normal variant of adult haemoglobin, having -chains
4 months after birth HbF is almost completely replaced instead of  (subunit composition 22; Fig. 17.2). In -
by HbA (Fig. 17.8). thalassaemia, concentration of this form of haemoglobin

Major site of
erythropoiesis Yolk sac Liver, spleen Bone marrow
50
a g
% of total globin synthesis

40

30

20
x b
10
e
0
0 2 4 6 8 2 4 6 8
Birth Age (months)
Pre-conceptual age Post-natal age
(HbF) (α2γ2) (HbA) (α2β2)

Fig. 17.8. Human haemoglobin polypeptide chains as a function of time during gestation and post-natal development.
 Oxygen Transporters: Haemoglobin and Myoglobin 359

is increased as a compensatory measure. Its pI value is Hb-Fe3+ Cyt-b5 Fe2+ NAD+

7.4, which is higher than that of HbA. Therefore, it shows
slower anodic mobility on electrophoresis.

Hb-Fe2+ Cyt-b5 Fe3+ NADH + H+

III. Haemoglobin Derivatives
Fig. 17.9. The methaemoglobin reductase system.
Haemoglobin derivatives are formed by:
 Change in oxidation state of haemoglobin, e.g. meth-  The methaemoglobin that is spontaneously pro-
aemoglobin, a non-functional oxidized form of haemo- duced can be reduced back to haemoglobin by the
globin. methaemoglobin reductase system present within the
 Combination of different ligands with the haem part RBCs (Fig. 17.9).
of the haemoglobin molecule. For example, carboxy This enzyme system uses NADH as the reductant.
haemoglobin is formed by binding of carbon monox- Cytochrome b5 and cytochrome b5 reductase are the
ide to the haem iron; sulph-Hb, by its binding with other essential components of this system. Impaired
hydrogen sulphide. function of this system leads to congenital methaemo-
globinaemia (Case 17.1).
Haemoglobin derivatives can be detected by their  The polypeptide chains of haemoglobin contain a
characteristic absorption spectra. protected pocket each between the E and the F heli-
ces, in which the haem molecule is tucked in. These
A. Methaemoglobin pockets offer protection against oxidation of haem.
This is evident from the fact that when the haem mol-
The haem iron of haemoglobin is present in the ferrous ecule is dissociated from the polypeptide chain, it
state. Its oxidation to the ferric form produces haemin rapidly gets oxidized to haemin.
from haem, and the haemoglobin molecule is now called The distal histidine residues appear to play an impor-
methaemoglobin. Methaemoglobin is ineffective as an tant role of creating a protective environment for haem.
oxygen transporter. Replacement of this histidine by a tyrosine residue,
Normally, about 1% of the circulating haemoglobin for example, favours production of methaemoglobin.
occurs in form of methaemoglobin. Certain chemicals Defect in the cytochrome b5 reductase system, or struc-
cause rapid ferrous to ferric conversion, resulting in pro- tured abnormality in the polypeptide chain(s), result in
duction of methaemoglobin. Examples include organic congential methaemoglobinaemia. It is a mild condition
and inorganic nitrites, aniline dyes, and aromatic nitro com- and does not manifest clinically under normal circum-
pounds. The methaemoglobin production by these agents stances. However, use of certain drugs, as mentioned ear-
leads to conditions, collectively referred to as acquired lier, results in excessive production of methaemoglobin
methaemoglobinaemias. In contrast to the acquired and the oxygen deficiency symptoms appear.
forms, the congenital methaemoglobinaemia results Treatment of methaemoglobinaemia involves conver-
mostly because of defect in the cytochrome b5 reductase sion of the ferric iron back to the ferrous state. Methylene
system, as discussed below. blue is commonly used for this purpose.

Methaemoglobin is a non-functional oxidized form of B. Carboxy Haemoglobin
haemoglobin formed by oxidizing agents that oxidize
the ferrous haem iron to the ferric state.
The toxic effects of carbon monoxide (CO) were
explained first by John Scott Haldane on the basis of
Since the methaemoglobin formation results in loss of oxy-
competition between carbon monoxide and oxygen for
gen carrying capacity, it is hazardous and must be prevented.
binding the haem iron. In fact, the affinity of carbon
Erythrocytes have certain defensive mechanisms to pre-
monoxide for Hb is 200 times more than that of oxygen.
vent excessive formation of methaemoglobin. They are
described below. Hb  CO  CO.Hb (Carboxy haemoglobin)
 Ascorbic acid and glutathione are reducing substances Since CO.Hb is unsuitable for oxygen transport, its
which destroy many oxidizing radicals, thus prevent- binding renders the haemoglobin functionally inactive
ing them to react with haemoglobin (Chapter 27). (Case 17.2).
360 Textbook of Medical Biochemistry

plus sodium thiosulphate, which converts haemoglobin


Haemoglobin can be poisoned by carbon monoxide to met-Hb. The latter then traps cyanide as cyanmet-Hb,
that occupies the oxygen-binding site on the haem iron. which is a non-toxic compound.

IV. Haemoglobinopathies
C. Glycated Haemoglobins
Haemoglobinopathies are a family of disorders resulting
These are the haemoglobin derivatives that are produced
due to mutations in genes for the haemoglobin chains.
by non-enzymatic post-translational modification (e.g.
The mutation may cause synthesis of defective globin
glycation) of the -chains. They make up about 4–6% of
chain or may even stop the synthesis altogether. More
the total haemoglobin in normal red blood cells. Being
than 400 different mutations have been described, which
present in very small amounts, the glycated haemoglo-
result in as many structural variants of the haemoglobin
bins are not pathological, but rather serve a useful pur-
polypeptide chains. Two types of defects arise due to
pose of monitoring patient’s compliance in diabetic state
these mutations: the qualitative defects and the quanti-
or even in diagnosis of diabetes (Chapter 15).
tative defects.
Qualitative defects lead to production of structurally
D. Sulfhaemoglobin abnormal haemoglobin molecules. These defects involve struc-
tural alteration in the polypeptide chain of the haemo-
It is a greenish compound produced by covalent attach- globin, leading to functional impairment. Replacement,
ment of sulphur to the porphyrin ring (not iron atom). addition and deletion of the amino acids are some com-
Sulfhaemoglobin cannot combine with oxygen. Exposure mon structural alterations.
to sulphur containing compounds, either occupationally A number of qualitative defects (over 300) have been
or from air pollution, or depsone (used to treat leprosy) identified, which may affect either of the two types of
can produce this compound. In poisoning by phenace- chains. For example, in sickle cell anaemia, the -chain
tin, acetanilide or sulfanilamides, sulfhaemoglobin is is affected, whereas in HbM Boston the -chain under-
produced. These drugs produce methaemoglobin also, goes structural alteration (Table 17.2).
so that sulfhaemoglobin and methaemoglobin appear Quantitative defects result in synthesis of the normal
together in these patients. haemoglobin molecules, but in insufficient quantities. Prime
example of this type is the thalassaemias in which either
the - or -chains are underproduced. Rarely both these
E. Cyanmethaemoglobin types of defects occur together in a single disorder.
A partial listing of haemoglobinopathies is given in
Haemoglobin cannot combine with cyanide, but methae- Table 17.2. By convention, the haemoglobinopathies are
moglobin reacts with cyanide forming cyanmethaemoglobin usually named with a capital letter (e.g. haemoglobin S),
(cyanmet-Hb). This property is used as a treatment modal- a geographic location (e.g. haemoglobin Seattle), or both
ity in cyanide poisoning. First, the patient is given nitrite (e.g. haemoglobin M Boston or D Punjab).

Table 17.2. Some mutated human haemoglobins

Mutated haemoglobin Affected chain Residue Substitution Notes
S  6 Glu  Val Decreased solubility of Hb. Sickling of RBCs.
C  6 Glu  Lys Decreased solubility. Sickling.
E  26 Glu  Lys
Zurich  63 His  Arg Affinity of Hb for O2 increased. Solubility decreased.
Seattle  70 Ala  Asp
Hiroshima  146 His  Asp High O2 affinity. Salt bridges stabilizing T form impossible to make.
Kansas  102 Asn  Thr Low oxygen affinity. Cyanosis common.
D Punjab  121 Glu  Gln Migrates similar to HbS on electrophoresis. Severe condition.
M Boston  58 His  Try Substitution in proximal and distal histidine results in increased
tendency of haem to get oxidized to haemin. Cyanosis common.
 Oxygen Transporters: Haemoglobin and Myoglobin 361

Diagnostic Analysis of Normal and arise from a specific amino acid change in a protein. Since
Mutant Haemoglobins then it has been so extensively studied, biochemically and
All significant Hb variants and many of the more com- biophysically, that it has virtually become a paradigm of
mon mutants, including HbS and HbC, can be separated a molecular disease.
by electrophoresis. The process is carried out at pH 8.6
at which these proteins are negatively charged, and there- Molecular Defect
fore, move towards anode. The sickle cell haemoglobin (HbS) arises out of a substi-
Change in amino acid composition of a globin chain tution of the glutamic acid residue in position 6 of the
may produce a haemoglobin with a different electropho- -chain with the valine residue (6Glu  Val). As noted
retic mobility. By examining the position and relative earlier, this substitution can be traced to an A  T trans-
amount of the haemoglobin bands, it is possible to diag- version in the -chain gene and results in formation of
nose the most common haemoglobinopathies. For exam- an abnormal haemoglobin of subunit composition 2s2
ple, in sickle cell anaemia, the predominant haemoglobin (Fig. 17.11). Both, homozygous and heterozygous forms
(85–95% of the total) is haemoglobin S (HbS); its posi- of disease have been described. The heterozygous form is
tion on electrophoretogram is shown in Figure 17.10. also referred to as sickle cell trait.
The HbS has lower anodic mobility because of removal of a
negative charge by the sickle cell mutation (Glu  Val). 
Likewise, any mutant exhibiting a net gain or loss of Sickle cell anaemia results because of a point mutation
charged residues when compared with HbA can be (A  T transversion) in the -chain gene. It leads to a
detected by this method. Glu  Val substitution in the -chain. The resultant hae-
The volume of blood sample required is less than moglobin S (HbS) is characterized by the subunit struc-
100 l, making this technique acceptable for even neo- ture 2s2.
nates. Quantification is performed by scanning densitome-
try. Another method, isoelectric focusing, also based on
differences in pI, separates the proteins rapidly in a pH
Sticky Patches and Tubular Polymer Formation
Replacement of a negatively charged glutamic acid with a
gradient generated by an electric field (see Chapter 4).
hydrophobic valine changes surface properties of the
molecule, especially its solubility. It becomes less soluble
A. Sickle Cell Anaemia in water in its deoxygenated form, although the oxygen-
binding affinity remains unaffected. In a chain compris-
It is the most commonly occurring haemoglobinopathy ing of 146 amino acids, a single substitution may appear
caused by an inherited structural abnormality in the  minor, but its effects are far from trivial. This is because
globin polypeptide. Originally described in 1910 by James the substituted valine forms a hydrophobic pocket, the
Herrick, the disease was the first inherited disorder shown to so called sticky patch, on the surface of the molecule
which is reactive to the nearby Hb molecules.
The X-ray structure of deoxyhaemoglobin S has
− + revealed that one mutant valine side chain in each HbS mol-
ecule fits into a sticky patch on the surface of another haemo-
S F A H
globin molecule. This intermolecular contact allows HbS
Fig. 17.10. The electrophoresis of haemoglobin. to form linear polymers of deoxy HbS. These polymers

Haemoglobins −S − Barth’s −H

α βs γ γ β β

βs α γ γ β β

α2βs γ4 β4

Polypeptide chain
Haem

Fig. 17.11. Structure and subunit composition of different haemoglobins: HbS, HbH and Hb Barth’s. HbS is the predominant
haemoglobin type in sickle cell anaemia, whereas HbH and Hb Barth’s occur in thalassaemia.
362 Textbook of Medical Biochemistry

distort the RBCs into sickle shape, which in highly vul- producing any clinical symptoms and compatible with a
nerable to lysis. normal life span. The red cells of these patients contain
The hydrophobic pocket on surface of the -subunit is approximately 70% HbA and 30% HbS (Case 17.3).
not seen in case of oxyhaemoglobin, therefore oxyhae-
moglobin S cannot polymerize. This also explains why Diagnosis
danger of polymerization is greatest in hypoxic conditions. Sickling test: A blood smear is exposed to low oxygen
tension by adding a reducing agent, e.g. sodium dithio-
 nite. The red cells respond by changing to sickle shape.
HbS has a normal oxygen binding affinity, but it has This is detected microscopically.
reduced water solubility in deoxygenated form. Deoxy- Electrophoresis: Electrophoresis remains the mainstay of
genated but not oxygenated HbS forms an insoluble diagnosis of sickle cell anaemia. Electrophoretic separa-
fibrous precipitate in the erythrocytes. tion is possible because the sickle cell mutation (Glu 
Val) removes a negative charge from the -chain result-
Clinical Features ing in a decreased anodal mobility (Fig. 17.10).
Sickle cell anaemia is a severe-haemolytic, painful, debili- Solubility test: A sample of haemolyzed red cells is
tating, and often fatal disease characterized by erythrocytes exposed to anoxic conditions by adding a reducing agent.
of distorted shapes. The stiff polymers form precipitate An opalescence is observed, which indicates presence of
in the erythrocyte, which causes distortion of shape of less soluble deoxy-HbS.
the RBCs into sickle shape (i.e. sickling). At first the sick-
ling is reversible, but after some time the oxygenation– Treatment
deoxygenation cycle permanently deforms the shape of 1. Initial management aims at avoiding hypoxia and
the erythrocyte. The deformed cells increase the viscosity dehydration and administration of cyanate, which
of blood and cause obstruction to blood flow in small increases the oxygen affinity by covalent modification
vessels and capillaries. The resulting vascular occlusion of the amino termini of the globin polypeptides.
may result in tissue death (infarction). Painful bone and 2. An alternative approach, which involves manipulations
joint infarctions are common; multiple renal infarcts can to increase the synthesis of fetal haemoglobin, has been
lead to renal failure; and many patients are crippled by found useful. Hydroxyurea has been found to increase
recurrent CNS infarctions. the fraction of cells containing fetal haemoglobin,
Recurrent attacks of sever pain in extremities, bones or although the mechanism whereby it acts is not known.
abdomen, known as painful crisis or sickling crisis are Fetal Hb is useful because of its high O2 affinity.
experienced by most patients. Others may experience 3. Repeated blood transfusions are required in severe
aplastic crisis (bone marrow failure), or sequestration cri- anaemic cases.
sis where sudden trapping of erythrocytes in the enlarged
spleen occurs; the latter is associated with high mortality. Protection from Tropical Malaria
Renal failure, cerebrovascular accidents, cardiac failure HbS gene occurs in India, Mediterranean region and
and liver diseases are the other important causes of death. Saudi Arabia. In some parts of Africa, up to 40 per cent of
the population carries the sickle gene. These areas have
 highest prevalence of malaria as well. Presence of the sickle
Haemoglobin S forms rigid fibres in the deoxy form gene has a survival value since the native Africans with
within the erythrocyte and distorts its shape into sickle sickle cell genes show a much lower susceptibility to
shape. The patient suffers from recurrent episodes of malaria. The mosquito borne parasite that invades the red
haemolytic and painful vaso-occlusive crisis. blood cells finds less favorable conditions for its growth
in the sickled cell compared to normal cells. Moreover,
Interestingly, the disease follows a milder course in increased lysis cause premature destruction of the parasite.
many HbS homozygotes because they express relatively
high levels of fetal haemoglobin, which contains -instead 
of defective -chain. Possession of the sickle gene confers significant survival
People with sickle cell trait (heterozygous form with about value in the areas where malaria is endemic and is fatal
40% HbS) also form insoluble fibres, though at a lower in childhood.
rate. They are asymptomatic in normal circumstances.
Their cells exhibit sickling only when pO2 is low, such as HbC Disease
at high altitudes or in chronic lung disorders. Therefore, The HbC is a haemoglobin with a 6Glu  Lys substitu-
sickle cell trait is a relatively benign condition as such not tion. Like HbS it is found in black population and is
 Oxygen Transporters: Haemoglobin and Myoglobin 363

related to sickle cell anaemia. Its symptoms, however, are In thalassaemia minor, anaemia may be present but is
less severe. Presence of HbC also appears to create a less very mild. In thalassaemia major, severe anaemia develops.
hospitable environment for the malaria, perhaps even
causing premature destruction of the parasitized cells. 
The thalassaemias are caused by underproduction of
 either the - or the -chain. The -thalassaemia is
It is now believed that HbS and HbC arose as a means caused most often by large deletions, and many differ-
of combating malaria. ent mutations can cause -thalassaemias.

B. Thalassaemias ␣-Thalassaemias
Genes for the -chains are clustered on chromosome 16.
Thalassaemias are a group of disorders characterized by Genome of an individual contains four copies (two cop-
insufficient production of structurally normal ␣- or ies from each parent) of the -globin genes; two copies
␤-chains. High prevalence of thalassaemias has been are located on each of the chromosome 16. Most patients
reported among the people from the regions around the with -thalassaemia have large deletions that remove one or
Mediterranean Sea (thalassa is the Greek word for sea). both of the -chain genes from a chromosome. Figure
Insufficient production of one of the globin chains of 17.12 shows the possible deletion types. Four types of
haemoglobin results in lack of coordination between the -thalassaemias corresponding to deletion of one, two,
synthesis of the - and the -chains. Normally, the sub- three, or all four of the -chain genes occur:
unit synthesis is coordinated in such a way that each
1. Silent carrier state (Fig. 17.12a) is due to deletion of one
newly synthesized -chain readily pairs with an -chain;
gene. It is a haematologically normal state; no clinical
and conversely, each newly synthesized -chain pairs
manifestations are observed in this condition.
with a -chain. The lack of coordination results in:
2. a-Thalassaemia trait involves deletion of two genes
 Formation of insoluble aggregates by the polypeptide from the same or different chromosomes (Fig. 17.12b
chains. The aggregaetes are damaging to the cell and and c). This results in mild physical manifestations,
reduce its lifespan. such as mild anaemia and slightly enlarged spleen.
 Impairment of the haemoglobin synthesis, so that the 3. Haemoglobin H disease involves deletion of three genes
erythrocytes are small and poorly filled with haemoglobin. (Fig. 17.12d). The condition is characterized by mild
to moderate haemolytic anaemia.
Molecular Defect 4. Homozygous a-thalassaemia (Fig. 17.12e) with four
Underlying cause of the underproduction of a globin deletions is always lethal, either in utero or at birth.
chain is a gene mutation. A number of mutations have The condition is also called hydrops foetalis or hae-
been reported, including gene deletion, gene substitution, moglobin Barth’s disease (Box 17.3).
or deletions of one to several nucleotides in the DNA.
Chromosome 16
Some mutations lead to: (a) (b) (c)
 Underproduction, abnormal processing and prema-
α1
ture degradation of mRNA, or
 Increased proteolytic degradation of the - or the
-chain. α2

Types of Thalassaemias
Depending on which chain is affected, the thalassaemias (d) (e)
are divided into two major classes: - and -thalassaemias.
In -thalassaemia relative or absolute deficiency of the
-chain occurs, whereas in -thalassaemia the -chains
are affected. Heterozygous forms of - or -thalassaemia are
called thalassaemia minor, and the homozygous forms are
termed thalassaemia major.
α Major-homozygous
Thalassaemias
Fig. 17.12. One or more genes for the -chains are deleted in
Minor-heterozygous
β Major-homozygous -thalassaemias. (a) Silent carrier, (b and c), -Thalassaemia trait,
Minor-heterozygous (d) Haemoglobin H disease, (e) Haemoglobin Barth’s disease.
364 Textbook of Medical Biochemistry

BOX 17.3
Types of Haemoglobins in Thalassaemic Patient
Though synthesis of the -chain is hampered in -thalassaemias, the -chain and the -chain synthesis proceeds as usual.
As a result, there is accumulation of certain abnormal proteins (Fig. 17.11). These are:
(i) -Tetramer, which is termed HbH. It is the predominant haemoglobin in patients with deletion of three -chain genes.
This aberrant haemoglobin is unstable; it gradually denatures to form inclusion bodies in the cells.
(ii) -Tetramer, termed Hb Barth’s, have tenfold higher oxygen affinity than haemoglobin A and, therefore, cannot deliver
the bound oxygen in tissues. -Tetramers cannot serve as effective oxygen carriers. Thus, tissue hypoxia is the major con-
sequence, and oxygen deficiency symptoms rapidly appear.

-Thalassaemias deformities (“chipmuk facies”). Extramedullary erythro-

Synthesis of b-chains decreases or stops altogether in the poiesis is seen in liver and spleen in some patients.
b-thalassaemias. In contrast to large deletions in -
thalassaemia, most patients with -thalassaemia have 
single base substitutions. More than 90 such substitu- Some of -thalassaemia mutations result in a complete
tions are known. Few cases of -thalassaemia are caused absence of the -chains in the homozygous state (°-
by large deletions. thalassaemia). Others cause a decrease in the -chain
Normally, there are only two genes responsible for the synthesis (-thalassaemia).
-globin chains, one from each parent. Each copy of
chromosome 11 has one gene for -globin chain. The muta- Treatment
tion in -thalassaemia may involve a single chromosome Untreated, the patients with thalassaemia major suffer
(i.e. the heterozygous state, called ␤-thalassaemia minor) recurrent infections and severe anaemia. With regular blood
or it is a homozygous state, involving both the chromo- transfusions, life expectancy is approximately 20 years of
somes (called ␤-thalassaemia major). age. Although blood transfusion is life saving, the cumu-
lative effect of the procedure is iron overload, a syndrome
1. b-Thalassaemia minor is benign and require any treat-
known as haemosiderosis. It has a high mortality, usually
ment. There is almost 50% decrease in -chain syn-
between the ages of 15 and 25 years. For this reason, the
thesis. A compensatory increase in production of the
patients are treated not only with regular blood transfu-
- and the -chains occurs. These chains combine
sions but also with desferrioxamine, an iron chelator that
with the -chains. Thus an increase in the amount of
is best administered through a subcutaneous infusion
haemoglobin A2(22) and F(22) occurs, which is a
pump. Desferrioxamine forms a soluble iron complex
distinguishing feature of -thalassaemias.
that can be excreted by the kidneys. Bone marrow replace-
-Thalassaemia minor is mostly asymptomatic and
ment therapy has evolved as a highly successful mode of
life expectancy is also normal since some amount of
treatment since the last decade or so. It has improved life
-chains are normally synthesized by these individuals.
expectancy significantly.
2. b-Thalassaemia major is the homozygous state, which is
most severe among all congenital haemolytic anaemias.
It is also called Cooley’s anaemia. The affected individ-
uals require regular blood transfusions for survival. V. Myoglobin
Clinical features of -thalassaemia major: Since the -chain Myoglobin is a relatively small molecule (MW 167,00),
begins to be synthesized at a later stage in fetal gestation, consisting of a single polypeptide chain, attached non-
the physical manifestations of -thalassaemia become covalently to a haem molecule (Fig. 17.13). It is mainly
apparent only after birth. The affected infants are normal located in cardiac and skeletal muscles, where it serves as
at the time of birth because of abundance of fetal hae- a reservoir for oxygen. In addition, it transports oxygen
moglobin, but a severe anaemia develops during the first to the mitochondria. The haem group is responsible for the
year of life and the haemoglobin level in most infants deep brown colour of myoglobin (and of haemoglobin as
falls below 5 gm/dL. The marrow responds to anaemia well). Myoglobin is particularly abundant in muscles of
and becomes over-active. Consequently, a massive expan- diving mammals such as whale and seal, whose muscles
sion of the red bone marrow occurs, leading to facial are so rich in myoglobin that they are brown coloured.
 Oxygen Transporters: Haemoglobin and Myoglobin 365

Haem group
100

% Saturation
50

0
10 20 30 40 50
pO2 (torr)
Myoglobin
Fig. 17.14. Oxygen dissociation curve of myoglobin.
Fig. 17.13. Structure of myoglobin.
to myoglobin is directed by mass action of oxygen. When
Storage of oxygen by muscle myoglobin permits these oxygen is plentiful, formation of oxygenated myoglobin
animals to remain submerged in water for long periods. occurs and when oxygen is very scarce, dissociation of
the oxygenated myoglobin occurs.
The oxygen dissociation curve of myoglobin is a sim-
A. Basic Structure ple hyperbolic curve (Fig. 17.14) in contrast to the sigmoid
shape of the oxygen dissociation curve of haemoglobin.
The single polypeptide chain of myoglobin has 153 amino
The curve is a molecular adaptation for its storage function.
acid residues. About 75% of the chain is in a -helical
conformation. There are eight major -helical segments,  High affinity of myoglobin for oxygen permits myo-
referred to as A, B, C, … H. Four of the these segments are globin to store oxygen even at the relatively lower par-
terminated by proline residues, whose rigid R group does tial pressure (40 torr) that exists in resting muscles.
not fit within the straight stretch of -helix. The non-  In exercising muscles, where the oxygen partial pres-
helical segments lie in between these helical portions. sure falls to about 20 torr, myoglobin is still over 95%
The polypeptide chain is so compactly folded that saturated. It unloads very little oxygen which makes it
there is no space in its interior. Most of the hydrophobic suitable for its role as a reservoir of oxygen.
R groups are in the interior of the molecule, hidden from  It is only during severe physical exercise—when pO2
water. They form a clustered structure stabilized by within the muscles falls to a low level ( 5 torr)—that
hydrophobic interactions. In contrast, the charged amino myoglobin releases a significant proportion of its
acids are located almost exclusively towards the exterior stored oxygen.
where they form hydrogen bonds with the surrounding
The myoglobin has high oxygen affinity, but does not
aqueous medium.
show Bohr effect, cooperative effect and 2,3-BPG effect.
 
The compact structure of myoglobin is stabilized by Myoglobin is a monomeric haem-containing muscle protein
hydrogen and ionic bonds as well as by the hydrophobic that reversibly binds a single oxygen molecule. Unlike hae-
interactions between the hydrophobic R groups. moglobin it lacks allosteric properties, but has far greater
affinity for oxygen.
The polypeptide chain of myoglobin is structurally
similar to the individual polypeptide chains of the hae- Properties of haem are different in haemoglobin, myo-
moglobin molecule. Thus, myoglobin provides a simpler globin and other haem-containing proteins (Box 17.4).
model for the study of complex oxygen-binding proper-
ties of haemoglobin.
VI. Anaemias
B. Oxygen-binding Characteristics of In our country, anaemia is one of the most common
Myoglobin medical problem. In this condition, the haemoglobin
concentration is reduced (normal is 13–16 g/dL in males;
Myoglobin has a very high affinity for oxygen; it is 50% and 12–15 g/dL in females).
saturated with oxygen at a partial pressure of just 1–2 torr. The commonest cause of anaemia in India is iron defi-
At about 20 torr, it is over 95% saturated. Binding of oxygen ciency. For clinical manifestation of the iron deficiency
366 Textbook of Medical Biochemistry

BOX 17.4
Haem Proteins
Haem proteins are a group of specialized conjugated proteins that contain haem as their prosthetic group. Most abundant
haem proteins in humans are haemoglobin and myoglobin. Cytochromes and catalase are other examples. Haem is tightly
bound to the apoprotein portion and performs different functions in different haem proteins. For example, haem group of
myoglobin and haemoglobin reversibly bind oxygen; the haem group of the cytochromes functions as an electron carrier;
whereas, that of the enzyme, catalase, forms part of the enzyme’s active site that catalyzes breakdown of hydrogen peroxide.

anaemia (see Case 19.1). The causes of anaemia are (i.e. heavy periods), bleeding peptic ulcer, or hookworm
given below: infection.

Decreased production of erythrocytes: Defective synthe-

sis of haemoglobin may lead to decreased production of Exercises
erythrocytes. A number of cofactors and mineral ele-
ments are required for this process, including copper, Essay type questions
iron, ascorbic acid, pyridoxal phosphate, and folic acid. 1. Draw a diagram of the sigmoidal- and hyperbolic-
Nutritional deficiency of these factors therefore results in oxygen dissociation curves of haemoglobin and
anaemia. myoglobin respectively. Describe how haemoglobin
effectively delivers oxygen to myoglobin in muscles.
Enhanced destruction of erythrocytes: Under normal cir- 2. Explain the structural and functional differences
cumstances a balance exists between the production and between haemoglobin and myoglobin.
the destruction of erythrocytes. Integrity of the erythro- 3. Explain the structural basis for cooperative oxygen
cyte structure is affected in a number of disorders, both binding to haemoglobin. What is the physiological
intracorpuscular and extracorpuscular. These disorders relevance of the Bohr effect?
result in excessive destruction of the cells (haemolysis), 4. How do BPG, carbon dioxide and pH value affect
resulting in (haemolytic) anaemia. Some of the common oxygen binding to haemoglobin?
conditions that lead to haemolytic anaemia are:
(a) Intracorpuscular defects are haemoglobinopathies Write short notes on
caused by enzyme deficiencies such as deficiency of glucose- 1. Thalassaemias
6-phosphate dehydrogenase or pyruvate kinase (Case 10.1). 2. Haemoglobin electrophoresis
(b) Extracorpuscular defects such as infections (malaria 3. Methaemoglobinaemias
or coxsackie virus infection), drug exposure (quinine or 4. Haemoglobin S
methyl dopa), autoimmune haemolysis or mismatched 5. CO poisoning
blood transfusion. 6. Bohr effect
7. Haemolysis in G6PD deficiency
Haemorrhage: Chronic blood loss may lead to anaemia. 8. Fetal haemoglobin
The commonest causes are haemorrhoides, menorrhagia 9. Sickle cell trait
 Oxygen Transporters: Haemoglobin and Myoglobin 367

CLINICAL CASES
CASE 17.1 Shortness of breath and cyanosis in a 1-year-old child
A 1-year-old had frequent episodes of headache, exer- Defective (or diminished) action of some enzyme protein
tional dyspnoea and cyanosis. The venous blood sample, was suspected and further investigations were conducted for
drawn for biochemical investigations, was chocolate-brown measuring intracellular levels of some major proteins of eryth-
in color but turned red on shaking. Addition of a few drops rocytes. When antibodies to the enzyme cytochrome b5 reduc-
of 10 per cent potassium cyanide to the blood resulted in tase were added, the precipitation was less than that expected
rapid production of a bright red colour. A number of bio- in a normal subject. It was thereby concluded that the level of
chemical tests were performed as an initial screen; no this enzyme protein within the erythrocytes was low.
abnormality was, however, detected.
Physical examination ruled out any cardiac or pulmo- Q.1. What is the probable diagnosis of this case?
nary disease and there was no history of any recent expo- Q.2. How do altered properties of the cytochrome b5
sure to any drugs or chemicals. Oxygen therapy was reductase account for the symptoms of this patient?
started but the cyanosis was not as promptly alleviated
with it, as would be expected in a normal subject.

CASE 17.2 Oxygen insufficiency following inhalation of automobile exhaust

A 48-year-old car mechanic was brought to the hospital Q.1. What is the rationale behind arriving at the above
emergency in a disoriented state. Earlier, he had felt con- diagnosis? What further tests are required to con-
fusion, throbbing headache and chest pain. These symp- firm this diagnosis?
toms seemed to have made sudden appearance, for he Q.2. What are the various factors that lead to tissue
had appeared his normal self at the time he reported for hypoxia in case of carbon monoxide poisoning?
duty early in the morning. Q.3. What is the rationale behind starting oxygen therapy
On examination, his blood pressure was 108/70 mmHg, immediately? Why was hyperbaric oxygen given?
pulse was 112/min, and respiration was shallow and fast Q.4. Has the smoking habit of the patient influenced
(34/min). Analysis of the blood sample showed increased development of the present condition?
concentration of carboxy-haemoglobin (39.6%). Q.5. In spite of oxygen insufficiency, the patient does not
The patient was put on oxygen therapy with a diagnosis present with any signs of cyanosis. Give reason.
of sub-acute carbon monoxide poisoning. Hyperbaric oxy- Q.6. Why is CO poisoning more serious in infants?
gen was given, to which he responded well.

CASE 17.3 A 10-year-old boy with breathlessness, pallor and persistent tiredness
A 10-year-old boy was brought to casualty with fever and analyzed by the staff-nurse in the side lab; presence of
breathlessness. Earlier he was seen by a general practitioner abnormally large amounts of urobilinogen was reported.
who had prescribed antibiotics with a diagnosis of upper The child was admitted for the treatment of respiratory
respiratory tract infection. Presently the child appeared out tract infection and for further investigations. Emergency
of breath and was complaining of aches and pains, and blood sample was sent to the biochemistry and haematol-
tiredness. On examination, he was clinically anaemic, icteric ogy laboratory. A sputum sample was also analyzed. The
and showed pallor and signs of retarded growth and devel- antibiotic was changed when pneumococci were isolated
opment. His blood pressure was 98/68 mmHg and pulse from the sputum sample. The boy responded well to the
was 100 beats per minute, with wide pulse pressure and new antibiotic and the fever rapidly subsided. However,
hyperdynamic precordium. The sclera was yellow, abdomen most other symptoms persisted as before. Results of some
was distended and the spleen was enlarged. Urine was of the blood tests are as here.
368 Textbook of Medical Biochemistry

Test Patient’s Reference range Electrophoresis of haemoglobin was performed to

reports detect haemoglobin structural variant, if any. It revealed
presence of HbS. Based on these tests, the child was diag-
Haemoglobin 5.2 g/dL 13–16 g/dL
nosed as having sickle cell anaemia.
Red Blood cells 12
2 ( 10 )/L 4.5–6.5 1012/L Peptide maps (or finger prints) of the trypsin digestion of
Platelets 190,000/ L 150,000–400,000/ L the haemoglobin of this child were obtained and compared
with that of a normal healthy child of the same age group.
Serum
4.8 mg/dL 0.1–1.0 mg/dL The two were found to be remarkably different.
Bilirubin
Alanine 48 U/L 10–40 U/L Q.1. How does the above diagnosis explain clinical fea-
transaminase tures of the child?
Alkaline 90 U/L 40–100 U/L Q.2. Paper electrophoresis of the haemoglobin obtained
phosphatase from the child’s parents was also carried out; the
Lactate 386 U/L 100–300 U/L results are shown below.
dehydrogenase
Sodium 138 mmol/L 132–144 mmol/L Anode
Potassium 5.5 mmol/L 3.6–5.0 mmol/L HbA
HbS
The van den Bergh test for water soluble (conjugated)
and water insoluble (unconjugated) bilirubin in the serum Control Child Parents
sample showed elevation of unconjugated bilirubin level.
Bilirubin was not detected in the urine sample. However, the Interpret the results and discuss whether the child’s
urine contained large amount of urobilinogen (i.e. urobiliru- parents also have clinical abnormalities like him?
binuria), as stated above. Q.3. Comment on the quantitative blood picture. What
A fresh blood smear contained a few crescent-shaped are other biochemical features of this disorder?
cells. However, after 24-hour incubation in a sealed wet Q.4. State principle of the peptide finger print test.
smear, nearly all the red cells assumed the shape of sickles. How does the finger print test of this child differ from
An increased reticulocyte count was also reported in this that of a normal subject? Give reason for your
smear. answer.