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Dr.S.Chakravarty MBBS,MD

HEMOGLOBIN- STRUCTURE AND FUNCTIONS. Dr.S.Chakravarty MBBS,MD. Learning objectives:. At the end of the session a student will be able to :- Describe the properties of globular proteins Describe the structure and function of haemoglobin and myoglobin

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Dr.S.Chakravarty MBBS,MD

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  1. HEMOGLOBIN- STRUCTURE AND FUNCTIONS Dr.S.Chakravarty MBBS,MD

  2. Learning objectives: • At the end of the session a student will be able to :- • Describe the properties of globular proteins • Describe the structure and function of haemoglobin and myoglobin • Analyse the oxygen dissociation curve and factors affecting oxygen dissociation curve • Differentiate the features of fetal and adult haemoglobin • List the various hemoglobin variants and hemoglobin derivatives • Describe the formation of Glycatedhemoglobin and its use in monitoring diabetes

  3. Features of Globular proteins: • Globular nature - tertiary structure. • Good water solubility – hydrophobic core and hydrophilic surface. • catalytic/regulatory/transport role i.e. a dynamic metabolic function • Exists as colloids in the medium. USMLE Concept!!—what type of amino acids will be present in the core? Valine, arginine or glutamic acid ?

  4. Hemoproteins = Heme containing proteins • HEME + PROTEINS Eg.Hemoglobin • (Porphyrin ring + Iron) + Globin chains • (protoporphyrin IX + Iron)

  5. SOME IMPORTANT HEMOPROTEINS Imp. Concept :- Many drugs will induce cyt.p450-Induction of heme synthesis. ( Look into Porphyrias later!!)

  6. Porphyrins • Porphyrins are cyclic compounds formed by linkage of four pyrrole rings joined together by methenyl bridges(=HC-)bridges. • Characteristic property :- They form compelxes with metal ions bound to the nitrogen atom of the pyrrole rings. • Eg. Hemoglobin with Iron. Chlorophyll with magnesium .

  7. NATURAL PORPHYRINS HAVE SUBSTITUENT SIDE CHAINS • THERE ARE CHARACTERISTIC SIDE CHAINS SUBSTITUTED FOR THE 8 HYDROGENS.

  8. Simplified Representation of Heme

  9. Porphyrins • Porphyrins are cyclic compounds formed by linkage of four pyrrole rings joined together by methenyl bridges(=HC-)bridges. • Characteristic property :- They form compelxes with metal ions bound to the nitrogen atom of the pyrrole rings. • Eg. Hemoglobin with Iron. Chlorophyll with magnesium .

  10. O2 N N N N • Fe2+ bound to 4 Ns, other 2 bonds perpendicular to plane of ring available for bonding • In Hb, one of these attached to N terminus of His, other binds O2. His

  11. Usmle loves proximal and distal Histidine!! • Proximal histidine (F8 chain) – binds iron of heme • Distal histidine (E7 chain) – stabilizes oxygen after binding to iron

  12. NATURAL PORPHYRINS HAVE SUBSTITUENT SIDE CHAINS • THERE ARE CHARACTERISTIC SIDE CHAINS SUBSTITUTED FOR THE 8 HYDROGENS.

  13. Normal Hemoglobin Structure M V M M V P P M Porphyrin ring

  14. Simplified Representation

  15. Quaternary structure: • TETRAMERIC SRTUCTURE •  chain- 141 AMINO ACIDS • β chain- 146 AMINO ACIDS • Joined by • H bonds • Salt bridges • Van Der Walls forces • Iron must exist in Fe2+ state A salt bridge is actually a combination of two noncovalent interactions: hydrogen bonding and electrostatic interactions

  16. HEME HEME 2,3BPG HEME HEME

  17. Binding sites of various molecules on Hb: USMLE concept!! • Oxygen and carbon monoxide – Fe2+ • 2,3 BPG – crevice in T-form of Hb • Carbon-di-oxide – amino terminal end of globins. • H+ ions – histidine 146 of beta subunit.

  18. Oxygenation is accompanied by conformational changes

  19. Change from T form to R form The α2β2 (green)rotates 15 degrees relative to α1β1

  20. Reasons for Co-operativity • The Iron moves into the plane of the heme on oxygenation

  21. T structure to R structure • b Breaking of salt bridges as more Oxygen is added. The binding of oxygen to one heme residue increases the affinity of remaining Heme residues.

  22. OXYGEN DISSOCIATION CURVE Myoglobin RETAINS OXYGEN • P50 is the partial pressure of O2 that half-saturates a given Hb Hemoglobin DELIVERS OXYGENtissues At point X Myoglobin can attain half saturation at a lower partial pressure of oxygen = more affinity! x x USMLE concept!

  23. Oxygen dissociation curves Hb F Hb A

  24. Questions on p50 Concept for USMLE! • P50 is the partial pressure of O2 that half-saturates a given Hb • REMEMBER :- lower values of P50 corresponding to high affinity and vice versa!! • Low P50High affinity = less shift to right or shift to left ! • High P50 Low affinity = more shifting of ODC to right!

  25. Questions on p50 Concept for USMLE! • REMEMBER :- lower values of P50 corresponding to high affinity and vice versa!! • Low P50High affinity = less shift to right • High P50 Low affinity = more shifting of ODC to right! Answer :-Polycythemia !! Decreased p50 = increased affinity

  26. Shifts of the Oxygen Dissociation Curve Protons!! :- oxygen saturation is increasing

  27. Sources of protons that lower the pH in the tissues: • Production of lactic acid in the tissues: • Production of carbonic acid due to increased production of carbon dioxide in the tissues. • Co2 + H20 ------- > H2CO3------ > H+ + HCO3- (Carbonic anhydrase)

  28. Bohr effect – transport of CO2 Increased concentrations of CO2 and H+ promote the release of O2 from hemoglobin in the blood. • Carbon dioxide +water carbonic acid dissociates into protons and bicarbonate ions. • Deoxyhemoglobin acts as a buffer by binding protons and delivering them to the lungs. • In the lungs, the uptake of oxygen by hemoglobin releases protons that combine with bicarbonate ion, forming carbonic acid, which when dehydrated by carbonic anhydrase becomes carbon dioxide, which then is exhaled.

  29. Chloride shift or Hamburger effect • Cl- ions move into RBCS as HCO3- ions to maintain electrical neutrality.This is CHLORIDE SHIFT!! • Causes RBC to swell in venous blood .

  30. Why oxygenation disrupts salt bridges ? No ionic interaction in R form ( breaking of salt bridges) Presence of “salt bridge” in T form (his 146 with asp 94 )

  31. The Haldane effect what happens in the Lungs !! • The Haldane effect describes how oxygen concentrations determine hemoglobin's affinity for carbon dioxide. For example, high oxygen concentrations enhance the unloading of carbon dioxide. The converse is also true: low oxygen concentrations promote loading of carbon dioxide onto hemoglobin. In both situations, it is oxygen that causes the change in carbon dioxide levels.The Bohr effect, on the other hand, describes how carbon dioxide and H+ affect the affinity of hemoglobin for oxygen. High CO2 and H+ concentrations cause decreases in affinity for oxygen, while low concentrations cause high affinity for oxygen

  32. Role of 2,3 bisphosphoglycerate Absent crevice Crevice where 2,3 BPG binds

  33. BPG preferentially binds to deoxy- haemoglobin &stabilizesT-form + + + -ve -ve + -ve -ve + + BPG Binds Hb in a pocket b/w Beta chains.On oxygenation this pocket collapses

  34. Adult Hb Types • Hemoglobin A (α2β2) - The most common with a normal amount over 95% • Hemoglobin A2 (α2δ2) - δ chain synthesis begins late in the third trimester and in adults, it has a normal range of 1.5-3.5% • Hemoglobin F (α2γ2) - In adults Hemoglobin F is restricted to a limited population of red cells called F-cells. • However, the level of Hb F can be elevated in persons with Chronic hemolyticanaemias like sickle-cell disease and beta-thalassemia .

  35. Hb F α2 γ2 Greater affinity for oxygen This greater affinity for oxygen is explained by the lack of fetal hemoglobin's interaction with 2,3-bisphosphoglycerate (2,3-BPG or 2,3-DPG).

  36. Modifications of hemoglobin: Hemoglobin derivatives • Carbamino Hb • Carboxy Hb • Met Hb • Sulf Hb • Glycated Hb

  37. Carbamino hemoglobin: • Carbon-di-oxide binds to Hbto form caramino hemoglobin. • The N-terminal amino group Val forms carbamino complex with CO2 RNH2+CO2RNHCOOH • Transport of carbon dioxide in blood: • HCO3 – 75% • Bound to Hb – 15% • Dissolved in plasma – 10%

  38. Carboxy hemoglobin: CARBON MONOXIDE POISONING • Carbon monoxide affinity(approx 250:1) compared to oxygen. • PROBLEM NO UNLOADING OF OXYGEN TO TISSUES • Earlier days :- Burning in fireplace with windows closed • Tobacco smoking (through carbon monoxide inhalation) raises the blood levels • Pollution from Automobiles • Sleeping inside the car with engine onin a Closed garage • Cherry red –appearance of blood.

  39. Carbon monoxide poisoning: • Decreases hemoglobin saturation • Shifts oxygen dissociation curve to the left

  40. Methemoglobin • Iron in the heme group is in the Fe2+ (ferrous) state, not the Fe3+ (ferric) of normal hemoglobin. • Normally 1-2% exists as met-Hb • Chocolate brown color of blood. • Hb cannot bind to - oxygen

  41. Met-hemoglobin reductase About 20% of the reducing activity is directly due to NADPH also. A glutathione dependent Met-Hbreductase is also described.

  42. Methemogobinemia • Congenital causes: • Cytochrome b5 reductase(methhemoglobinreductase) deficiency • G6PD deficiency – def of NADPH • Hemoglobin M • Pyruvate kinasedeficiency – def of NADH 2. Acquired causes: • Environmental – aromatic amines, nitrates, chlorobenzene. • Drugs – sulpha drugs, nitroglycerine, chloroquine.

  43. Clinical features: • shortness of breath, cyanosis, mental status changes, headache, fatigue, exercise intolerance, dizziness and loss of consciousness. Lab analysis :- Absorption spectra at 633nm Treatment:IV methylene blue.

  44. Cyanmet-Hb • Cyanide poisoning • Lab Hb estimation by Drabkin’s reagent-cyanmetHb method

  45. Treatment of cyanide poisoning • Cyanide has affinity for heme proteins mainly cytochrome oxidase inhibition of cellular respiration . • Treatment:-The United States standard cyanide antidote kit first uses a small inhaled dose of amyl nitrite, followed by intravenous sodium nitrite, followed by intravenous sodium thiosulfate. • Amyl nitrite + sodium nitrite  forms met Hb. Cyanide has more affinity for met Hb forming cyanmeth Hb. • Sodium thiosulfate  reacts with cyanmeth Hb to form sodium thiocyanate (water soluble) • Methylene blue to revert Met Hb. 4. Vit B12 in the form of Hydroxycobalamin – forms cyanocobalamin – water soluble

  46. Glycation and glycosylation: • Attachment of sugars to protein: • Glycation – Nonenzymatic, slower process • Glycosylation – enzymatic and fast process

  47. Glycated hemoglobin • Glycationis a haphazard process that impairs the functioning of biomolecules – irreversible. • Sugars are highly reducing substances. • Binds to Hb of RBCs – stays until the death of RBCs. • Indicates the level of blood glucose. • Used in monitering Diabetes mellites.

  48. Heme “Proximal” histidine “Distal” histidine Structure of Myoglobin

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