This document discusses various hemoglobin derivatives that are formed due to ligands binding to the heme part of hemoglobin or changes in the iron oxidation state. It specifically describes carboxyhemoglobin which is formed when hemoglobin binds to carbon monoxide, preventing oxygen transport. Other derivatives discussed include methemoglobin and sulfhemoglobin. The document also examines hemoglobinopathies such as sickle cell anemia, caused by a single amino acid substitution, and thalassemias which involve impaired globin chain synthesis. Clinical manifestations and treatments for various hemoglobin derivatives and disorders are summarized.

1. Haemoglobin Derivatives
Gandham. Rajeev

2. • Hemoglobin derivatives are formed by the
combination of different ligands with the
heme part, or change in the oxidation state
of iron.

3. Carboxy-Hemoglobin (CO-Hb)
• Hemoglobin binds with carbon monoxide
(CO) to form carboxy-Hb.
• The affinity of CO to Hb is 200 times more than
that of oxygen.
• It is then unsuitable for oxygen transport.

4. • When one molecule of CO binds to one
monomer of the hemoglobin molecule, it
increases the affinity of others to O2; so that
the O2 bound to these monomers are not
released.
• This would further decrease the availability
of oxygen to the tissues.

5. Carbon Monoxide Poisoning
• CO is a colorless, odorless, tasteless gas
generated by incomplete combustion.
• CO poisoning is a major occupational hazard
for workers in mines.
• Breathing the automobile exhaust in closed
space is the commonest cause for CO
poisoning

6. • The carboxy-Hb level in normal people is
0.16%.
• An average smoker has an additional 4% of
CO-Hb.
• One cigarette liberates 10–20 ml carbon
monoxide into the lungs.

7. Clinical Manifestations
• Clinical symptoms manifest when carboxy-Hb
levels exceed 20%.
• Breathlessness, headache, nausea, vomiting,
& chest pain.
• At 40-60% saturation, death can result.
• Administration of O2 is the treatment.

8. Methemoglobin (Met-Hb)
• When the ferrous (Fe2+ ) iron is oxidized to
ferric (Fe3+) state, met-Hb is formed.
• Small quantities of met-Hb formed in the RBCs
are readily reduced back to the ferrous state
by met-Hb reductase enzyme systems.
• About 75% of the reducing activity is due to
enzyme system using NADH & cytochrome b5

9. Methemoglobinemias
• Normal blood has only less than 1% of
methemoglobin.
• It has markedly decreased capacity for
oxygen binding and transport.
• An increase in methemoglobin in blood,
(methemoglobinemia) is manifested as
cyanosis.
• Causes may be congenital or acquired.

10. Congenital Methemoglobinemia
• Presence of Hb variants like HbM can cause
congenital methemoglobinemia.
• Cytochrome b5 reductase deficiency is characterized
by cyanosis from birth.
• 10-15% of hemoglobin may exist as methemoglobin.
• Oral administration of methylene blue, 100-300
mg/day or ascorbic acid 200-500 mg/day decreases
met-Hb level to 5-10% and reverses the cyanosis.

11. Acquired or Toxic Methemoglobinemia
• Met-hemoglobinemia may develop by intake
of water containing nitrates or due to
absorption of aniline dyes.
• Drugs which produce met-hemoglobinemia -
acetaminophen, phenacetin, sulphanilamide,
amyl nitrite, & sodium nitroprusside.

12. Sulf-hemoglobinemia
• When hydrogen sulfide acts on oxy-Hb, sulf-hemoglobin
is produced.
• It occur in people taking drugs like
sulphonamides, phenacetin, acetanilide,
dapsone, etc.
• It cannot be converted back to oxy-hemoglobin.

13. Hemoglobinopathies
• Abnormal hemoglobins are the resultant of
mutations in the genes that code for α or β
chains of globin
• As many as 400 mutant hemoglobins are
known.
• About 95% of them are due to alteration in
single amino acid of globin

14. Types of abnormal Hb
• Two types:
• If the mutation affects structural gene, it
results in replacement of a single amino acid
in Hb by some other amino acid resulting into
abnormal Hb.
• E.g: Hb-S, Hb-M, Hb-C, Hb-D & others.

15. • If the mutation affects the regulator gene,
which affects the rate of synthesis of
peptide chains, the amino acid sequence
remains unaffected.
• E.g: Thalassaemias

16. Globin synthesis
• The globin genes are organised into two gene
families or clusters
• α-Gene family:
• There are 2 genes coding for α-globin chain
present on each one of chromosome 16.
• The ζ (zeta)-gene, other member of a-gene
cluster is also found on chromosome 16 & is
active during the embryonic development

17. • β-Gene family:
• The synthesis of β-globin occurs from a single
gene located on each one of chromosome 11.
• This chromosome also contains four other
genes.
• One ε-gene expressed in the early stages of
embryonic development.

18. • Two γ-genes (Gγ & Aγ) synthesize γ-globin
chains of fetal hemoglobin (HbF).
• One δ-gene producing δ-globin chain found in
adults to a minor extent (HbA2).

19. Sickle-cell anemia (HbS)
• Sickle-cell anemia (HbS) is the most common
form of abnormal hemoglobins.
• Erythrocytes of these patients adopt a sickle
shape (crescent like) at low oxygen
concentration
• It primarily occurs in the black population.

20. Molecular basis of HbS
• The glutamic acid in the 6th position of β chain
of HbA is changed to valine in HbS.
• This single amino acid substitution leads to
polymerization of hemoglobin molecules
inside RBCs.
• This causes a distortion of cell into sickle
shape

21. Normal & HbS

22. • The substitution of hydrophilic glutamic acid
by hydrophobic valine causes a localized
stickiness on the surface of the molecule
• The deoxygenated HbS may be depicted with
a protrusion on one side and a cavity on the
other side, so that many molecules can
adhere and polymerize

23. • The sickled cells form small plugs in
capillaries.
• Occlusion of major vessels can lead to
infarction in organs like spleen.
• Death usually occurs in the second decade of
life.

24. Homozygous and heterozygous HbS
• Sickle cell anemia is said to be homozygous, if
caused by inheritance of two mutant genes
(one from each parent) that code for β-chains.
• In case of heterozygous HbS, only one gene (of
β-chain) is affected while the other is normal

25. • The erythrocytes of heterozygotes contain
both HbS & HbA & the disease is referred to as
sickle cell trait.
• The individuals of sickle-cell trait lead a normal
life, & do not usually show clinical symptoms.

26. Abnormalities associated with HbS
• Life-long hemolytic anemia:
• The sickled erythrocytes are fragile & their
continuous breakdown leads to life-long
anemia.
• Tissue damage and pain:
• The sickled cells block the capillaries resulting
in poor blood supply to tissues.
• This leads to extensive damage & inflammation
of certain tissues causing pain.

27. • Increased susceptibility to infection :
• Hemolysis & tissue damage are accompanied
by increased susceptibility to infection &
diseases.
• Prematured eath:
• Homozygous individuals of sickle-cell anemia
die before they reach adulthood (< 20 years)

28. Mechanism of sickling in sickle-cell anemia
• Glutamate is a polar amino acid & it is
replaced by a non-polar valine in sickle-cell
hemoglobin.
• This causes a marked decrease in the solubility
of HbS in deoxygenated form
• Solubility of oxygenated HbS is unaffected

29. Sticky patches & formation of
deoxyhemoglobin fibres
• The substitution of valine for glutamate
results in a sticky patch on the outer surface
of β-chains.
• It is present on oxy- & deoxyhemoglobin S
but absent on HbA.
• There is a site or receptor complementary to
sticky patch on deoxyHbS.

30. • The sticky patch of one deoxyHbS binds with
the receptor of another deoxyHbS & this
process continuous resulting in the formation
of long aggregate molecules of deoxyHbS
• The polymerization of deoxy-HbS molecules
leads to long fibrous precipitates.

31. • These stiff fibres distort the erythrocytes into
a sickle or crescent shape
• The sickled erythrocytes are highly
vulnerable to lysis.
• ln case of oxyHbS, the complementary
receptor is masked, although the sticky patch
is present.

32. HbS gives protection against malaria
• HbS affords protection against Plasmodium
falciparum infection
• Hence the abnormal gene was found to offer
a biologic advantage.

33. Sickle cell trait protects from malaria

34. Diagnosis of sickle cell anemia
• Sickling test:
• A simple microscopic examination of blood
smear prepared by adding reducing agents
such as sodium dithionite.
• Sickled erythrocytes can be detected under
the microscope

35. Electrophoresis
• Electrophoresis at alkaline pH shows a slower
moving band than HbA.
• At pH 8.6, carboxyl group of glutamic acid is
negatively charged.
• Lack of this charge on HbS makes it less negatively
charged, & decreases the electrophoretic mobility
• At acidic pH, HbS moves faster than HbA.
• In sickle cell trait, both the bands of HbA and HbS can
be noticed

36. Electrophoresis at pH 8.6

37. Management of sickle cell disease
• Administration of sodium cyanate inhibits
sickling of erythrocytes
• Cyanate increases the affinity of O2 to HbS &
lowers the formation of deoxyHbS
• It causes certain side effects like peripheral
nerve damage
• In severe anemia, repeated blood transfusion
is required.
• It result in iron overload & cirrhosis of liver

38. Hemoglobin C disease
• Cooley's hemoglobinemia (HbC) is characterized by
substitution of glutamate by lysine in the sixth position
of β-chain.
• Due to the presence of lysine, HbC moves more slowly
on electrophoresis compared to HbA and HbS.
• HbC disease occurs only in blacks.
• Both homozygous & heterozygous individuals of HbC
disease are known.
• It is characterized by mild hemolytic anemia.
• No specific therapy is recommended.

39. Hemoglobin D
• Caused by the substitution of glutamine in
place of glutamate in the 121st position of β-
chain.
• Several variants of HbD are identified from
different places indicated by the suffix.
• For instance, HbD (Punjab)
• HbD, on electrophoresis moves along with
HbS.

40. Hemoglobin E
• Most common abnormal hemoglobin after HbS.
• lt is estimated that about 10% of the population in
South-East Asia (Bangladesh, Thailand, Myanmar)
suffer from HbE disease.
• In India, it is prevalent in West Bengal.
• HbE is characterized by replacement of glutamate by
lysine at 26th position of β-chain.
• The individuals of HbE (either homozygous or
heterozygous) have no clinical manifestations

41. Thalassemias
• Thalassemias are a group of hereditary
hemolytic disorders characterized by
impairment/imbalance in the synthesis of globin
chains of Hb
• Thalassemias (Greek: thalassa-sea) mostly
occur in the regions surrounding the
Mediterranean sea, hence the name.
• Also prevalent in Central Africa, India.

42. Molecular basis of thalassemias
• Hemoglobin contains 2α & 2β globin chains.
• The synthesis of individual chains is so
coordinated that each α-chain has a β-chain
partner & they combine to finally give
hemoglobin (α2β2).
• Thalassemias are characterized by a defect in
the production of α-or β-globin chain

43. • Thalassemias occur due to a variety of
molecular defects
• Gene deletion or substitution,
• Underproduction or instability of mRNA,
• Defect in the initiation of chain synthesis,
• Premature chain termination.

44. α-Thalassemiasas
• α-Thalassemias are caused by a decreased
synthesis or total absence of α-globin chain of
Hb.
• There are four copies of α-globin gene, two on
each one of the chromosome 16.
• Four types of α-thalassemias occur which
depend on the number of missing α-globin
genes

45. Salient features of different α -thalassemias
• Silent carrier state is due to loss of one of the
four α -globin genes with no physical
manifestations.
• α -Thalassemia trait caused by loss of two genes
(both from the same gene pair or one from each
gene pair).
• Minor anemia is observed

46. • Hemoglobin H disease, due to missing of three
genes, is associated with moderate anemia
• Hydrops fetalis is the most severe form of α-
thalassemias due to lack of all the four genes.
• The fetus usually survives until birth & then dies.

47. β-thalassemias
• Decreased synthesis or total lack of the
formation of β-globin chain causes β-
thalassemias.
• The production of α-globin chain continues to
be normal, leading to the formation of a globin
tetramer (α4) that precipitate.
• This causes premature death of erythrocytes.
• There are mainly two types of β-thalassemias

48. β-Thalassemia minor
• This is an heterozygous state with a defect in
only one of the two β-globin gene pairs on
chromosome 11.
• Also known as β -thalassemia trait, is usually
asymptomatic, since the individuals can make
some amount of β-globin from the affected
gene

49. β-Thalassemia major
• This is a homozygous state with a defect in
both the genes responsible for β-globin
synthesis.
• The infants born with β-thalassemia major
are healthy at birth since β-globin is not
synthesized during the fetal development

50. • They become severely anemic and die within
1-2 years.
• Frequent blood transfusion is required for
these children.
• This is associated with iron overload which in
turn may lead to death within 15-20 years

51. References
• Text book of Biochemistry – U Satyanarayana
• Text book of Biochemistry – DM Vasudevan
• Text book of Biochemistry – MN Chatterjea

52. Thank You