Haemoglobin chemistry

A comprehensive presentation on Hemoglobin chemistry for medical ,dental ,biotechnology ,Life sciences ,& pharmacology students. Presentation includes structure & functions of a normal hemoglobin molecule.Bohr's effect along with allosteric modulators of hemoglobin for oxygen transport are illustrated.Molecular changes ,types,diagnosis, Management & inheritance of Sickle cell anemia is described .Types , mutations involved ,diagnosis ,inhertance & Management of Thalassemia disease is presented here . Presentation also involves other hemoglobinopathies Hb C/D/E /Lepore/Wyane etc.Changes in oxygen carrying capacity of hemoglobin after formation of Carboxy Hemoglobin is illustrated . Formation of Meth-Hb in vivo & in vitro is described along with its genetic & diagnostic aspects.Unstable variants & chronic Heinz body anemia are described briefly .Text is supported by Google images.

1. HEMOGLOBIN CHEMISTRY
DR ROHINI C SANE

3. Structural Aspects of hemoglobin
•Molecular weight of hemoglobin (human)—67000 Dalton
•Normal concentration of hemoglobin(male)—14-16 gm%
•Normal concentration of hemoglobin(female)—13-15gm %
•Hemoglobin =heme+ globin (TOTAL 574 amino acids)
•Normal hemoglobin 97% HbA+2% Hb A2 + 1% HbF
•Globin Structure of HbA(97% ) -2 alpha chains + 2 beta chains
•Alpha chain (each )–141 amino acids
•Beta chain (each ) -146 amino acids
•HbF ( < 2% ) = 2 ALPHA chains +2 GAMMAchains
•HbA2( < 5% ) = 2 ALPHA chains +2 deltachains
•Subunits held by non covalent interactions ,hydrophobic interactions
,ionic interactions

8. Structural Aspects of hemoglobin
Type Composition & symbol% Total haemoglobin
HbA1 α2β2 97%
HbA2 α2δ2 5%
Hb F α2γ2 2%
HbA1 C α2β2 (GLYCATED HB ) <5% ( prognosis of
Diabetes Mellitus )
38 Histidine in one Hb molecule facilitates buffering action.

9. Structural Aspects of hemoglobin
Polypeptide chain typesymbol N TERMINAL END C TERMINAL END
Alpha α VALINE Not specific
Beta β VALINE HIS
Gamma γ GLYCINE HIS
Delta δ VALINE HIS

10. Structural Aspects of hemoglobin
1.Alpha chain gene 2 genes on chromosome 16
2.Beta ,gamma ,delta gene a single gene on chromosome 11
3.Delta gene active during embryonic development
4.2 gamma genes ( G γ-Grover /Aγ) responsibleforsynthesisofHb F
5.2WEEKSOFGESTASTION CONC OF Hb F startsincreasing
6.Concentration of Hb F is 80% at birth ,6months after birth
concentration decreases less than 3%
7.Alpha gene 7 helical segments
8.Beta gene 8 helical segments
9.38 Histidine molecules impart BUFFERING action

12. Structural Aspects of hemoglobin
•Isoelectric p H of HbA6.85
•Isoelectric p H of HbA27.4
•During electrophoresis at p H 8.6( OF BARBITONE BUFFER ) both
HbA& HbA2 carry positive charge move towards negatively charged
electrode ( cathode )
•Hb A moves faster to cathode than HbA2

15. Abnormal hemoglobin variants
•Alpha chain mutation
•Beta chain mutation
•Hemoglobin variants
•αgene family -2 genes on chromosome 16
•δgene active in embryonic development
•βgene family –single gene on chromosome 11(tandomgenes )
•εgene embryonic development
•2 γgenes ( Gγ& A γ) synthesis of HbF
•2 Weeks after gestation -80% at birth HbF ,
•decrease in HbF after birth-upto6 months 3 % retained
•δ gene (δ globin ) HbA2

17. HaemoglobinType Gene assembled
Grover II α₂ ε₂
Grover I ζ₂ ε₂
HbF α₂ γ₂
Hb A2 α₂ δ₂
Hb A α₂ β₂

19. Hemoglobinopathies–400 mutant of hemoglobin
1.Synthesis of abnormal hemoglobin
2.Production of insufficient quantities of normal hemoglobin
(decreased synthesis of Beta chain in beta thalassemia )
3. Both

22. Structure of Hemoglobin
•4 PYRROLE RINGS + 4 METHYL GROUPS + 2 VINYL GROUPS
•METHYL –CH3
•VINYL –CH3CH2
•PROPINOYL –CH3CH2 CH2
•METHYL –CH3
•METHYLENE –CH2
•METHENYL -=CH

31. Porphyrin( C20H14N4 )
•Cyclic compounds 4 pyrrole rings held by methylene bridges ( -CH-)
•Metal ion + Nitrogen atom of pyrrole ring to form complex Metallo
porphyrin
•8 hydrogen atoms substituted
•Pyrrole ring  4 closed brackets 4 substitutes positions
•Type I porphyrin symmetrical arrangement of substituent groups on all
8 positions ( eg.UroporphyrinI )
•Type III porphyrin asymmetrical arrangement of substituent groups on all 8
positions ( eg.UroporphyrinIII ) Fisher IX

32. Porphyrin( C20H14N4 )

33. Hans Fischer Model
of
4 pyrolerings
( porphyrins)
of Hemoglobin

34. ←Oxygenated hemoglobin withO₂
Oxidized hemoglobin has Ferric ( Fe ⁺³ oxidized form of iron atom )
Deoxy Hb -O ₂ CARRYING CAPACITY LOST

35. HEME GROUP
•Iron atom of heme:
Ferrous state (Fe ⁺² -reduced form of iron)
Attached to Six coordinated bonds =
4 coordinated bonds planer
+ 1 coordinated bond linked to O₂
+ 1 coordinated bond linked to His (64 ) of αor βglobin chain
Undergoes Fe ⁺²reduced form↔ Fe ⁺³ oxidized form
Heme is a constituent of Hemoglobin, catalase ,cytochromes
,chlorophylls ,Tryptophan pyrrolase.

36. Alpha chain of hemoglobin
Alpha chain
1.38 Histidine residues –( buffering action )
2.58 th distal Histidine
3.87 th proximal Histidine ( lies near Fe²⁺atom )
4.Forces holding alpha & beta chains together are
a)Van der Waals forces
b)Hydrogen bonds
c)Inter& intra electrostatic bonds

37. Transport of oxygen by hemoglobin
•Oxygenation :α₂β₂subunits slip over each other ( 2x ( alpha –beta ) –
salt bridges broken
•Oxy –Hb -Relaxed form ( R ) -salt bridges broken on oxygenation
•De -oxy –Hb Tight form ( T ) –( 2 x α) + ( 2 x β)

39. Taut (T ) & Relax ( R ) forms of hemoglobin
•T conformation –electrostatic forces between COO-& NH2 group
•( taut tense ) Deoxy haemglobin
•Hydrogen bonds & ionic bonds limit movement of monomers low
affinity for monomers
•R conformation –salt bonds broken HIGH AFFINITY FOR OXYGEN
•( 2x alpha )+(2xbeta) 2x (alpha –beta )
•Deoxy hemoglobin oxy hemoglobin
•OXYGEN breaks salt bridges ( R form –high oxygen affinity )
•ALLOSTERIC BEHAVIOUR OF HAEMOGLOBIN

40. Deoxy –Hb HbO₂ Hb O₄ Hb O₆ Hb O₈
T form ↓ ↓ ↓ ↓ ↓
↑
R form
Oxygenation of hemoglobin

42. CO-OPERATIVE BINDING OF OXYGEN TO HEMOGLOBIN
•Binding of oxygen to Heme will increase binding of oxygen to other heme
•Affinity of oxygen for hemoglobin
•Last oxygen binds with affinity 100 time greater than first oxygen
•Heme –Heme interaction ( cooperative binding of oxygen to Heme )
•Release oxygen from one Heme will release oxygen from other
•As there is communication between Heme groups of hemoglobin
•Myoglobin is reservoir (transient )& supplier of oxygen
Lung Tissue
Oxygen concentration high Oxygen concentration low
Oxygen binds to hemoglobin Oxygen is released to tissue

43. Structural changes in hemoglobin on oxygen binding
•Using x ray crystallographic study
•Homotropic effectbinding of oxygen to hemoglobin
•Heterotrophic effect binding of 2,3BPG to hemoglobin
•Distance between two beta chain decreases oxygenation from 4nm to 2nm
•Increasing affinity for oxygen with addition every molecule of oxygen
•On oxygenation iron moves in plane of Heme
•Decrease in diameter of iron (movement of iron accompanied by pulling of
proximal Histidine
•affinity of hemoglobin for last oxygen first oxygen ( 100 times greater )
•Cooperative binding of oxygen to hemoglobin OR Heme –Heme interaction
•Release of oxygen from one Heme Release of oxygen from other Heme
•Therefore communication between Heme groups of hemoglobin

44. Structural changes in Hb on oxygen binding
•Structural change in one subunit of hemoglobin on oxygenation is
communicated to other subunits
•Binding of oxygen to one Heme distorts globin chain to which it is
attached distortion in neighboring chain oxygen binds more
easily.

46. On oxygenation iron moves in plane of Heme decrease in diameter of Iron movement
of Fe Is accompanied by pulling of proximal site primary event of Heme –Heme interaction
of Heamoglobin

49. Difference between oxygenation and oxidation of Hemoglobin
OXYGENATION OXIDATION
IRON(Fe +2) IN FERROUS STATE IRON(Fe +3) IN FERRIC STATE
CARRIER OF OXYGEN OXYGEN CARRYING CAPACITY IS LOST

51. Transport of oxygen and carbon dioxide
by hemoglobin

52. Transport of oxygen and carbon dioxide
by hemoglobin

56. Binding of carbon dioxide to Hemoglobin
•Hb –NH2 + CO2 Hb-NH –COO -+ H +
•OXY –HAEMOGLOBIN 0.15 moles OF CO2 of /mole of heme
•DEOXY –HAEMOGLOBIN 0.40 moles OF CO2 of /mole of heme
•CO2 Stabilizes the T form formation of Deoxy hemoglobin 
decreased oxygen affinity for hemoglobin

57. Transport of carbon dioxide in human body
•200 ml of CO ₂/min produced in body
In aerobic metabolism
O₂ (1 mole ) utilized CO₂ (1 mole ) liberated
1.( 15% ) of CO₂ transport ( dissolved form)by Hb
CO₂ + H₂O H ₂CO₃HCO₃¯ + H⁺
2. ( 85% ) of CO₂ transport in Bicarbonate form
CO₂ + uncharged αamino acids of Hb CarbamylHb
Hb -NH₂+ CO₂↔Hb –NH-COO¯ + H⁺ ( BUFFERED BY Hb-Haldane effect )

58. Transport of carbon dioxide by Hemoglobin
•Hb -NH₂ + CO₂ ↔ Hb-NH -COO¯ ( Carbamoyl Hb)+ H⁺
•CO ₂ stabilizes the T form →decreased oxygen affinity for hemoglobin
formation of Deoxy Hb
HEMOGLOBIN CONCENTRATIONOF CO₂
Oxy -Hb 0.15 mmol of CO₂ / moles of Heme
Deoxy –Hb 0.40 mmol of CO₂ / moles of Heme

59. (
1

62. Transport of oxygen to tissue
Myoglobin : Reservoir (transient ) & supplier of oxygen
Lung Tissue
PO ₂ High low
Oxygen binds hemoglobinOxygen released to tissue

64. Transport of oxygen by hemoglobin
1.Bind & transport large quantity of oxygen by Histidine
2.greater solubility
3.Powerful buffer
4.Release of oxygen at appropriate pressure

65. Oxygen dissociation curve (ODC )
•Graphic representation of binding ability of haemoglobinwith oxygen
at different partial pressure of oxygen
•Ability of hemoglobin to load & unload oxygen at physiological p O2
( partial pressure of oxygen)

68. Transport of oxygen by haemoglobin
P O₂ (mm) of Hg% saturation
Inspired air 158
Alveolar air 100 97%
lung 90
Capillary bed 40 60%
37% -40% O ₂ release of oxygen at tissue level

73. Bohr’s effect :
(1) binding of oxygen decreases with increase in concentration of hydrogen
ions ( decrease in pH )
(2) Increase in concentration of carbon dioxide decrease in pH ( increase
in hydrogen ion concentration)bindingof oxygen to hemoglobin
decreases release of oxygen to tissue
(3) Shift of oxygen curve to right (with increase in concentration of ,hydrogen
ions ( decrease in pH ), carbon dioxide ,2,3 BPG ,Chloride ions ,temperature
release of oxygen decrease in % saturation of Hb with oxygen
(4 ) RESPONSIBLE FOR RELEASE OF OXYGEN FROM OXY HAEMOGLOBIN TO
TISSUE (with increase in concentration of ,hydrogen ions ( decrease in p H ),
carbon dioxide ,2,3 BPG ,Chloride ions ,temperature)

75. Bohr’s effect :
Increase in concentration of hydrogen
( lower pH )
Binding of oxygen to hemoglobin decreases
release of oxygen to tissue
Shift of oxygen dissociation curve to right

77. Shift of curve towards right % saturation decreases ( oxygen released from Heme )
BOHR’ S EFFECT -responsible for release of oxygen from oxy hemoglobin to the tissue
( increase in p CO₂ & decrease in pH ) is observed during metabolism of cell.
Increase in Conc of 2,3 BPG & CHLORIDE
(Cl ¯ )Shift of curve towards right
Allosteric effectors : interact with
Hb & release O ₂ from oxy-Hb
A. 2,3 BPG
B. CO₂
C. H ⁺
D. Cl¯

78. Bohr’s Effect :

79. Mechanism of Bohr’s Effect
•Caused by binding of hydrogen & CO2 TO HEMOGLOBIN
•Aspartic acid ( 94 ) is in close proximity with his 146 of beta chain of
hemoglobin
•Binding of hydrogen to Histidine is promoted by negative charge on
aspartic acid
•Ionic bond formed between negatively charged aspartic acid &
positively charged Histidine formation of salt bridges
•OXY –Hb( R-form ) DEOXY –Hb ( T-form )

82. Oxy -hemoglobin Deoxy -hemoglobin
pI6.6 pI6.8
More negatively charged CATIONS REQUIRED TO REMOVE
EXTRA NEGATIVE CHARGE .
OXY –Hb + H ⁺HHb+ O ₂( released to tissue )
H ⁺-trapped
One proton 2 oxy molecule released
Lung –oxygen concentration high
4 O₂bind to one hemoglobin therefore 4x 0.6 = 2.4 protons
released
H-Hb+ 4 O₂ Hb(O₂ ) + 2.4 H ⁺
One mill mole of Deoxy –Hb
take up 0.6 mequ from 0.6
mequ of H ₂CO ₃

87. TISSUE LUNG
CO2 HIGH CO2 LOW
HYDROGEN ION CONC HIGH HYDROGEN ION CONCENTRATION LOW
CONCENTRATION OF OXYGEN LOW CONCENTRATION OF OXYGEN HIGH
FORMATION OF DEOXY HAEMOGLOBIN FAVORED FORMATION OF OXY HAEMOGLOBIN FAVORED
HISTIDINE PROTONATED HISTIDINE DEPROTONATED
AFFINITY FOR OXYGEN DECREASES AFFINITY FOR OXYGEN INCREASES (HIGH PO2 )
Hb O 2 + H+ HbH+ + O2
EQULLIBRIUM TOWARDS RIGHT EQULLIBRIUM TOWARDS LEFT
CO2 BINDS ( Carbamoyl hemoglobin formation )OXYGEN BINDS ( OXY Hb formation )
Removal of hydrogen ion from terminal amino group Removal of CO2 from
Stabilizes Hb in T form(co2 binding releases oxygen to
tissue )
CO2 BINDS LOOSELY TO R FORM

88. Role of chloride in oxygen transport
•Chloride bind to Deoxy hemoglobin with affinity greater than oxy
hemoglobin
•When chloride bind to Deoxy hemoglobin there is release of oxygen
•Influx of chloride into cell cytosol of RBC in peripheral tissue is
accompanied by efflux of bicarbonate ions
•Influx of bicarbonate ions into cell cytosol of RBC is accompanied by
exfluxof chloride in lung tissue
•concentration of chloride ions

89. ISOHYDRIC TRANSPORT OF CO₂ & CHLORIDE SHIFT

90. Role of Chloride ( Cl¯ ) in oxygen transport
Chloride ( Cl¯ ) binds to de-oxy Hb
(1) Chloride ( Cl¯ )binding to de-oxy Hb release of oxygen
Deoxy –Hb
chloride ion
release of O₂

91. CHLORIDE SHIFT : Hamburger effect
HCO₃¯ freely moves out
TISSUE RBC: HCO₃¯ freely moves out & chloride enters to
maintain electrical neutrality -Chlorideshift–RBC Of venous
blood bulge
CHLORIDE ION ( Cl¯ )
CONCENTRATION OF CHLORIDE IONS IS GREATER IN VENOUS BLOOD THAN ARTERIAL BLOOD

92. CHLORIDE SHIFT : Hamburger effect
HCO₃¯
LUNG RBC:chloride freely moves out &
HCO₃¯enters to maintain electrical
neutrality -Reversal of chloride shift–
RBC Of venous blood bulge
CHLORIDE ION ( Cl¯ )

93. Oxygen released
CHLORIDE ENTERS RBC
ERYTHROCYTE IN TISSUE CAPILLARY : CHLORIDE SHIFT

94. ERYTHROCYTE IN LUNG CAPILLARY : CHLORIDE SHIFT
→ TO EXPIRED AIR
CHLORIDE LEAVES RBC ←
HCO ₃¯
ENTERS RBC
OXYGEN ENTERS

96. Hb acts as buffer
Hb act as buffer
For every 2 protons bound to
Hb
4O ₂ released
CARBONIC UNHYDRASE
FOUND IN RBC.

97. Significance of 2,3 BPG (Bi Phospho glyceride)
Increased stability Deoxy Hb confirmation by 2,3 BPG ( Mammals )

98. Effect of 2,3 BPG on oxygen affinity of Hb
•Most abundant phosphate in RBC
•Molar concentration of 2,3 BPG = Molar concentration of Hb
•Synthesis ( synthesis through Rapport Leuberingcycle)
2, 3 BPG mutase ( Glycolysis )
1,3 BPG 2,3 BPG
•Retinholds& Ruth Benesch’s(1967 )2,3 BPG decreases affinity of Oxygen to
Hemoglobin
•2,3 BPG regulates the binding of oxygen
•1mole of 2,3 BPG binds to 1mole of Deoxy Hb not to oxy –Hb
•Molecular concentration 2,3 BPG = Molecular concentration OF hemoglobin
•HbO₂ + 2,3 BPG Hb 2,3 BPG+ O₂ ( release of O₂ )
( Oxy –Hb ) ( De-oxy Hb)
•At partial pressure of O₂ in tissue 2,3 BPG shift curve towards right

109. 2,3 BPG & Hemoglobin
•2,3 BPG decrease p H 6.95 ( intracellular in RBC )
•Binding of 2,3 BPG to Deoxy Hb-stabilization of T confirmation
•Biding of 2,3 BPG stabilizes Deoxy Hb
•Hb + 2,3 BPG Hb2.3 BPG ( Deoxy Hb bound to 2,3BPG )+ O₂
(release of O₂ to the tissue )there fore 2,3 BPG regulates binding of
oxygen to Deoxy Hb

110. Clinical significance of 2,3 BPG
•Release of oxygen to tissue ( supply of oxygen to tissue )
•To cope with oxygen demand varied concentration of 2,3 BPG
1. Hypoxia: concentration of 2,3 BPG in RBC increases in chronic
hypoxic conditions
Adaptation to high altitude
Obstruction to pulmonary odema ( air flow in bronchial blocked )
•2. Anemia: concentration of 2,3 BPG in RBC increases in chronic
anemic conditions to cope with O₂demand of body even at low
Hb concentration

111. Clinical significance of 2,3 BPG
•3. Blood Transfusion: storage of blood in acid citrate dextrose decrease
in concentration of 2,3, BPG ( O₂ remains bound to Hb )
•Blood stored in ACD fails to supply O₂ to tissuewith 24-48 hrs2,3 BPG
restored
•O₂ supply /tissue O₂ demand is met adequately after 24-48 hrs.
•Blood with (ACD )+ Inosine ( Hypoxanthine Ribose ) prevent decrease in
2,3, BPG
•Inosinephosphorylation of tissueentry into HMP shunt get
converted to 2,3, BPG increase in Conc in 2,3, BPG release of oxygen

114. Myoglobin
1.Monomeric O₂ binding protein
2.Molecular weight -17000
3.Concentration in Heart & skeletal muscles: 2.5gm/100gm
4.Single polypeptide with 153 amino acids
5.pI= 6.5
6.Reservoir for O₂ ( carrier of O₂ is His 92 of Heme )
7.90 % saturation at 30nm pO₂( Hb 50% saturated )
8.Binding of O₂ to Hb ( one Hb 4 Heme 4 O₂ ,one myo1 heme
1 O₂ molecule )
9.Absorbance spectra –oxy Hb 582,542 nm

120. Oxygen dissociation curve
Myoglobin : hyperbolic
Hb-sigmoid
Affinity for O₂ of Myoglobin> Hb
Half saturation (50% )-myoglobin is at 1mm &
for Hb 26 mm
No Bohr’ s effect
No cooperative binding
No 2,3 BPG effect
Mb + O₂ ↔ Mb O₂
HbO₂O₂ Mb O₂ O₂ CELLS ( FOR RESPITATION)
Severe exercise PO₂ 5mm Hg release of oxygen

123. NORMALHAEMOGLOBIN DERIVATIVES
HAEMOGLOBIN
DERIVATIVES
COLOR CONCENTRATION
Oxy –Hb Red 97%
Deoxy –Hb purple Cyanosis > 5%
CO –Hb Cherry red 0.16%
Sulph–Hb Green High in cockroaches

125. Abnormal derivatives
1.Meth Hb ( synthesis in living system by H₂O₂, drugs ,free radicals
2. Carboxy Hb

126. Meth Hemoglobin –Meth-Hb
•Concentration of serum Meth-Hb= ( < 1% )
•Brown color of dried blood ( Meth –Hb ) & meat ( meth-myoglobin )
Normal Hemoglobin Meth-Hb
synthesis by oxygenation synthesis by oxidation ( BY H2O2 ,free radicals
,drugs )
O ₂ loosely binds Fails to bind to O ₂ ( H₂O molecule occupies O ₂
site in Heme )
Fe ²⁺ state ( Ferrous ) –no oxidation of Fe
²⁺ ( ferrous ) to ferric ( Fe³⁺ )
Fe ²⁺ Fe³⁺

129. Fe ²⁺
Fe ³⁺

131. Meth –Hb reductase
75 %
NADH DEPENDENT
20 %
NADPH
DEPENDENT
5%
GLUTATHIONE
DEPENDENT

132. Concentration of serum Meth Hb > 1%
(normal < 1% )
Decrease capacity for oxygen binding therefore
transport
Increase concentration of Meth –Hb (Cyanosis )
Meth Hemoglobinaemia ( acquired or congenital)

133. Congenital Meth Hemoglobinaemia
•Hemoglobin M ( proximal or distal Histidine of αor βglobin chain
replaced by Tyrosine
•Deficiency of cytochrome b5 reductase
•10-15% Hb as Meth –Hb ( normal < 1% )

134. Histidine -----------------------------Histidine
•58 distal 87 proximal
Histidine ------------------------------Histidine
•63 distal 92 proximal
αGlobin chain
βGlobin chain
Mutation in hemoglobin-Histidine to Tyr ( formation of Meth-Hb )

135. Acquired or Toxic Meth Hemoglobinaemia
1.Drinking of water contain Aniline dyes or nitrates
2.Drugs –Acetaminophen, Phenacein, Sulphanilamide,Amyl nitrite ,
Na-nitroprusside
3.Person with G-6 –PD deficiency

136. NADPH synthesis
↓
Decrease dependent Meth–Hbreductase (Normal-75%)
Deficient HMP
Person with G-6 –PD deficiency

137. MANIFESTATION OF DISEASE EASILY
TREATMENT –SMALL DOSES OF REDUCING AGENTS
DECREASE IN METH-Hb Hb ( inadequate )
5% NADPH dependent Meth Hb reductase

138. Meth –Hemoglobinaemia
Treatment of Acquired Meth –Hemoglobinaemia
2mg/ body Kg weight intravenous leucomethyleneblue substitute
for NADPH
Preparation of Meth –Hb in laboratory
5 drops of blood + sodium Ferri-cyanide (oxidizing agent )formation
of Meth hemoglobin (brown )-dark band at 633 nm (red region )
Preparation of Reduced –Hb in laboratory
5 drops of blood + Sodium dithionite reduced Hb ( purple )
reversible reaction ( reversed by atmospheric oxygen )

139. Meth Hemoglobinemia

140. •( a )
Ascorbic
acid
•(200-500
mg/day)
•(b )
Methylene
blue
•-200-500
mg/day
•Gene
therapy
Treatment of Meth Hemoglobinaemia
Decrease level of Meth –Hb to 5-10% (cyanosis reversed)

141. Carboxy –Hb (CO-Hb )
Carbon monoxide ( CO )
1.produced by incomplete combustion –occupational hazard
2.Colorless
3.Odorless
4.Tasteless
5.Toxic industrial pollutant
6.Affinity of CO for Hemoglobin is 200 more than that for Oxygen( O₂ )
7. NORMAL INDIVIDUAL SMOKER
CONCENTRATION OF CO –Hb < 0.16 gm% > 4 gm%
One cigarette 10-20 ml of CO in Lungs

142. Heme of
hemoglobin
Heme of
myoglobin
Heme of
cytochrome
CO

143. Clinical manifestation of increased CO
1.Conc of CO-Hb> 20 gm%
2.Head ache
3. Nausea
4.Vommitting
5. Breathlessness
6. Irritability
7. 40-60 % saturation of Hb with CO DEATH
8.

145. Identification of CO-Hbby absorption spectroscopy
•Band pattern for normal Hb & CO –Hb similar ( band 580 & 540nm )
Normal Hb
Reduced Hb
Oxy –HbDeoxy Hb
Entry of O₂ Oxy –Hb
reformed (reversible )
Carboxy Hb
Fails to form Reduced Hb
Carboxy Hb
(CO high affinity for Hb )
Na-Dithionite
vigorous shaking

146. Sickle cell anemia

148. SICKLE CELL ANAEMIA-SICKLE CELL HAEMOGLOBIN
•1957-first sickle cell hemoglobin (Hb S )
•FIRST MOLECULAR DISEASE ONE GENE ONE PROTEIN( Beadle
& Taum)
Crescent shape ( low hemoglobin content )

149. Occurrence of sickle cell anemia
•Tropical area –black population25% population Heterozygous ,central part
of & east part of India (scheduled tribe =ST)

151. MOLECULAR BASIS OF SICKLE CELL ANAEMIA
Linus Pauling ( 1954 Noble prize ) reported abnormal electrophoretic mobility & peptide mapping
Glutamic acid ( sixth position on beta globin chain ) replaced by Valine (Recessive Mutation )
Hb A & Hb F PREVENT SICKLING

154. Sickle cell disease
1.Glutamic acid Valine (HbS)—hydrophilic to hydrophobic amino
acid
2.Stickiness on surface of a Hb molecule
3.polymerization of Hb in RBC Distortion of RBC into sickle shaped
4.Deoxy HbS–protrusion on one side and cavity on other side
5.Many molecule adhere together
6. Deletion of HbStemp & pH dependent
7. Solubility is minimal at pH 6.35
8. Solubility increases with increase in pH

155. Sickle cell disease

156. Sickle cell disease
•solubility is minimum at pH 6.35
•solubility increases with increase in pH
•decrease in oxygen saturation & Hb concentration
•increase in proportion of polymeric & soluble
molecules

157. Sickle cell disease
•HbSbind & transport oxygen
•Decrease in oxygen saturation & Hb concentrationrelative
proportion of polymeric & soluble molecules
•Deoxygenated state sickling viscosity of blood increases slows
down the circulation decrease in oxygen tension-further sickling
•Vicious cycle

158. DE OXYGENATED STATE
SICKLING
VISCOCITY OF BLOOD INCREASES
SLOWS DOWN CIRCULATION
OXYGEN TENSION DECREASES
FURTHER SICKLING

159. DE OXYGENATED
STATE
SICKLING
VISCOCITY OF
BLOOD
INCREASES
SLOWS DOWN
CIRCULATION
OXYGEN TENSION
DECREASES
FURTHER
SICKLING
Viscous cycle of sickling

162. Mechanism of sickling in sickle cell anemia
•Glutamic acid replaced by Valine on beta chain at sixth position
•Decrease in solubility of HbS(Deoxy HbS)—T form
•Solubility of HbS( OXY Hb S ) unaffected
•HbAlack sticky patches
•Formation of long aggregates of Deoxy HbSpolymerization of HbS–
(Deoxy )fibrous PPTStiff fibers distorts RBC (SICKLE )LYSIS
•Sickle cells plug capillaries occlusion of major vessels infarction
of organ ( spleen ) death occurs in second decade of life

163. Formation of long aggregates of Deoxy HbS
Sticky patches of one HbS( Deoxy Hb)+ receptors of another HbS( DEOXY ) 
AGGREGATE
polymerization of HbS–(Deoxy )
Fibrous precipitate
Stiff fibresdistorts RBC ( SICKLE )
Lysis

165. Sickle cell
Plug in capillaries
Occlusion of major vessels
Interaction in organ (spleen )
Death occurs in
second decade of life

172. HbSgives protection against plasmodium falciparum causative of malaria
•Normal RBC (malaria parasite enters) multiplies RBC lysishemolytic
anemia
•RBC with sickle cell trait malarial parasite enters could not multiply 
no malaria, no RBC lysisnormal health
1. Shorter life span of RBC carrying HbSinterrupts parasite cycle
Malaria parasite increase in acidity( decrease in pH )increase in sickling
RBC to 40% (normal 2% )lysisof RBC
2. Low potassium level in sickled cellsunfavorable for parasite
sickle cell traitis an adaptation for survival of individual in malarial infested
region
Life span of sickle cell (homozygous )< 20yrs

174. Sickle cell anemia
Homozygous
1.Two mutant genes (one from each
parent)that code for beta chain
2.RBCs contain HbS
3.Sickle cell disease
4.Life span < 20years
Heterozygous
1 .one gene of beta chain is affected other gene
normal
2.RBCs contain Hbs& HbA
3.Sickle cell trait
4.Normal life –no clinical symptoms

177. Abnormalities associated with HbS
1. Life long hemolytic anemia-RBC fragile continuous hemolysis
2.Tissue damage & pain sickle cells block capillaries poor blood
supply to tissueextensive damage inflammationpain
3.Increased susceptibility to infection
4. Premature Death -Homozygouslife span < 20yrs

178. Diagnostic of sickle cell anemia
1.Sickling test: blood smear + reducing agent ( sodium dithionite )
microscopic examination
Normal individual-sickle cells < 2% , sickle cell patient -sickle cell > 2%
2.solubility test : hem lysate in presence of reducing agentopalescence in
hemolysate(presence of Deoxy HbS)
3. ELECTROPHORESIS OF Hb:
Glutamic acid (-vecharged ) Valine ( neutral ) decreased mobility
towards anode
4.Finger printing technique –Ingram
5.Sourthen blot

179. Management of sickle cell disease
1.Repeated blood transfusions iron overload ( Iron chelaterDes ferroxamine)
cirrhosis
2.Treatment( anti sickling agents )
a)Urea
b)*Cyanates(0.1 N) increase affinity for oxygen toHbSDecrease
Deoxy HbS
c)Aspirin
•INTERFERE WITH POLYMRIZATION inhibit sickling
3. sodium butyrate : induce HbF production CLINICAL IMPROVEMENT
4.Gene therapy
5.Family counselling
*side effects of cyanatesnerve damage

181. 25% HbA,50 % heterozygous Hb AS SA ,25%Homozygous

182. Inheritance of Hb variants

183. AA SA SC
25% 25% 25% DOUBLE 25% HETEROZYGOTE
HETEROZYGOTE

186. Hemoglobinopathies
TYPE OF
HEMOGLOBINOPATHIES
Mutation Amino acid
substitution
Codon
Hb S Beta 6 GluVal GAGGUG
Hb C Beta 6 GluLys GAGAAG
Hb E Beta 26 GluLys GAGAAG
Hb D (Punjab ) Beta 121 GluGln GAGCAG
Hb O (Arab ) Beta 121 GluLys GAGAAG
Hb SM PROXIMAL or distal
Histidine in Alpha or Beta
chain
His Tyr CACUAC
HbM The first abnormal Hb
( Saskatoon )1948

187. Mutations of Hemoglobin
Abnormal -Hb Amino acids Code
change
m-RNA Type of Mutation
1HbS GluVal GAG 
GUG
CTC CAC partially acceptable –
Transvers
2Hb-M HisTyr CAUUAU Missense -NON PROTEIN –
PROPERTIES CHANGED
3Hb Wyne Met SerCysLys
↓
Met LeuAlaLys
AUG UCU UGA AAA
↓
AUG CUU GAA AAA
Frame shift
4 Tyr Termination of
polypeptide
UACUAA Non sense –PREMATURE
TERMINATION –βThalessemia
5Hb constant spring Termination Gln UAA CAA Nonsense –chain elongation
6Hb-P,HbQ , Hb –N,Hb-JGluAsp
Hb Wyne Met SerCysLys
↓
Met LeuAlaLys
AUG UCU UGA AAA
↓
AUG CUU GAA AAA
Frame shift

188. Hb electrophoresis for
Heamoglobinopathies
Hb C/E / S/ H /
beta Thalessemiatrait

189. Hb S /D/A2/C/ E -SAME ELECTROPHORETIC
MOBILITY
Hb E /HbC–Heterozygous
Hb E /HbC–Homozygous

191. ELECTROPHORETIC MOBILITY TOWARDS ANODE SLOWER THAN Hb A & Hb S

192. Hemoglobin D ( Punjab )
1.Most common Hb variant in Punjab
2.GluGln( 121 thposition on beta chain )
3.No sickling
4.Severe condition

194. Hb E SIMILLAR MOBILITY AS A₂

196. Unstable Hb variants
Increased tendency to denature ( molecular aggregates within cells )
↓
Increased hemolysis

197. Chronic Heinz body Anemia ( CHBA )
unstable variants of HbHemoglobinopathy
αchain unstable variants Hb Torino
βchain unstable variantsHb Belefast
γchain unstable variantsHb F Poole

198. Unstable Hb variants
Hemichrome
( denatured Hb )
Membrane bound Heinz bodies –trapped
in spleen hepatosplenomeghaly–help
in identification ( hemolysis )

199. Hemoglobin Abnormal chainCodon & AA at position Resultant abnormality
Hb constant spring αchain UAA CAA
(itermGlu)
Chain is stopped only at next stop
signal –extra 31 amino acids
Hb Icaria αchain UAA AAA
(itermLys )
Chain is stopped only at next stop
signal –extra 31 amino acids
Hb Wayne βchain Frame shift mutation 137 AA onwards changed

200. -----------------------------------------------------------← UAA
↓
--------------------------------------------------------------------------
↑
CAA
Chain elongation
-------------------------------------------------------------------
↑
CAA
↑
---------------------------------← UAAPremature termination

201. 137
137 UAA
FRAME SHIFT MUTATION -WAYNE

202. Body CHBA (Chronic Heinz Body anemia)
1.Autosomal dominant inheritance
2.Moderate –severe hemolytic anemia
3.Hemolytic jaundice(spleenomeghaly)
4.Diagnosis supravitalstaining microscopic examinationcresy
violet ( Bronze bodies indentation trapped in spleen
hemolysis
5.Electrophoresis presence of abnormal band
6.No specific treatment

203. Hb variants with increased Oxygen affinity
•αchain variants (Hb Chesapeake )
•β chain variants (Hb Olympia )

204. Hb binds to oxygen
But difficulty in unloading
Tissue hypoxia
Increased hypoxia
Increased erythropoiesis
Erythrocytosis
Individuals are asymptomatic
CHBA (Chronic Heinz Body anemia)

205. CHBA (Chronic Heinz Body anemia)
1.Decreased cooperative effect
2.Oxygen dissociation curve (ODC )
shifted towards left
3. Diminished Bohr’s effect
4.Decreased interaction with 2,3 BPG
5.Autosomal dominant inheritance

206. Hb variants with decreased Oxygen affinity
Hb Kanas
Cyanosis
Hb Hope
1.No hemolytic anemia
2.No Meth hemoglobinomia
3.unstable

207. Hemoglobin M ( Hb M )
•Autosomal dominant inheritance
cyanosis
Oxygen binding is decreased
Met-Hb
Hemin
Hb gets oxidized
Proximal or distal Histidine of αor βchain replaced

208. Hemoglobin M ( Hb M )
1.Alpha 58 HisTyr ( Hb M Boston)
2.Beta 92His Tyr ( Hb M Hyde park )
3.Most common Hb variant
4.Single base substitution or point mutation
5.Terminator colon mutation ( Constant spring /Constant Icaria )
6.Frame shift mutation Wyne137 amino acid changed
altered amino acids after 138 amino acids abnormal

209. Terminator codon mutation
1.Elongated polypeptide
2.Premature chain termination
3.Frame shift mutation Hb Wayne 137 th amino acid onwards
abnormal synthesis up to 147 amino acids ( due to deletion of
one base pair )

213. Fetal hemoglobin ( Hb F )
•Hb 2 αchains & 2 delta chains ( delta chain 146 amino acids ,
39amino acids differ from beta chain )
Physical chemical properties of Hb F
1.Increased solubility of Deoxy HbF
2.slower electrophoretic mobility
3.Increased resistance of Hb F to alkali denaturation
4.Decreased interaction with 2,3 BPG
5.Hereditary persistence of HbF ( HPF )increased HbF without
Thalassemia, no DELTA BETA gene switching
6.Kleihourstaining for Hb F detection

214. Fetal hemoglobin ( HbF )
•HbF has γglobin less positive amino acids
•HbF weak binding to 2,3 BPG
•HbF has higher affinity for O₂ compared to adult Hb
•Binding affinity for O₂ of HbF > HbA( transfer of O₂ from maternal
blood to fetus by HbFO₂)
•Delivery of O₂to fetus

216. Embryonic Hb -3-8 weeks
Grover I -zeta₂Epsilon ₂(ζ₂ε₂)
Grover II -ALPHA ₂ zeta ₂( α₂ε₂)

222. Fetal hemoglobin ( Hb F )
•Hb F –(2 α2 γ)
•αglobin not synthesized
•Synthesis γ& βchain continues Tetramers (γ₄ )—Hb Bart
•β₄Tetramers (β ₄ )—Hb H
•HbHlack Heme –Heme interaction

223. Hb H & Hb Bart
Hb lack Heme –Heme interaction
Oxygen dissociation curve -Hyperbolic
No delivery of sufficient oxygen to tissue
Fetal death

225. Thalassemia
•Thalassasea
•Hereditary
•hemolytic disorder
•Impairment /imbalance in synthesis of globin chains of globin
•Mediterranean sea /Central Africa /India /Far east
•Deficit of gene functions, amino acid sequence normal

228. Molecular basis of Thalassemia
•Normal hemoglobin = α₂β₂
•αThalassemia (cause : decrease synthesis of αglobin chain/s)
•βThalassemia (cause : decrease synthesis of βglobin chain/s)
1.Gene deletion & substitution
2.Under production or instability of mRNA
3.Defect in the initiation of chain synthesis
4.Premature chain termination

232. Thalassemia
αThalassemia
1.αdeletion are rare
2.Hb A decreased
3.Hb F increased
4.Hb A₂ increased
δ₂β₂(delta beta Thalassemia)
1.Hb Lepore
2.Hereditary Persistence of HbF

237. BetaThalassemia
Decreased synthesis or lack of the beta chain
Production of alpha chain continues
α₄(tetramer )
Premature death of RBC

238. BetaThalassemia
MINOR
HETEROZYGOUS/TRAIT
DEFECT IN SYNTHESIS OF ONE OUT OF TWO BETA
GENES ON CHROMOSOME 11
Asymptomatic
Some amount of beta globin from
affected gene
MAJOR
HOMOZYGOUS
DEFECT IN SYNTHESIS OF BOTH BETA
GENES ON CHROMOSOME 11
Healthy at birth anemia ,hypertension
,hepatospleenomeghaly
Beta globin is not synthesized during
fetal development

249. TYPE Number of missing genesNumber of missing genes
1.Normal Nil Nil
2. Silentcarrier 1 No symptoms
3.αThalassemia Trait 2 Minor anemia
4. Hemoglobin H 3 Mild /moderate anemia
/normal life
5. Hydrops Fetalis 4 Fetal death occurs at birth
Alpha Thalassemia

257. Treatment of Thalassemia
1. Repeated blood transfusion
2.Spleenectomy decrease lessen anemia
3. Bone marrow transplantation ( as bone marrow skull expands 
skull bone “ Hair on end appearance ”
4.Chemotherapy : Azacytidine
5 .Gene therapy /stem cell therapy on the way of success ?

258. Azacytidine
Activates dormant gene for γglobin-temporarily
Base is incorporated into repressed γglobin-
No methylation
Non methylated gene can be expressed
ExpressionofHbF
Treatment of Thalassemia-chemotherapy
CHEMOTHERAPY HAS LIMITED SUCCESS

259. 1.Repeated blood transfusion 2.

261. •Transfusions:
•Regular blood transfusions to ensure non-anemic states and prevent some of the disease complications (Target Hb
90-100 g/L)
•Leukodepletiontechniques are used to ensure less alloimmunizationand non-hemolytic transfusion reactions.
•Testing for viruses is done to reduce transfusion transmitted infections
•Iron chelation:
•Deferoxamine/deferipronework by binding serum iron and clearing it via the urine.
•Deferipronehas been shown to improve cardiac functioning (left ventricular ejection fraction; LVEF) in patients with
thalassemia major.
•Endocrine therapy:
•Administration of the deficient hormones (sex hormones and thyroid hormones)
•Use of fertility agents to induce spermatogenesis and achievement of pregnancy
•osteoclast inhibitors (bisphosphonates) to prevent osteopenia and osteoporosis.
•Splenectomyand cholecystectomy:
•Splenectomiesoften assist with reducing transfusion requirements
•Cholecystectomies are often required to the presence of bilirubin stones in the gallbladder.
TREATMENT OF THALESSEMIA PATIENTS

269. Hemoglobin Lepore
•Hemoglobin with 2 α+ 2 δchimeric chains
•δ(delta )chain β( beta ) chain
•Homologous crossing over of chromosome chimeric

270. Hemoglobin Lepore

272. Hereditary persistent fetal Hb ( HbF )
1.Increase in HbF
2.no clinical symptoms
3.Failure to switch over γgene to βgene

282. Inheritance of Hb variants
•αchain inheritance
•αgenes 4 (less likely to produce impairment )
•β genes 2 (β gene variant more common &severe than αchain
inheritance
•αchain variant constitute only 25%