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Hb chemistry and disorders
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- 2. Hemoproteins (heme asprosthetic gr.) Hemoglobin – RBC’s Myoglobin – Muscles Cytochromes – heme gr serves as e- carrier Catalase – it catalyzes H2O2 Heme gr. serves to reversibly bind oxygen Dr.N.Sivaranjani
- 3. Hemoglobin isthe red blood pigment (Hemoprotein) present in the RBC’s. Heme is synthesized from Reticuloendothelial cells. • Heme activates synthesis of globin in nucleated reticulocytes of bone marrow cells. • Two chains of globin are formed independently at same rate. (15% a.a from daily protein intake ) Normal level in blood: 14-16 g/dl in males 13-15 g/dl in females Dr.N.Sivaranjani
- 4. Biomedical Importance • Transportof respiratory gases – O2 from lungs to tissues. H+, CO2 from tissues to lungs. • Major blood buffer - 36 histidine residues in Hb • Change in the structure of hemoglobin can give rise to disorders . Sickle Cell Anemia Thalassemia Met-hemoglobinemia Dr.N.Sivaranjani
- 5. First proteinto have its molecular mass accurately determined First protein to be characterized by Ultracentrifugation First protein to be associated with a specific physiological function (O2 transport ) & in Sickle cell anemia First protein in which a point mutation was determined to cause a single amino acid change First protein (Hb & Mb) were X ray structure was elucidated. Dr.N.Sivaranjani
- 6. Structure of Hemoglobin Conjugated globular Tetrameric protein Non protein component : HEME (4) Protein component : GLOBIN (4) Molecular weight – app. 65,000 daltons Chromo protein (heme - red color) Example of quaternary structure of protein Ex of Globular protein Dr.N.Sivaranjani
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- 8. Structure of globin •Consist of 4 polypeptide chains : 2 α and 2 β ( α1, α2 and β1, β 2) • 4 polypeptide chain are held together by non-covalent bonds. • Each subunit contains a heme group. • Adult Hb (HbA1) contains - 2 α & 2 β chains • Fetal Hb(HbF) contains – 2 α & 2 γ chains • HbA2 has – 2 α & 2 δ chains Dr.N.Sivaranjani
- 9. • Normal adultblood contains : 97 % HbA1, 2 % HbA2 1% HbF. • α chain gene is on chromosome 16 • β,γ,δ chains are the products of tandem genes on chromosome 11. Dr.N.Sivaranjani
- 10. Embroynic Hb • SeveralHb are found at fetal life but absent in adult life since it undergoes complex changes during development. Hb Gover 1 – ζ2ε2 Hb Gover 2 – α2ε2 Hb Portland – ζ22 by the end of the first trimester ζ and ε replaced by α and - HbF β subunits synthesis begins in the third trimester Dr.N.Sivaranjani
- 11. 2 1 1 2 Bonds Structure ofGlobin α1 – β1 and α2 – β2 interact extensively by hydrophobic, hydrogen bonds and salt bridges. In contrast there are only polar type interactions between like subunits i.e. α1 – α2 and β1 – β2 2 identical dimer – (αβ)1 & (αβ)2 Dr.N.Sivaranjani
- 12. A E F B C D G H Structure of βchain Chain – 7 helices Chain – 8 helices Polypeptide chains amino acids chain 141 chain 146 Hydrophilic amino acids: outer surface - solubility Hydrophobic amino acids : inner surface (Heme binds) except E7 and F8 histidine residues Dr.N.Sivaranjani
- 13. A E F B C D G H Heme Heme pocket: E& F helices Heme pocket Location of Heme Dr.N.Sivaranjani
- 14. 2 1 Heme 1 2 Heme HemeHeme Location of Heme 4heme groups / Hb Dr.N.Sivaranjani
- 15. Function of Globin– Forms a hydrophobic pocket - protects iron in Fe+2 state , prevents oxidation . Thus , allows reversible binding of O2 with heme. Iron in Fe+2 state binds O2 reversibly. Fe+3 state it does not bind O2 . Dr.N.Sivaranjani
- 16. Iron ( Fe++) + Protoporphyrin (IX) ring Structure of heme Heme is a metalloporphyrin Dr.N.Sivaranjani
- 17. Cyclic compound formedby fusion of four Pyrrole rings linked by methenyl bridges (=CH-). Four pyrrole rings - Roman numbers I, II, III and IV. Methenyl bridges – Greek numbers α, β, γ and δ. Protoporphyrin Dr.N.Sivaranjani
- 18. I II IIIIV α β γ δ N N N N I II III IV Structure ofPorphyrin 1 2 3 4 56 7 8 1 2 3 4 5 67 8 Usual substitution are - Methyl, Propionyl, Vinyl & acetyl grDr.N.Sivaranjani
- 19. Based on presenceof side chain groups Uroporphyrin Coproporphyrin Protoporphyrin acetate and propionate methyl and propionate Methyl, Propionate & Vinyl Dr.N.Sivaranjani
- 20. Based on arrangementof side chain groups Type I Symmetric Type III Asymmetric – most predominant in biological system Dr.N.Sivaranjani
- 21. I II III IV A A A A P P P P I II III IV A A A A P P P P Structure of Uroporphyrin UroporphyrinI Uroporphyrin III Dr.N.Sivaranjani
- 22. I II III IV M M M M P P P P I II III IV M M M M P P P P Structure of Coproporphyrin CoproporphyrinI Coproporphyrin III Dr.N.Sivaranjani
- 23. I II III IV M M M M V V P P I II III IV M M M M V V P P Structure of Protoporphyrin ProtoporphyrinI Protoporphyrin III Dr.N.Sivaranjani
- 24. α β γ δ Fe++ I II IIIIV N N N N M VM M M PP V Structure ofHeme I II III IV M M M M V V P P Fe++ Protoporphyrin IX + Iron ( Fe++ ) Dr.N.Sivaranjani
- 25. Properties of Porphyrins Solubility - polar side chains - more soluble in blood Non polar side chains – less soluble Light absorption - All porphyrins absorb light maximally at 400 nm. When porphyrin combines with metal its absorption changes. Protoporphyrin IX absorbs light at 645 nm Heme (protoporphyrin IX + Fe) absorbs light at 545 nm Photosensitivity which is one of clinical symptom seen in some porphyrias is due to light absorption property of porphyrins All porphyrins are colored compounds Fluorescence - Porphyrins show fluorescence in organic solvents when they are exposed to UV-light. It is used to detect porphyrins in biological fluids. Dr.N.Sivaranjani
- 26. Fe++ N N N N NH ( Proximal F8) Histidine NH(Distal E7) Histidine O2 I II III IV Structure of Heme Fe++ ion has 6 valencies : 4 linked with N of pyrrole ring 5th - imidazole N of proximal histidine In OxyHb , 6th binds to O2 In deoxy Hb, water molecule present b/w Fe2+ and distal histidine Dr.N.Sivaranjani
- 27. Each Hemoglobin binds4 molecules of O2 Hb tetramer – highly soluble individual globin – insoluble unpaired globin precipitates – damage the RBC 1 chain Quaternary structure of Hb Heme Heme Heme 1 chain 2 chain 2 chain Heme Dr.N.Sivaranjani
- 28. Structural organization ofHb Primary structure – α globin chain – 141 a.a β,,δ chain – 146 a.a Secondary structure – each globin chain contains several helical segments β,,δ – 8 helices - A B C D E F G & H α – 7 helices - A B C E F G & H , lacks D Tertiary structure – globin chain is looped about itself to form a pocket for heme binding (prosthetic gr) Quaternary structure – Hb is tetramer – 2 pairs of globin chain (α2β2) with 4 heme. Subunits are held by non covalent bonds. Dr.N.Sivaranjani
- 29. Heme α chain -141 a.a β chain – 146 a.a α chain - 7 helices β chain – 8 helices Dr.N.Sivaranjani
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- 31. Hb is anIdeal respiratory pigment 1. Transport large quantities of O2 2. Great solubility 3. Take up & release O2 at appropriate partial pressures 4. Powerful buffer Hemoglobin is an Allosteric protein 1. Multimeric 2. Forms • Inactive / Tform (Deoxy-Hb) • Active/ R form (Oxy-Hb) 3. Regulated by allosteric modulators Dr.N.Sivaranjani
- 32. O2 dissociation curve(ODC): • The binding ability of Hb with O2 at different partial pressures of oxygen (pO2) can be measured by a graphic representation known as ODC. • Ability of Hb to unload & load O2 at physiological pO2 is shown by ODC. Dr.N.Sivaranjani
- 33. Steep slope (increased affinity of Hb to O2) Slow rise ( low affinity of Hb to first O2) Plateau ( Saturation of Hb ) Oxygen Dissociation Curve for Hemoglobin Sigmoid shaped curve Curve indicates that oxygen binding by Hb depends on partial pressure of O2Dr.N.Sivaranjani
- 34. • In pulmonaryalveoli - Hb is 97% saturated with O2 -Since O2 partial pressure is more in lungs • In tissue capillaries - where pO2 is 40mm of Hg, the Hb is about 60% saturated. • So physiologically - 40 % of O2 is released Tissues Lungs Sigmoid curve explains how Hb transports O2 from the lungs to tissues. • In tissue - O2 is liberated from Hb • In lung capillaries - O2 is taken by Hb. Dr.N.Sivaranjani
- 35. • Sigmoid shapeof ODC is due to allosteric effect or co-operativity. • Binding of oxygen to one heme increases the binding of oxygen to other hemes - Homotropic interaction. This is called positive cooperativity. • Thus the affinity of Hb for the last O2 is about 100 times greater than binding of first O2 to Hb. Dr.N.Sivaranjani
- 36. Quaternary structure ofHb exist in two alternate conformations : • T state (tense) – with low affinity for O2 – deoxy state • R state (relaxed) – with higher affinity for O2 – oxy state • In T subunits – the binding sites are closed. • In R state – the binding sites are open. • With successive addition of O2 , T shifts the equilibrium towards R state. Dr.N.Sivaranjani
- 37. Deoxy-Hb (Inactive / Tform) ↓O2 affinity Tissues Oxy-Hb ( Active/ R form) ↑O2 affinity Lungs Allosteric inhbition Favor release of O2 from Hb Allosteric activator Favor binding of O2 to Hb O2 O2 Oxygenation Deoxygenation Dr.N.Sivaranjani
- 38. Salt bridges Forms ofHemoglobin 1 Heme 2 1 Heme HemeHeme Deoxy-Hb ( T form) Salt bridges Restrict movement of subunits Heme pocket not accessible for O2 Decreased affinity for O2 2 Dr.N.Sivaranjani
- 39. Molecular changes duringOxygenation Rupture of salt bridges • Movement of iron atom into the plane of porphyrin ring. • Rotation of 22 subunits through 15º. Oxy-Hb( R form) 1 2 2 O2 O2 O2 O2 1 Dr.N.Sivaranjani
- 40. N N Fe++ Histidine NHHistidine (Distal E7) O2 Deoxy-Hb 0. 6nm NH (Proximal F8 ) N N NHHistidine (Distal E7) NN N NH Fe++ N ( Proximal F8 )Histidine Oxy-Hb Dr.N.Sivaranjani
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- 42. 1 2 1 2 Oxy-Hb (R state ) 1 2 2 1 15º 15ºO2 Rotation α1β1 and α2β2 dimers rotate 15⁰ towards another Deoxy Hb ( T state ) Dr.N.Sivaranjani
- 43. 1 Heme 2 1 2 Heme HemeHeme Deoxy-Hb T form (+) O2 Ruptureof salt bridges Dr.N.Sivaranjani
- 44. 1 2 2 1 Heme HemeHeme (+)O2 Hb-O2 O2 Dr.N.Sivaranjani
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- 47. 1 2 12 O2 O2 O2 O2 Hb-O8 (Rform) Cooperativity Heme-heme interaction Deoxy-Hb Hb-O2 Hb-O4 Hb-O8Hb-O6 O2 O2 O2O2 (2)(1) (4) (18) Dr.N.Sivaranjani
- 48. O2 Oxy Hb Rform LungsTissues O2 The binding and release of O2 is due to transition of Hb between T and R form Deoxy Hb T form Dr.N.Sivaranjani
- 49. Partial pressure ofo2 at which half of the Hb molecules are saturated with O2 P50 Significance Indicates the affinity of molecule to O2 (inversely related) Inc. P50 – dec. O2 affinity Dec. P50 – inc. O2 affinity Dr.N.Sivaranjani
- 50. Left shift ↓ P50 Rform ↑ P50 Right shift T form At low temperature, due to release of less O2 to the tissues Febrile condition : increased need of O2 . Dr.N.Sivaranjani
- 51. Factors affecting OxygenDissociation Curve Allosteric Modulators • 2,3 BPG • H+ concentration • CO2 • Temperature Dr.N.Sivaranjani
- 52. Effect of 2,3bisphosphoglycerate (2,3 BPG) Binds to deoxyHb and stabilizes it by formation of salt bridges (Negative cooperativity). Oxygen released to tissues hence it significantly reduces the affinity of Hb for O2 This reduced affinity allows Hb to release O2 efficiently at partial pressure found in tissues by shifting the O2 dissociation curve to right. Dr.N.Sivaranjani
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- 54. 2 1 2 1 Deoxy-Hb BPG pocket Mechanismof action of 2,3 BPG One molecule of 2,3 BPG binds in a pocket formed by globin chains in central cavity of deoxygenated T-form of Hb tetramer. On oxygenation this pocket collapse. Dr.N.Sivaranjani
- 55. Biomedical significance - Levelof RBC 2,3-BPG are related to tissue demands of O2 supply - ↑ in chronic hypoxia – high altitude , COPD. ↑ in severe Anemia - ADAPTATION to supply O2 Blood Transfusion – blood stored in acid citrate-dextrose -↓ 2,3 BPG in RBC. Rx - addition of inosine (hypoxanthine-ribose) prevents the dec.of 2,3-BPG Fetal Hb (HbF) – binding ability of BPG to HbF is LOW . This facilitates trans placental O2 transfer Dr.N.Sivaranjani
- 56. ↑ 2,3 BPG ↓2,3 BPG Significance • Hypoxia ↑2,3BPG due to deprivation of O2 favors unloading of O2 to tissues • Fetal blood ↓affinity of HbF to 2,3BPG, favors O2 to be transferred from maternal blood to fetal blood. Dr.N.Sivaranjani
- 57. ↑ H+ concentration ↓H+ concentration ↑ Partial pressure of CO2 ↓ Partial pressure of CO2 Tissues Effect of pH and pCO2 pH ↑ pH Lungs Dr.N.Sivaranjani
- 58. • Bohr Effect •Influence of pH and pCO2 to facilitate oxygenation of Hb in lungs and deoxygenation at the tissues is known as Bohr effect. Bohr effect - changes in H+ ions and CO2 conc. promote release of O2 in tissues. Haldane effect - inc. in conc. of CO2 will displace O2 from Hb & binding of O2 to Hb will displace CO2 from blood Dr.N.Sivaranjani
- 59. Red Blood CellCapillary H2CO3 Carbonic Anhydrase H++ HCO3 - HCO3 - Cl- Metabolically active tissue CO2 + H2OCO2 O2 utilized Bohr effect HbO8 Oxyhemoglobin H+ Hb 4O2 Deoxyhemoglobin pH dec Haldane effect cl- shift / Hamburger effect Buffer Dr.N.Sivaranjani
- 60. Effect of H+concenteration / Partial pressure of CO2 Metabolically active tissues Increased CO2 production Increased H+ concentration (Increased carbonic anhydrase activity) O2 delivered to metabolically active tissues Stabilizes deoxy Hb (low affinity / T form) by formation of salt bridges Dr.N.Sivaranjani
- 61. Red Blood CellCapillary O2+ HHb HbO2 Lungs O2 +H+ H2CO3 Air CO2 + H2O CO2Exhaled Carbonic Anhydrase Bohr effect HCO3 - Cl-HCO3 - pH inc. reversal of cl- shift C A Dr.N.Sivaranjani
- 62. Effect of pH,pCO2 - BOHR Effect Shift to right Dr.N.Sivaranjani
- 63. Method Of Transport% Carried • Dissolved form CO2 + H2O → H2CO3 → HCO3 + H+ • Carbamino hemoglobin without much change in pH. R–NH2 + CO2 --------- R–NH–COOH • As bicarbonate (HCO3 -) Isohydric transport-minimum change in pH during the transport. 5-10 10-15 80-90 Transport of carbon dioxide in blood Dr.N.Sivaranjani
- 64. Red Blood CellCapillary Metabolically activetissue CO2 Transport of carbon dioxide CO2 5-10% Hb-NH2 + CO2 Hb-NHCOOH CO2 10-15% H2O+ H2CO3 Carbonic Anhydrase H+ + HCO3 -HbO8 HHb4O2 CO2 80-90% Dr.N.Sivaranjani
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- 67. Left shift ↓ 2,3BPG ↓ H+ concentration ↓ pCO2 ↓ Temp. ↑ 2,3 BPG ↑ H+ concentration ↑ pCO2 ↑ Temp. O2 affinity is decreased O2 affinity is increased Tissues Lungs Right shift Dr.N.Sivaranjani
- 68. Combination of differentligands with heme part or change in oxidation state of Fe. • Oxy-Hb – Cherry red • Deoxy Hb – Purple • Met Hb – Dark brown • CO-Hb – Cherry red • Sulf-Hb – Green • Oxy Hb & Deoxy Hb are common derivatives present in blood. Hemoglobin Derivatives Dr.N.Sivaranjani
- 69. Detection of hemoglobinderivative Spectroscopic examination – when colored solutions of Hb derivatives are viewed through a spectroscope (simple device that resolves white light into 7 colors 400nm-700nm ) , dark lines or bands are seen in different spectrum of light. Microscopic examination of acid hematin Dr.N.Sivaranjani
- 70. Oxy Hb- shows 2 bands b/w D & E line First band – 540 nm – green region broad band Second band – 580 nm – yellow region narrow band Deoxy Hb – shows 1 band b/w D & E line, one band – 565 nm – green region Sodium hydrosulphite – deoxygenation Reoxygenation by vigorous shaking Carbamino Hb – CO2 with Hb Sulf Hb – sulfur containing compounds (sulfonamides) with Hb. cyanosis – impaired oxygenation of Hb. Cyan met Hb – Met Hb + cyanide. (abs. max at 540nm) Non-toxic , used in treating cyanide poisoning. Administration of NaNO2 - induces production of Met Hb, combines with CN- - CNMetHb – non toxic form – Excreted. Na thiosulphate – combines with CN- Dr.N.Sivaranjani
- 71. Met-hemoglobin Formation: Oxidation of Fe++(ferrous)toFe+++ (ferric) oxidized state Mechanism In Met-Hb the O2 cannot bind to Fe+++ Tissue hypoxia Symptoms Dr.N.Sivaranjani
- 72. Fe+++ N N N N NH ( Proximal F8) Histidine NH(Distal E7) Histidine O2 I II III IV Met-hemoglobin Dr.N.Sivaranjani
- 73. Hb-Fe+++ Hb-Fe++ Cyt b5 -Fe++ Cytb5 -Fe+++ NAD+ NADH+H+ Cytochrome b5 NADH met-Hb reductase system 75 % Normal level :<1% readily reduced Oxidants 20 % of reducing system – NADPH dep 5 % - Glutathione dependent Met-Hb reductase Dr.N.Sivaranjani
- 74. Met-hemoglobinemias Condition in whichlevel of met-hemoglobin in blood is high Types Congenital met-hemoglobinemias Acquired met-hemoglobinemias Dr.N.Sivaranjani
- 75. Congenital met-hemoglobinemias Acquiredmet-hemoglobinemias Due to inherited defect – mutation in globin chain eg: HbM Inherited deficiency of Cytochrome b5 NADH met-Hb reductase Dec. availability of NADPH due to inherited deficiency of G6PDH Chemicals: Nitrites Aniline dyes Aromatic nitro compounds Drugs: Sodium nitroprusside Acetaminophen Sulphanilamide Dr.N.Sivaranjani
- 76. Clinical features Cyanosis More than30% of met Hb- life threatening cond. Lab diagnosis • Colour of the blood – dark brown • Spectroscopic examination of blood- 2 bands 1 in 542 nm - green region and 2 in 633 nm - red region of spectrum Treatment Methylene blue (reducing agent) – regenerate NADPH Ascorbic acid Dr.N.Sivaranjani
- 77. Binding of COto one monomer of Hb (not reversible) Increases the affinity of other monomers to oxygen O2 bound to other monomers are not released Tissue hypoxia Affinity of CO to Hb is 200 times more than O2 Formation Binding of carbon monoxide to Hb Mechanism Carboxy hemoglobin Dr.N.Sivaranjani
- 78. Carbon monoxide poisoning Causes •Smoking • Inhalation of automobile exhaust fumes in closed space (commonest) • Exposure to Coal mining Clinical features Depends on amount of carbon monoxide saturation, Normal 0.1 % 5-10% Asymptomatic 30% toxic symptoms - Breathlessness, cyanosis, weakness, headache, vomiting , pain in chest & abd. > 50% life threatening - Coma & death Dr.N.Sivaranjani
- 79. Diagnosis • Colour ofblood- cherry red • Spectroscopic examination of blood- Absorption band similar to oxy Hb • Sodium dithionate convert oxy to deoxy • Fails to convert CO Hb Treatment • Administration of O2 - under severe case -O2 under high pressure (hyperbaric O2). • Blood transfusion Dr.N.Sivaranjani
- 80. Acid hematin /Ferri heme chloride • Fe is oxidized to ferric ,has net +ve charge which Can combine with –ve charge cl- to form hemin or hematin chloride. • Hemin crystals can be prepared from very old blood strains in medico- legal cases • Blood or eluted blood stains heated with Nippe’s fluid(1% KCL,KBr & KI in glacial acetic acid) over a glass slide. Dark brown rhombic crystals are seen under microscope. very sensitive but answered by heme part of blood of all species. Dr.N.Sivaranjani
- 81. Hemoglobinopathies Are a groupof genetic diseases due to mutations in the genes that code for globin chain or alpha chain (major variants) of Hb resulting in abnormal Hb or decreased amount of normal Hb . - Due to mutations in α or β globin chains - - α gene – 2 genes in 2 pairs on Chr.16 - β gene – 1 gene in 2 pairs on Chr.11 Dr.N.Sivaranjani
- 82. Hemoglobinopathies Type of defects 1.Qualitativedefects Mutations resulting in the production of structurally abnormal Hb molecules. Ex- Sickle cell Anaemia 2. Quantitative defects Mutations resulting in the production of decreased synthesis of normal Hb molecules. Ex -Thalassemia Dr.N.Sivaranjani
- 83. Sickle Cell Anemia/ HbS / Sickle cell Hb Most common hemoglobinopathy African American population Autosomal recessive –2 mutant gene are inherited Molecular defect : Presence of abnormal Hemoglobin- HbS Dr.N.Sivaranjani
- 84. HisVal ThrLeu GluProGlu 1 2 3 4 5 6 7 normal RBC ValProthrLeuHisVal Glu 1 32 4 5 6 7 β chain HbS ( Abnormal hemoglobin) Sickle shaped RBC GAG GUG β chain HbA ( normal hemoglobin) Point mutation in DNA due to substitution T for A Dr.N.Sivaranjani
- 85. sticky patch onsurface of HbS deoxy HbS has protrusion on one side & cavity on other Many molecules Adhere & Polymerize to form long fibers at low oxygen tension – distort cell shape Pathophysiology Glutamic acid Valine (hydrophobic)β6 HbA & HbF prevent sickling Dr.N.Sivaranjani
- 86. Sickle Cell Anemia OxyHbS Deoxy HbS Complementary site to sticky patch Sticky patch Oxy Hb Deoxy Hb HbS has dec. solubility in deoxy state. Dr.N.Sivaranjani
- 87. Polymerization of deoxyHbS Sticky patch of 1 deoxyHbS binds with complementary site of another deoxy HbS leading to polymerization of deoxy HbS to form gelatinous network of long fibrous polymer– Distort shape of RBC – sickle shape. Sickling occurs under deoxygenated state Dr.N.Sivaranjani
- 88. normal RBC Sickleshaped RBC Dr.N.Sivaranjani
- 89. Normal RBC flowfreely in blood vessels Rigid sickle shaped RBC block the blood flow in blood vessels Dr.N.Sivaranjani
- 90. Sickle cell anemiaSickle cell trait • Individual has received 2 mutant genes, one from each parent • Homozygous •Hbs only • Clinical symptoms •Individual has received 1 mutant gene from parent and normal gene from other parent • Heterozyous (AS) cond. •HbA + HbS in equal amounts •No clinical symptoms •At high altitudes – symptoms Dr.N.Sivaranjani
- 91. Sickle shape RBC’s Rigid Plugsin capillaries Occlusion of b.v – leads to infarction Organ damage (spleen, CNS, Bone, renal, liver, lungs etc)-Tissue damage & pain. Sickle cell crisis / vaso occlusive crisis – fever, acute pain, tenderness, anxiety, rpt episodes of pul. Inf, leg ulces, stroke, Gall stones & Jaundice Fragile Hemolysis (life span <20 days) Life long Hemolytic Anemia – pallor mucous memb, fatigue & dyspnea Increased susceptibility to infection Early death Homo-20yrs Clinical manifestation Dr.N.Sivaranjani
- 92. Normal RBC’s squeeze through bloodvessels Sickle shaped RBC’s block the Blood vessels Normal hemoglobin Deoxy HbS polymerisation Dr.N.Sivaranjani
- 93. • Hb Sgives protection against MALARIA • Malaria is caused by parasite- Plasmodium falciparum • Major cause of death in tropical areas (black Africans) • These malarial parasite spends a part of its life cycle in RBCs. Factors which interrupt the parasites cycle : Inc. lysis of sickled cells - shorter RBC life span. Parasites cause acidity of RBC – inc. sickling K+ conc. is low in sickled cell – parasite cannot survive • Sickle-cell trait (heterozygous with 40% HbS) provides resistance to malaria - Adaptation for the survival of the individuals in malaria infested regions • Homozygous - cannot live beyond 20 years. Dr.N.Sivaranjani
- 94. • Electrophoresis • Atalkaline pH – HbA move faster than HbS lack of negative charge on HbS , dec electrophoretic movement • At acidic pH – HbS move faster than HbA • Sickling test Diagnosis Dr.N.Sivaranjani
- 95. Point of application + Electrophoresisat pH 8.6 HbA HbS Normal Sickle cell trait Sickle cell anemia Dr.N.Sivaranjani
- 96. • Blood smearprepared • Reducing agent sodium dithionite added • Blood smear examined under microscope Sickling test Dr.N.Sivaranjani
- 97. • Anti sicklingagents – Hydroxyurea – inc. production of chain. • Sodium butyrate – induce HbF production • Cyanate – carbomylates N terminal a.a of Hb, eliminates salt bridges, inc. O2 affinity to Hb. Effective but toxic side effects – cataract , PNS damage. • 5-Azacytidine – inc. HbF – not used due to toxicity • Acute painful crisis – Hydration & Analgesics, Antibiotics • Repeated Blood transfusions – severe anemia Emerging treatments • Gene therapy , stem cell transplantation , inducing HbF expression Management Dr.N.Sivaranjani
- 98. TyrosineHemoglobin M Fe+++ N N N N I II III IV NH (Proximal F8 ) Histidine CAC UAC Histidine Tyrosine Dr.N.Sivaranjani
- 99. Examples of qualitativedefects Abnormal Hb Affected chain Base Substitution / A.A substitution Clinical features Movement on electrophoresis HbS Beta 6 GAG GUG Glu Val AS – asymp. SS - Hemolytic anemia slow movement than HbA HbC - Cooley`s Hb. Seen in black race Beta 6 GAG AAG Glu Lys AC – asymp. CC – hemolytic anemia SC – mod. disease slow movement than HbA HbE Second most prevalent West Bengal Beta 26 GAG AAG Glu Lys Hetero – asymp. Homo – dec. Hb Similar mobility as HbA2 Dr.N.Sivaranjani
- 100. Abnormal Hb Affected chain Base Substitution/ A.A Substitution Clinical feature Movement on electrophoresis HbD Punjab Beta 121 GAG CAG Glu Gln It migrates similar to HbS. HbM Alpha 58 Beta 92 CAC UAC His Tyr HbM Boston His Tyr Hb M Hyde Park Heme is oxidized to hemin , O2 binding is dec. Dr.N.Sivaranjani
- 101. Thalassemias Inherited Mediterraneanregion & Africa , India. DEFECT – - Insufficient synthesis - Total absence of either of α / β chains . Genes – α gene – 4 copies of genes (2 on each chr.16) β gene – 2 copies (1 on each Chr.11) Mutations – Deletions of one or more genes Underproduction or instability of mRNA Defect in initiation of chain syn.- mutation in promoter region Defect in protein synthesis – premature chain termination Dr.N.Sivaranjani
- 102. Types thalassemias: Insufficientproduction of globin chain thalassemias: Insufficient production of globin chain • β thalassemia is more common than α . • Thalassemia major – homozygous- severe form • Thalassemia minor – heterozygous –minimal symptoms Lack of coordination in α & β chains Impaired Hb synthesis. Formation of insoluble aggregates of excess chain damages RBC – Hemolytic Anemia. Dr.N.Sivaranjani
- 103. thalassemias Types Missing genes Symptoms Silenttrait 1 No symptoms thalassemias trait 2 Mild anemia – similar to β thalassemia minor Hb H disease – Heterozygous 3 Moderate anemia Hb barts (4tetramers) Hydrops fetalis 4 High O2 affinity – No. O2 to fetal tissue Tissue asphyxia, Edema, CCF & Fetal death Dr.N.Sivaranjani
- 104. thalassemias Types Missing genes Symptoms thalassemias minor / Heterozygous form / beta (+) 1 No symptoms thalassemias major / Homozygous form / beta (o) 2 Severe hemolytic Anemia, leg ulcers, hepatosplenomegaly, CCF, susceptibility to infection and Death within 2 yrs. Children – Chipmunk faces due to maxillary marrow hyperplasia, fontal bossing. co-existence of HbS & beta thalassemia trait is fairly common. Dr.N.Sivaranjani
- 105. α-globin chainsyn. is normal – α4 precipitates - No complementary chains to bind – ppt – shorter RBCs life span – Hemolytic anemia COMPENSATORY ↑ in γ,δ chains - ↑ HbA2, HbF. Diagnosed by – Smear – inclusion bodies – leads to membrane damage & destruction of red cells X ray – Hair- on- end appearance Rx Blood transfusion - but iron overload – Death15-20 yrs Splenectomy - lessen the anemia. Marrow transplantation Dr.N.Sivaranjani
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- 107. Myoglobin • Skeletal andheart muscle. • Single polypeptide chain with single heme moiety. Protein component GLOBIN (1) Non protein component HEME (1) Conjugated globular Monomeric protein Dr.N.Sivaranjani
- 108. Structure of Myoglobin Cterminus N terminus globin Heme Dr.N.Sivaranjani
- 109. A E F B C D G H Hydrophilic aminoacids: outer surface Hydrophobic amino acids : inner surface except E7 and F8 histidine residues amino acids = 153 Helices = 8 Globular structure Structure of globin Tertiary structure Dr.N.Sivaranjani
- 110. A E F B C D G H Heme Heme pocket :E & F helices Location of Heme Heme pocket 1 molecule of Mb combines with 1 molecule of O2 1 heme group/ Mb Dr.N.Sivaranjani
- 111. 10 20 30 40 50 60 70 80 90 100 Myoglobinsaturation% pO2 in mmof Hg Oxygen dissociation curve Rectangular hyperbolar curve 5 Excerising Muscle Resting Muscle •Vigorous exercise – O2 used up by muscle – PO2- falls •Mb releases O2 •Suitable for storage •Unsuitable for transport While Bohr effect, co-operative & 2,3 BPG effect are ABSENT. Dr.N.Sivaranjani
- 112. Lungs Muscle O2 Arteries Hemoglobin Function of Myoglobin Functionboth as Stores of O2 & readily releases it at pO2 of 5mmHg (exercising muscles) for mitochondrial synthesis of ATP myoglobin 20mmHg 5mmHg O2 Mitochondria Dr.N.Sivaranjani
- 113. Myoglobin in Urineand Blood Myoglobinuria – Mb (Small MW) is excreted through urine – dark red. Severe crush injury Myocardial infarction (MI) – serum Mb estimation is useful in early detection of MI. Dr.N.Sivaranjani
- 114. Difference Hemoglobin Myoglobin LocationRBC’s Muscles Polypeptide chains 4 1 Heme groups 4 1 Binds to oxygen 4 molecules 1 molecule Function Transport O2 and CO2 Store O2 ODC Sigmoid hyperbolar Co-operativity Present Absent 2,3 BPG effect Present Absent Bohr effect Present Absent Dr.N.Sivaranjani