Haemoglobin chemistry

HEMOGLOBIN CHEMISTRY

DR ROHINI C SANE

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

• Hb F ( < 2% ) = 2 ALPHA chains +2 GAMMA chains

• Hb A2 ( < 5% ) = 2 ALPHA chains +2 delta chains

• Subunits held by non covalent interactions ,hydrophobic interactions ,ionic interactions


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.


Polypeptide chain type symbol N TERMINAL END C TERMINAL END

Alpha α VALINE Not specific

Beta β VALINE HIS

Gamma γ GLYCINE HIS

Delta δ VALINE HIS


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 /A γ) responsible for synthesis of Hb F

5. 2WEEKS OF GESTASTION CONC OF Hb F starts increasing

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


• Iso electric p H of HbA 6.85

• Iso electric p H of HbA 27.4

• During electrophoresis at p H 8.6( OF BARBITONE BUFFER ) both HbA& HbA 2 carry positive charge move towards negatively charged electrode ( cathode )

• Hb A moves faster to cathode than HbA 2

Abnormal hemoglobin variants

• Alpha chain mutation

• Beta chain mutation

• Hemoglobin variants

• α gene family -2 genes on chromosome 16

• δ gene active in embryonic development

• β gene family –single gene on chromosome 11(tandom genes )

• ε gene embryonic development

• 2 γ genes ( G γ & A γ ) synthesis of HbF

• 2 Weeks after gestation -80% at birth HbF ,

• decrease in HbF after birth- upto 6 months 3 % retained

• δ gene (δ globin ) HbA2

Haemoglobin Type Gene assembled

Grover II α ₂ ε ₂

Grover I ζ ₂ ε ₂

HbF α ₂ γ ₂

Hb A2 α ₂ δ ₂

Hb A α ₂ β ₂

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

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

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 .Uroporphyrin I )

• Type III porphyrin asymmetrical arrangement of substituent groups on all 8 positions ( eg .Uroporphyrin III ) Fisher IX

Porphyrin ( C20H14N4 )

Hans Fischer Model of 4 pyrole rings( porphyrins ) of Hemoglobin

←Oxygenated hemoglobin with O₂

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

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 α or β globin chain

Undergoes Fe ⁺² reduced form ↔ Fe ⁺³ oxidized form

Heme is a constituent of Hemoglobin, catalase ,cytochromes ,chlorophylls ,Tryptophan pyrrolase .

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

Transport of oxygen by hemoglobin

• Oxygenation :α₂β₂ subunits 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 )

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

Deoxy –Hb Hb O₂ Hb O₄ Hb O₆ Hb O₈T form ↓ ↓ ↓ ↓ ↓

↑R form

Oxygenation of hemoglobin

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

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

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.

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

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

Transport of oxygen and carbon dioxide by hemoglobin

Transport of oxygen and carbon dioxide by hemoglobin

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

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 + H ₂O H ₂CO₃ HCO₃¯ + H⁺

2. ( 85% ) of CO₂ transport in Bicarbonate form

CO₂ + uncharged α amino acids of Hb Carbamyl Hb

Hb -NH ₂+ CO ₂↔Hb –NH-COO¯ + H⁺ ( BUFFERED BY Hb-Haldane effect )

Transport of carbon dioxide by Hemoglobin

• Hb -NH₂ + CO ₂ ↔ Hb-NH -COO¯ ( Carbamoyl Hb)+ H⁺

• CO ₂ stabilizes the T form →decreased oxygen affinity for hemoglobin formation of Deoxy Hb

HEMOGLOBIN CONCENTRATION OF CO₂

Oxy -Hb 0.15 mmol of CO₂ / moles of Heme

Deoxy –Hb 0.40 mmol of CO₂ / moles of Heme

(1

Transport of oxygen to tissue

Myoglobin : Reservoir (transient ) & supplier of oxygen

Lung Tissue

PO ₂ High low

Oxygen binds hemoglobin Oxygen released to tissue

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

Oxygen dissociation curve (ODC )

• Graphic representation of binding ability of haemoglobin with oxygen at different partial pressure of oxygen

• Ability of hemoglobin to load & unload oxygen at physiological p O2 ( partial pressure of oxygen)

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

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) binding of 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 ,temperaturerelease 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 )

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

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 withHb & release O ₂ from oxy-Hb A. 2,3 BPGB. CO₂ C. H ⁺D. Cl¯

Bohr’s Effect :

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 )

Oxy -hemoglobin Deoxy -hemoglobin

pI 6.6 pI 6.8

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 O₂ bind 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

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

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 exflux of chloride in lung tissue

• concentration of chloride ions

ISOHYDRIC TRANSPORT OF CO₂ & CHLORIDE SHIFT

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

CHLORIDE SHIFT : Hamburger effect

HCO₃¯ freely moves out

TISSUE RBC : HCO₃¯ freely moves out & chloride enters to maintain electrical neutrality - Chloride shift – RBC Of venous blood bulge

CHLORIDE ION ( Cl ¯ )

CONCENTRATION OF CHLORIDE IONS IS GREATER IN VENOUS BLOOD THAN ARTERIAL BLOOD

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 ¯ )

Oxygen released

CHLORIDE ENTERS RBC

ERYTHROCYTE IN TISSUE CAPILLARY : CHLORIDE SHIFT

ERYTHROCYTE IN LUNG CAPILLARY : CHLORIDE SHIFT

→ TO EXPIRED AIR

CHLORIDE LEAVES RBC ←

HCO ₃¯ ENTERS RBC

OXYGEN ENTERS

Hb acts as buffer

Hb act as buffer

For every 2 protons bound to Hb

4O ₂ released

CARBONIC UNHYDRASE FOUND IN RBC.

Significance of 2,3 BPG (Bi Phospho glyceride)

Increased stability Deoxy Hb confirmation by 2,3 BPG ( Mammals )

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 Leubering cycle)

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

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

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 O₂ demand of body even at low Hb concentration

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 hrs 2,3 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

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 heme1 O₂ molecule )

9. Absorbance spectra –oxy Hb 582,542 nm

Oxygen dissociation curve

Myoglobin : hyperbolic Hb- sigmoidAffinity for O₂ of Myoglobin> Hb Half saturation (50% )-myoglobin is at 1mm & for Hb 26 mm

No Bohr’ s effectNo cooperative binding No 2,3 BPG effectMb + O₂ ↔ Mb O₂Hb O₂ O₂ Mb O₂ O₂ CELLS ( FOR RESPITATION)Severe exercise PO₂ 5mm Hg release of oxygen

NORMAL HAEMOGLOBIN 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

Abnormal derivatives

1.Meth Hb ( synthesis in living system by H₂O₂, drugs ,free radicals

2. Carboxy Hb

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⁺

Fe ²⁺

Fe ³⁺

Meth –Hb reductase

75 %

NADH DEPENDENT

20 %

NADPH DEPENDENT

5%GLUTATHIONE DEPENDENT

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)

Congenital Meth Hemoglobinaemia

• Hemoglobin M ( proximal or distal Histidine of α or β globin chain replaced by Tyrosine

• Deficiency of cytochrome b5 reductase

• 10-15% Hb as Meth –Hb ( normal < 1% )

Histidine -----------------------------Histidine

•58 distal 87 proximal

Histidine ------------------------------Histidine

•63 distal 92 proximal

α Globin chain

β Globin chain

Mutation in hemoglobin- Histidine to Tyr ( formation of Meth-Hb )

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

NADPH synthesis

↓

Decrease dependent Meth–Hb reductase (Normal-75%)

Deficient HMP

Person with G-6 –PD deficiency

MANIFESTATION OF DISEASE EASILY

TREATMENT – SMALL DOSES OF REDUCING AGENTS

DECREASE IN METH-Hb Hb ( inadequate )

5% NADPH dependent Meth Hb reductase

Meth –Hemoglobinaemia

Treatment of Acquired Meth –Hemoglobinaemia

2mg/ body Kg weight intravenous leucomethylene blue 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 )

Meth Hemoglobinemia

• ( 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)

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

Heme of hemoglobin

Heme of myoglobin

Heme of cytochrome

CO

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.

Identification of CO-Hb by 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

Sickle cell anemia

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 )

Occurrence of sickle cell anemia

• Tropical area –black population25% population Heterozygous ,central part of & east part of India (scheduled tribe =ST)

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

Sickle cell disease1. 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

Sickle cell disease

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

Sickle cell disease

• HbS bind & 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

DE OXYGENATED STATE

SICKLING

VISCOCITY OF BLOOD INCREASES

SLOWS DOWN CIRCULATION

OXYGEN TENSION DECREASES

FURTHER SICKLING

DE OXYGENATED STATE

SICKLING

VISCOCITY OF BLOOD

INCREASES

SLOWS DOWN CIRCULATION

OXYGEN TENSION DECREASES

FURTHER SICKLING

Viscous cycle of sickling

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

• HbA lack 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

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 fibres distorts RBC ( SICKLE )

Lysis

Sickle cell

Plug in capillaries

Occlusion of major vessels

Interaction in organ (spleen )

Death occurs in second decade of life

HbS gives protection against plasmodium falciparum causative of malaria

• Normal RBC (malaria parasite enters) multiplies RBC lysishemolytic 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 HbS interrupts parasite cycle

Malaria parasite increase in acidity( decrease in pH )increase in sickling RBC to 40% (normal 2% ) lysis of RBC

2. Low potassium level in sickled cells unfavorable for parasite

sickle cell trait is an adaptation for survival of individual in malarial infested region

Life span of sickle cell (homozygous )< 20yrs

Sickle cell anemia

Homozygous1.Two mutant genes (one from each

parent)that code for beta chain

2.RBCs contain HbS

3.Sickle cell disease

4.Life span < 20years

Heterozygous1 .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

Abnormalities associated with HbS

1. Life long hemolytic anemia-RBC fragile continuous hemolysis

2.Tissue damage & pain sickle cells block capillaries poor bloodsupply to tissue extensive damage inflammation pain

3.Increased susceptibility to infection

4. Premature Death -Homozygous life span < 20yrs

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 (- ve charged ) Valine ( neutral ) decreased mobility towards anode

4. Finger printing technique –Ingram

5.Sourthen blot

Management of sickle cell disease 1.Repeated blood transfusions iron overload ( Iron chelater Des 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

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

Inheritance of Hb variants

AA SA SC 25% 25% 25% DOUBLE 25% HETEROZYGOTE

HETEROZYGOTE

Hemoglobinopathies

TYPE OF HEMOGLOBINOPATHIES

Mutation Amino acid substitution

Codon

Hb S Beta 6 Glu Val GAGGUG

Hb C Beta 6 GluLys GAGAAG

Hb E Beta 26 GluLys GAGAAG

Hb D (Punjab ) Beta 121 GluGln GAGCAG

Hb O (Arab ) Beta 121 GluLys GAGAAG

Hb SM PROXIMAL or distal Histidine in Alpha or Beta chain

His Tyr CACUAC

HbM The first abnormal Hb( Saskatoon )1948

Mutations of HemoglobinAbnormal -Hb Amino acids Code

change m-RNA Type of Mutation

1 HbS Glu Val GAG GUG

CTC CAC partially acceptable –Transvers

2 Hb- M His Tyr CAU UAU Missense -NON PROTEIN –PROPERTIES CHANGED

3 Hb Wyne Met Ser Cys Lys↓

Met Leu Ala Lys

AUG UCU UGA AAA↓

AUG CUU GAA AAA

Frame shift

4 Tyr Termination of polypeptide

UAC UAA Non sense –PREMATURE TERMINATION –β Thalessemia

5 Hb constant spring Termination Gln UAA CAA Nonsense –chain elongation

6 Hb-P,Hb Q , Hb –N,Hb -J Glu Asp

Hb Wyne Met Ser Cys Lys↓

Met Leu Ala Lys

AUG UCU UGA AAA↓

AUG CUU GAA AAA

Frame shift

Hb electrophoresis for HeamoglobinopathiesHb C/E / S/ H /beta Thalessemia trait

Hb S /D/A2/C/ E -SAME ELECTROPHORETICMOBILITY

Hb E /HbC – HeterozygousHb E /HbC – Homozygous

ELECTROPHORETIC MOBILITY TOWARDS ANODE SLOWER THAN Hb A & Hb S

Hemoglobin D ( Punjab )

1. Most common Hb variant in Punjab

2. Glu Gln ( 121 th position on beta chain )

3. No sickling

4. Severe condition

Hb E SIMILLAR MOBILITY AS A₂

Unstable Hb variants

Increased tendency to denature ( molecular aggregates within cells )

↓

Increased hemolysis

Chronic Heinz body Anemia ( CHBA )

unstable variants of Hb Hemoglobino pathy

α chain unstable variants Hb Torino

β chain unstable variants Hb Belefast

γ chain unstable variants Hb F Poole

Unstable Hb variants

Hemichrome

( denatured Hb )

Membrane bound Heinz bodies –trapped in spleen hepato splenomeghaly –help

in identification ( hemolysis )

Hemoglobin Abnormal chain Codon & AA at position Resultant abnormality

Hb constant spring α chain UAA CAA(iterm Glu )

Chain is stopped only at next stop signal –extra 31 amino acids

Hb Icaria α chain UAA AAA(iterm Lys )

Chain is stopped only at next stop signal –extra 31 amino acids

Hb Wayne β chain Frame shift mutation 137 AA onwards changed

----------------------------------------------------------- ← UAA↓

--------------------------------------------------------------------------

↑CAA

Chain elongation

-------------------------------------------------------------------

↑CAA

↑

--------------------------------- ← UAA Premature termination

137

137 UAA

FRAME SHIFT MUTATION -WAYNE

Body CHBA (Chronic Heinz Body anemia)

1. Autosomal dominant inheritance

2. Moderate –severe hemolytic anemia

3. Hemolytic jaundice(spleeno meghaly )

4. Diagnosis supravital staining microscopic examinationcresyviolet ( Bronze bodies indentation trapped in spleenhemolysis

5. Electrophoresis presence of abnormal band

6. No specific treatment

Hb variants with increased Oxygen affinity

• α chain variants (Hb Chesapeake )

• β chain variants (Hb Olympia )

Hb binds to oxygen

But difficulty in unloading

Tissue hypoxia

Increased hypoxia

Increased erythropoiesis Erythrocytosis

Individuals are asymptomatic

CHBA (Chronic Heinz Body anemia)

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

Hb variants with decreased Oxygen affinity

Hb Kanas

Cyanosis

Hb Hope

1. No hemolytic anemia

2. No Meth hemoglobinomia

3. unstable

Hemoglobin M ( Hb M )• Autosomal dominant inheritance

cyanosis

Oxygen binding is decreased

Met-Hb

Hemin

Hb gets oxidized

Proximal or distal Histidine of α or β chain replaced

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 changedaltered amino acids after 138 amino acids abnormal

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 )

Fetal hemoglobin ( Hb F )

• Hb 2 α chains & 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. Kleihour staining for Hb F detection

Fetal hemoglobin ( HbF )

• HbF has γ globin 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 maternalblood to fetus by HbFO ₂)

• Delivery of O₂ to fetus

Embryonic Hb - 3-8 weeks

Grover I -zeta ₂ Epsilon ₂ (ζ₂ ε₂ )

Grover II -ALPHA ₂ zeta ₂ ( α₂ ε₂ )

Fetal hemoglobin ( Hb F )

• Hb F – (2 α 2 γ )

• α globin not synthesized

• Synthesis γ & β chain continues Tetramers (γ ₄ )—Hb Bart

• β ₄ Tetramers (β ₄ )—Hb H

• HbH lack Heme –Heme interaction

Hb H & Hb Bart

Hb lack Heme –Heme interaction

Oxygen dissociation curve -Hyperbolic

No delivery of sufficient oxygen to tissue

Fetal death

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

Molecular basis of Thalassemia

• Normal hemoglobin = α₂β₂

• α Thalassemia (cause : decrease synthesis of α globin chain/s )

• β Thalassemia (cause : decrease synthesis of β globin 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

Thalassemia

α Thalassemia

1. α deletion 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

Beta Thalassemia

Decreased synthesis or lack of the beta chain

Production of alpha chain continues

α₄ (tetramer )

Premature death of RBC

Beta Thalassemia

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

TYPE Number of missing genes Number of missing genes

1.Normal Nil Nil

2. Silent carrier 1 No symptoms

3. α Thalassemia Trait 2 Minor anemia

4. Hemoglobin H 3 Mild /moderate anemia /normal life

5. Hydrops Fetalis 4 Fetal death occurs at birth

Alpha Thalassemia

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 ?

Azacytidine

Activates dormant gene for γ globin-temporarily

Base is incorporated into repressed γ globin-

No methylation

Non methylated gene can be expressed

Expression of HbF

Treatment of Thalassemia-chemotherapy

CHEMOTHERAPY HAS LIMITED SUCCESS

1.Repeated blood transfusion 2.

•Transfusions:• Regular blood transfusions to ensure non-anemic states and prevent some of the disease complications (Target Hb

90-100 g/L) • Leukodepletion techniques are used to ensure less alloimmunization and non-hemolytic transfusion reactions.• Testing for viruses is done to reduce transfusion transmitted infections

•Iron chelation:• Deferoxamine/deferiprone work by binding serum iron and clearing it via the urine.• Deferiprone has 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.

•Splenectomy and cholecystectomy:• Splenectomies often assist with reducing transfusion requirements• Cholecystectomies are often required to the presence of bilirubin stones in the gallbladder.

TREATMENT OF THALESSEMIA PATIENTS

Hemoglobin Lepore

• Hemoglobin with 2 α + 2 δ chimeric chains

• δ (delta )chain β ( beta ) chain

• Homologous crossing over of chromosome chimeric

Hemoglobin Lepore

Hereditary persistent fetal Hb ( HbF )

1. Increase in HbF

2. no clinical symptoms

3. Failure to switch over γ gene to β gene

Inheritance of Hb variants

• α chain inheritance

• α genes 4 (less likely to produce impairment )

• β genes 2 (β gene variant more common &severe than α chain inheritance

• α chain variant constitute only 25%

Health & Medicine

Haemoglobin chemistry