This document provides information on hemoglobin structure and function. It discusses the history of hemoglobin discovery and describes the globin and heme components. Hemoglobin transports oxygen from the lungs to tissues and carbon dioxide from tissues to the lungs. Varieties include adult, fetal, and other variants like hemoglobin S, C, and E. Derivatives such as oxyhemoglobin, carbaminohemoglobin, and methemoglobin are also reviewed. Hemoglobinopathies include structural variants and thalassemias. Sickle cell anemia results from substitution of one amino acid, causing red blood cells to sickle in low oxygen conditions. Thalassemias are globin chain synthesis defects.

1. HAEMOGLOBIN STRUCTURE
AND FUNCTION
PRESENTOR : Dr.K.AMUDHA LAKSHMI M.B.B.S.,D.O.,M.D

2. INTRODUCTION
HISTORY
STRUCTURE OF HEMOGLOBIN
FUNCTIONS OF HEMOGLOBIN
VARIETIES OF HAEMOGLOBIN
SUMMARY
REFERENCES

3. INTRODUCTION
The cytoplasm of erythrocytes- oxygen-
binding protein- Haemoglobin.
Erythrocyte precursors- synthesizes
Mature erythrocytes- lose the property
The inclusion of haemoglobin within the
erythrocytes-most effective-functional purposes

4. HISTORY
Hemoglobin was
accidentally discovered
in Earthworm blood by
HUNEFELD in 1840

5. Hemoglobin was named by
HOPPE SEYLER in
1864

6. RBC was described by
JAN SWAMMERDAM

7. RBC complete account made
by ANTHONY VAN
LEEUWENHOEK

8. STRUCTURE OF HAEMOGLOBIN
Haemoglobin is a globular molecule
Molecular weight- 68,000.
Protein- Globin
Iron containing pigment called Haem

9. STRUCTURE OF GLOBIN
Common type- Adult haemoglobin-HbA
Four polypeptide chains
Two α chains-141 amino acids
Two β chains-146 amino acids
HbA (α2β2)

10. STRUCTURE OF HAEM
Iron–protoporphyrin IX
Porphyrin nucleus and iron

11. PORPHYRIN NUCLEUS
Four pyrrole rings- I, II, III and IV-
Tetrapyrroles
Joined together - Methine bridges (=CH—).
Carbon atoms- labelled α, β, γ and δ.
Eight side chains-1 to 8
Four methyl (H3C)- 1, 3, 5 and 8
Two vinyl (CH.CH2)- 2 and 4
Two propionic acid (CH2.CH2.COOH)- 6
and 7.

12. THE IRON
Iron- ferrous (Fe2+) form
Attached to the Nitrogen atom- Pyrrole ring.
Bond is available for loose union
In oxyhaemoglobin, O2 is attached,
In carboxyhaemoglobin, CO is attached & so
on

13. ATTACHMENT OF HAEM TO GLOBIN.
1 molecule of haemoglobin- 4 units of haem
Each attached to one of the four
polypeptide chains
4 iron atoms- 4 Oxygen molecules- 8
atoms of oxygen

14. FUNCTIONS OF HAEMOGLOBIN
1. Transport of O2 from lungs to tissues
Lungs-1 molecule of O2 is attached-
loosely and reversibly-6th covalent bond of each
iron atom- Oxyhaemoglobin- HbO2
Affinity of haem for oxygen- increases
Affinity of haemoglobin for the 4th oxygen-
more

17. Haemoglobin reacts with oxygen-rapid- less
than 0.01 sec
Deoxygenation of haemoglobin- very rapid
Oxygen–haemoglobin dissociation curve-
sigmoid shaped
Affinity of haemoglobin for oxygen is influenced
by
pH
Temperature
Concentration of 2,3-Diphospho-glycerate
or Biphosphoglycerate

18. 2,3-BPG rises- affinity of Hb for oxygen falls and
the oxygen–haemoglobin dissociation curve-
shifted to the right
more oxygen is released by blood to the tissues
At high altitude- 2,3-BPG increases by 50%-
more oxygen is available to the tissues.

19. 2,3-BPG accumulates- depresses hexokinase
and 2,3- BPG mutase activity- negative
feedback control.
Glucose metabolism- related to oxygen transport
-2,3-BPG concentration.
Stored blood- loses 2,3-BPG concentration-
oxygen affinity increases- less release of
oxygen.
Addition of inosine and adenosine to stored
blood- increases 2,3-BPG formation- restores
normal affinity of haemoglobin for oxygen.

21. Deoxygenated Hb forms carbamino-
haemoglobin more readily than oxygenated Hb
Venous blood more suitable for
transport of CO2 from the tissues to the lungs.
2. Transport of CO2 from the tissues to the lungs.
CO2 from the tissues- combines with amino
acids of the globin part

22. 3. Control pH of the blood.
Most important acid–base buffer system of
blood.
Concentration of Hb- twice the concentration
of plasma proteins
Hb has six times the buffering capacity

24. 4.Imparts Red colour to Blood

25. DEVELOPMENT OF HAEMOGLOBIN
First appear- 6 weeks after gestation.
Embryonic haemoglobin- Portland, Gower I
and Gower II
10–11 weeks- fetal HbF-prominent.
38 weeks- adult HbA
F cells- small pool of immature committed
erythrocyte precursor- produce Hb F

27. The human haemoglobin is encoded by
gene clusters (α and β).
α-like gene clusters- chromosome 16
β-like- chromosome 11
The locus control region (LCR) element-
control expression of each cluster

28. Normal blood haemoglobin
The normal blood haemoglobin concentration
at different ages

29. In fetus
Haemoglobin from the umbilical cord - 16.5
to 18.5 g/dL.
After birth
Haemoglobin concentration- more- 23 g/dL
Transfusion of cells from the placenta to
infant
Haemoconcentration by reduction of plasma
volume.

30. At the end of 3 months.
After 2 days of birth- Hb levels start falling
and stabilize- 3 months- 10.5 g/dL.
At 1 year of age
Concentration rises to 12 g/dL
In adult males
Mean blood haemoglobin (Hb) concentration
is 15.5 g/dL (range 14– 18 g/dL)
In adult females
Mean Hb concentration is 14 g/dL (range 12–
15.5 g/dL)

31. The normal haemoglobin becomes 100%
saturated when blood is equilibrated with 100%
oxygen (PO2 760 mmHg)
One gram of haemoglobin- fully saturated-
1.34 ml oxygen
Hb concentration- index of oxygen carrying
capacity of blood
Oxygen carrying capacity
males is 1.34 × 15.5 = about 21 ml/dl
females is 1.34 × 14 = about 18.5 ml/dl
Clinically, irrespective of the age, a level of
14.8 g/dL is considered as 100% haemoglobin

32. VARIETIES OF HAEMOGLOBIN
Haem moiety- remains the same.
Polypeptide chains of the globin part of the
haemoglobin- differs
Physiological varieties of haemoglobin
Haemoglobinopathies.

33. PHYSIOLOGICAL VARIETIES OF
HAEMOGLOBIN
ADULT HAEMOGLOBIN:
It is of two types.
Haemoglobin A [HbA (α2β2)]- normal adult
haemoglobin
Globin part consists- two alpha and two beta
polypeptide chains
It is a spheroidal molecule
molecular weight- 68,000.

34. ii. Haemoglobin A2 [HbA2 (α2δ2)]
Minor component- 2.5% of the total Hb in
normal adults.
Globin part-two alpha and two delta
polypeptide chains.
Delta chains- out of 146, 10 amino acids are
different compared with β chains

36. FETAL HAEMOGLOBIN:
Haemoglobin F [HbF α2γ2] - fetal RBCs
It gradually disappears 2–3 months after birth

37. Amount of HbF and HbA at Various Stages in
Human Beings

38. Structure of HbF
Globin part- two alpha and two gamma
polypeptide chains
Gamma chains -146 amino acids, 37
amino acids are different than that of beta
chains.

39. SPECIAL FEATURES
Affinity for oxygen of HbF is more- even
at low oxygen pressure.
Poor binding of 2,3-BPG by the gamma
polypeptide chain
Movement of oxygen from maternal to fetal
circulation is facilitated.
At PO2 20 mmHg, HbF is 70% saturated
while HbA is only 30–35% saturated.

40. Resistance to action of alkalies is more in HbF
than HbA.
Used in a photoelectric colorimetric method
to estimate HbF in the presence of HbA.
Lifespan of HbF- 1–2 weeks
HbA- 120 days

41. OTHER HEMOGLOBIN VARIANTS
i. Haemoglobin E(HbE) α2β2 (26glu lys)
Most common in United States.
Hb E homozygous- asymptomatic.
Microcytosis, hypochromia
Hb levels are rarely less than 10 g/dL.
ii. Haemoglobin C (HbC)
Substitution of lysine for glutamic acid at β
6 position
No sickling of RBCs
HbC -precipitate in polymer formation in
association with Hb S.

42. Homozygous HbC disease
mild haemolytic anaemia
splenomegaly
mild jaundice
pigment gall stones (Ca-bilirubinate
stones)
iii. Haemoglobin lupore: HbL iα2(δβ2)
globin is synthesized poorly
present like β thalassemia 2–20% HbL.

43. iv. Haemoglobin H disease:
Marked haemolytic anaemia
Haematocrit- 22–32%
MCV is markedly low (60–70 µm3)
Peripheral blood smear- abnormal cells
with hypochromia, microcytosis, target cells and
poikilocytosis.
Peripheral smear stained with supravital
stain detects HbH
v. Hb J and HbM
Cause haemolytic anaemia.

44. DERIVATIVES OF HAEMOGLOBIN
Haemoglobin readily react with any gas or
other substance to form-derivatives of
haemoglobin
1. Oxyhaemoglobin.
Haemoglobin reacts readily with oxygen to
form oxyhaemoglobin
unstable and reversible compound
iron remains in ferrous state.

45. 3. Carbamino-haemoglobin is a compound of Hb
with carbon dioxide
2. Reduced haemoglobin or deoxygenated
haemoglobin is formed when oxygen is released
from the oxyhaemoglobin
(CARBAMINO HAEMOGLOBIN)

46. 4. Carboxyhaemoglobin or carbon monoxy
haemoglobin
compound of Hb with carbon monoxide (CO)
CARBOXY HAEMOGLOBIN
The affinity of haemoglobin for CO is much
more (200–250 times) than its affinity for oxygen
CO displaces oxygen from haemoglobin-
reducing the oxygen carrying capacity of blood.

47. 5. Methaemoglobin.
Haemoglobin is treated with an oxidizing
agent, e.g. potassium ferricyanide, the ferrous
(Fe2+) is oxidized to ferric (Fe3+)
Sixth bond is attached to OH to form the
compound methaemoglobin- HbOH.
Disadvantages
It cannot unite reversibly with gaseous
oxygen
When present in large amount- produces
cyanosis

48. Normally, some methaemoglobin
(HbOH) does occur
But the methaemoglobin formed is soon
converted back to haemoglobin by the enzyme
NADH – methaemoglobin reductase present
in the RBCs
Congenital absence of this enzyme-
Hereditary methaemoglobinaemia

50. 6. Sulphaemoglobin
Combination of haemoglobin with hydrogen
sulphide (H2S).
Irreversible
7. Nitrous oxide haemoglobin
Combination haemoglobin with nitrous oxide.
8. Glycosylated haemoglobin
Derivative of haemoglobin A present in very
small amounts Haemoglobin A1C (HbA1C)-
glucose is attached to terminal valine in the β
chains
Glycosylated haemoglobin increases- poorly
controlled patients of diabetes mellitus.

51. Normally- Fraction of Hb glycosylated- 5%
Proportionate to blood glucose level.
Half-life of an erythrocyte- 60 days- HbA1C
reflects the mean blood glucose concentration
over preceding 6–8 weeks.
Estimation of HbA1C- valuable information
of management of diabetes mellitus.

52. DERIVED PRODUCTS OF HAEMOGLOBIN.
Iron containing
Iron free
1. Iron-containing derived products of
haemoglobin
Acid haematin or alkali haematin is
formed by the action of acid or alkali with
haemoglobin.
Haemochromogen is obtained by reduction
of alkali haematin.
Cathaemoglobin- haemoglobin containing
ferric ion with denatured globin.
Haem

53. Iron-Free Derived Products of Haemoglobin
Haematoporphyrin
Haempyrrole
Haematoidin
Bilirubin

54. DEGRADATION OF HAEMOGLOBIN
Tissue macrophages (Reticuloendothelial
system)- lining the hepatic and splenic
sinusoids- phagocytize old RBCs
Haemoglobin is broken down
Iron released- enters the plasma
Bilirubin- enters bile canaliculi

55. HAEMOGLOBINOPATHIES
Abnormal formation of haemoglobin occurs
due to disorders of globin synthesis
Haem synthesis - normal.
Classification:
5 major classes of haemoglobinopathies
Structural haemoglobinopathies- gene
mutation-change in amino acid sequence in
globin chain

56. Sickle cell anaemia- HbS
Thalassaemia syndromes- mutation that impairs
the production of globin mRNA- deficient or
absent globin biosynthesis.
Thalassaemic haemoglobin variants
Thalassaemia and structural
haemoglobinopathies, e.g. Hb E, Hb C, Hb L
and Hb J
Hereditary persistence of fetal haemoglobin
(HPFH): Increased fetal haemoglobin in adult
life.

57. Acquired Haemoglobinopathies
Modification of Hb molecule by toxins, e.g.
methaemoglobin, sulfhaemoglobin,
carboxyhaemoglobin and haemoglobin H in
leukaemia.

58. Sickle cell haemoglobin or Haemoglobin S
(HbS)
Most important haemoglobinopathy.
10–20% of Negroes
Sickle cell gene- Originated in the black
population in Africa.
HbS- substitution of valine for glutamic
acid at position 6 in the beta chain of HbA.
When HbS in low O2 tension or pH at tissue
level is low- less soluble and precipitates into
crystals within the RBCs.

59. Crystals elongate- change in shape of the
cells- biconcave to sickle-shaped (sickling)
RBC cells- less flexible- blockade of
microcirculation.
Increase blood viscosity- decreasing the
blood flow to tissues
More fragile
Very liable- Haemolysis producing the so-
called sickle cell anaemia.

60. Sickle trait inherited- Mendelian dominant
Full-blown disease is Autosomally
recessive.
Heterozygous individual with sickle cell trait
rarely has severe symptoms
Homozygous develop full- blown disease.
Resistance to one type of malaria.

63. Treatment of sickle cell anaemia
Presence of HbF in RBCs decreases the
polymerization of deoxygenated HbS
Drugs- 5-azacytidine and hydroxyurea,
which lead to formation of HbF in children and
adults-shown good results
Bone marrow transplantation

64. THALASSAEMIA (MEDITERRANEAN
ANAEMIA)
Defect in the synthesis of polypeptide chain
α and β of HbA
Types
α thalassaemia- α chain defect
β thalassaemia- β chain defect

65. α Thalassaemia trait
Asymptomatic or silent carrier state
Homozygous state of α thalassemia
Excess of γ globin form- Hb Barts (ϒ4)-
very high oxygen affinity
No delivery of the oxygen to fetal tissues-
asphyxia, oedema (Hydrops foetalis), congestive
heart failure and death in utero.
β Thalassaemia
More common
Thalassaemia major & thalassaemia
minor.

66. β THALASSEMIA
MAJOR
Homozygous
transmission
Complete absence of β
chain synthesis
Moderate to severe
anaemia
HbF- markedly
increased
Short lifespan
Treatment required
β THALASSEMIA
MINOR
Heterozygous
transmission
Partial absence of β
chain synthesis
Mild anaemia
HbF- normal or slightly
elevated
Long survival
No treatment

67. DETECTION AND CHARACTERIZATION OF
HAEMOGLOBINOPATHIES
Routine investigations
Complete haemogram
Peripheral blood smear
Screening and Quantification
Haemoglobin electrophoresis
Isoelectric focusing
High-pressure liquid chromatography
(HPLC).

68. Function assays for HbS
Sickling test
Solubility test
Precipitation test
Oxygen affinity (P50)- detect low/high
affinity of oxygen.
Spectrophotometry
Detect % of abnormal Hb e.g.
methaemoglobin and carboxyhaemoglobin.

69. For complete characterization of
haemoglobinopathy
Amino acid sequencing
Polymerase chain reaction (PCR)
Allele specific oligomerotyde hybridization
Automated DNA hybridization
Automated DNA sequencing

70. SUMMARY

71. REFERENCES
• Guyton and Hall- Text Book of Medical Physiology-13th
International edition.
• Ganong ‘s Review of Medical Physiology 26th edition.
• Text book of medical Physiology- Indhu Khurana 3nd
edition
• Comprehensive Text Book of Medical Physiology- G.K.
Pal 2nd edition
• Berne & Levy-Text book of Medical Physiology 1st
edition
• Boron-Text book of Medical Physiology 3rd edition
• Sharada Subramaniam- Text book of Medical
Physiology 7th edition