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Hemoglobin – Structure, Function and Hemoglobinopathies

Hemoglobin – Structure, Function and Hemoglobinopathies. CONTENTS Introduction Biological importance Biomedical importance Structure function Hemoglobin derivatives Hemoglobinopathies. Introduction. Hemoglobin is a Hemoprotein ,

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Hemoglobin – Structure, Function and Hemoglobinopathies

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  1. Hemoglobin – Structure, Function and Hemoglobinopathies

  2. CONTENTS • Introduction • Biological importance • Biomedical importance • Structure function • Hemoglobin derivatives • Hemoglobinopathies

  3. Introduction Hemoglobin is a Hemoprotein, i.e., conjugated proteins that contain heme as prosthetic group. Heme is a Complex of porphyrin ring with an atom of iron in the ferrous (Fe++) form at its center.

  4. Biological importance • Hemoglobin (Hb) and myoglobin (Mb) – • Most abundant heme proteins in humans. • Hb - red pigment present in RBC’s / blood and functions mainly in the transport of O2, while • Mb - stores O2 in the muscles. • Other examples for hemeproteins are • cytochromes (of ETC) and • enzymes - catalase, tryptophan pyrrolase, etc.

  5. Biomedical Importance • Hemoglobin (Hb) : • Concentration of Hb : Human blood has approximately 15 g of Hb /dL blood. • Males : 14 – 16 g of Hb /dL blood • Females : 13 - 15 g of Hb /dL blood • Anemia : Decrease in concentration of Hb

  6. Biomedical Importance • Transport function • In mammals , only Hb can transport of O2 • Hb transports H+, CO2 and NO as well. • CO2 is transported as carbaminoHb attached to globin (15%). • NO is also transported through globinthiol group • Hb acts as a blood buffer.

  7. Biomedical Importance • Respiratory poisons - Cyanide and carbon monoxide kill • Reason : they disrupt the physiologic function of the heme proteins cytochromeoxidase and Hb, respectively

  8. Clinical Importance • Hemoglobinopathy : • Definition : Mutations in the genes that encode either one or both chains of Hb compromising on the biological function. • E. g., Sickle cell anemia (HbS) and Thalessemia.

  9. Structure of Hb • Human Hb is a conjugated protein. i.e., Globin (apoprotein ) + Heme (prosthetic group). • There are 4 chains with 4 heme moieties

  10. Primary Structure of Hb • The globin chains are of two types . i.e., • (i)  - chain (141 amino acids) - common in all forms of Hb and • (ii) Any one of the following •  Beta •  Gamma •  Delta •  Epsilon Chromosome 16 (146 amino acids) Chromosome 11

  11. Higher levels of Hb structure Secondary structure • The globin chain have helical segments interrupted by non-helical segments, which permit the polypeptide chains to fold into tertiary structure. Tertiary structure The polypeptide chains fold upon themselves to form a coiled structure, with a central hydrophobic core that acts as a heme pocket. Quaternary structure • Globin or the four polypeptide chains or the subunits are linked to one another by ionic bonds.

  12. Shape : nearly globular (spherical) molecule • Molecular weight : 67,000 daltons (67 Kda) Structure of Hb • Configuration of Globin part of Hb : a tetramer • i.e., made up of 4 polypeptide chains • Each polypeptide chain has a heme moiety attached to it

  13. Hemoglobin Valencies 6thvalency O2 in Hb O2 and H2O in deoxyHb linked to 4 Valencies - Nitrogens of the pyrrole rings 5hvalency His

  14. Hemoglobin Valencies • The iron in the heme - Fe++ form which has six valencies • 4 valencies - the four pyrrolenitrogens • 5thvalency - imidazole ring of a histidine residue of the globin chain and • 6thvalency - • O2 in oxyhemoglobin and by • H2O in deoxyHb. linked to

  15. Role of Iron in hemoproteins • (i) bind to O2 – either for transport (as in case of Hb ) • for storage (myoglobin ) • The iron atom in the Hb is in the ferrous state (Fe++) and can bind to O2 reversibly • Ferric (Fe+++) state cannot bind oxygen.

  16. Role of Iron in hemoproteins • (i) bind to O2 – either for transport (as in case of Hb ) • for storage (myoglobin ) • The iron atom in the Hb is in the ferrous state (Fe++) and can bind to O2 reversibly • Ferric (Fe+++) state cannot bind oxygen. • (ii) as a carrier of electron where it oscillates between Fe+++ (oxidised) and Fe++(reduced) states Fe+++ Fe++ e- • (as in cytochromes)

  17. Principal forms of Hb in adult blood • HbA (adult Hb) • HbA2 and • HbF (foetalHb) • The different forms of Hb differ in the 2 other chains each of these have different subunit make-up.

  18. Forms of Hb – subunit make up and amount in blood Form Chain Composition Fraction of Total Hb HbA22 97 % HbA222 2 % HbF22 1 % In each form of Hb the subunits are held by weak non covalent forces – Vander Waals forces, Hydrogen bonds and Ionic bonds

  19. Gene expression of hemoglobin before and after birth

  20. HbA • is the major form of Hb in adults • 97% of the total Hb in blood • Under physiological conditions, HbA is slowly and non-enzymaticallyglycosylated. • i.e., HbA is modified by the covalent addition of hexoses • the extent of glycosylation is proportionate to the plasma concentration of a particular hexose.

  21. GlycosylatedHb - HbA1c • HbA1c has glucose residues attached to -globin chains of Hb in RBCs. - non enzymatically • 2 2 - glucose [normal <5.5 % 7% adequate control 9% poor control] • Clinical importance : • HbA1c could be used to monitor the control of the blood glucose level during the last 10 – 12 weeks for diabetic patients

  22. Structure of myoglobin Myoglobin is • a monomeric protein, i.e., contains only • one polypeptide chain (146 amino acids) and • one heme moiety • Can only store O2 and not transport

  23. Functions of Hb • 1) Main function : Transport of respiratory gases - • A) O2 in blood from the lungs to the tissues. • B) CO2 from tissues to the lungs. • 2) Hb acts as an important blood buffer. • Each Hb molecule binds 2 protons generated from CO2 and H2O.

  24. Functions of Hb • Each globin chain of Hb can bind one heme moiety • Hence each Hb molecule has 4 heme groups • and can bind a maximum of 4 molecules of O2 at a time. • The phenomenon is called as ‘cooperative binding of oxygen’

  25. States of Oxy and DeoxyHb

  26. Mechanism of Cooperative binding and release of O2 O2 O2 O2 O2 Confirmational change in one of the subunits which binds to oxygen O2 O2 O2 O2 O2 DeoxyHb ‘T’ form (taut form) O2 O2 O2 O2 O2 O2 O2 O2 O2 Oxy Hb (HbO2) ‘R’ form (Relaxed form) Confirmational change in one of the subunits which releases oxygen

  27. Factors Affecting the O2 delivery • Factors which affect the oxygen unloading from oxyHb at the peripheral tissues are 1) pH 2) pCO2 and 3) 2, 3 bisphosphoglycerate (2, 3 BPG)

  28. Oxygen Dissociation Curve (ODC)

  29. Mechanism of the factors • *Low pH, • **High pCO2 • 2, 3 BPG. • *In the peripheral tissues pH is low because of production of organic acids and CO2 by tissues. • **Effect of pCO2 is indirect by its effect on pH. Increase of pCO2 lowers the pH. stabilize deoxyHb in T state  the affinity of oxygen for Hb favors unloading of oxygen

  30. Action of 2, 3 Bisphospoglycerate (2,3 BPG) 2,3 BPG shunt (Glycolysis) HbO2 • NOTE :  - chains of HbF cannot bind 2,3 BPG . • HbF has high affinity for O2 even in presence of 2,3-BPG. Significance : This allows fetal blood to extract O2 from maternal blood. 2, 3 BPG  In conditions : Hypoxia, High altitudes  Peripheral tissues O2 2, 3 BPG - Hb

  31. HCO3 – Plasma Transport of CO2 Tissues Dissolved in plasma (10%) (75%) CO2 + H2O H2CO3 H + + HCO3 – HbO2 HHbO2 HHb RBC Carbonicanhydrase CO2 Metabolism Carbamino compounds (25%) Isohydric shift Release of O2 Cl- O2 HCO3- Cl-

  32. 2) Transport of CO2 • Production : About 200 ml of CO2 is produced /minute in the tissues at rest. • Transported in 3 different forms: • i) 75% of CO2 are transported as bicarbonate (HCO3–). • ii) 15% of CO2 produced is transported as carbaminoHb * • iii) About 10% of this is transported as dissolved form. • *CO2 binds to the free – NH2 groups at the N-terminal of the globin chains in Hb to form carbamino compounds.

  33. 3) Buffering Action • Hb transports CO2 from tissues to the lungs as HCO3- with minimum change in pH.

  34. HCO3 – Plasma Transport of CO2 Lungs HHb RBC O2 O2 • HHbO2 100 mmHg • H+ + HbO2 HCO3- CO2 Reverse Isohydric shift H2CO3 Carbonicanhydrase Cl- H2 O +CO2 Release of CO2 HCO3- Cl-

  35. Hemoglobin Derivatives – Methemoglobin and Carboxyhemoglobin Methemoglobin Hb with Fe3+ ions is known as methemoglobin (met-Hb). Formation of Met-Hb : Fe2+ Fe3+ 1. Oxidizing agents - H2O2, free radicals or oxidant drugs (such as sulfonamides), etc., 2. Hereditary - Congenital Methemoglobinemia.

  36. Carbon MonoxyHb / Carboxy – Hb (CO-Hb) Formation : CO + Hb CO-Hb Hb has 200 times more affinity for CO than O2. Normal levels : 0.16% of CO-Hb. An average smoker has an additional 4% of CO-Hb. Clinical symptoms manifest when CO-Hb levels exceed 20%. At about 40-60% saturation, coma and death can occur.

  37. Buffering Action……. • Each Hb molecule binds 2 protons generated from CO2 and H2O. Carbonic Anhydrase (in RBC) CO2 + H2O H2CO3 H+ + HCO3– (Peripheral tissue) HHb Hb

  38. TESTS TO DETECT HEMOGLOBIN / BLOOD : o Benzidine test for blood in urine (Qualitative) o Hemin crystals o Spectroscopy : absorption spectra used for clinical and forensic applications Hemoglobin in blood by Sali’s method (Quantitative))

  39. Hemoglobin Variants Over 900 mutant human Hb’s are known. However, they are rare and benign showing no clinical abnormalities. • Hemoglobinopathies Definition : A condition when a mutant Hb causes clinical abnormality (loss of biological function)

  40. Hemoglobinopathies The genetic defect (mutation) may result in abnormal Hb : synthesis of structurally abnormal globin (altered primary structure of or chain) with altered function. E.g., : Sickle cell anemia (HbS), hemoglobin C disease (HbC) or (ii) synthesis of globin chains in insufficient quantities E.g., Thalassemia.

  41. Sickle Cell Hemoglobin (HbS) and Sickle Cell Anemia • Occurrence : is the most common form of mutant/ abnormal Hb. • Sickle cell anemia is largely confined to • Tropical areas of the world - primarily in black population. • Tribal population in various parts of India.

  42. HbS - Defect Mechanism of Sickling : Hydrophilic polar amino acid Hydrophobic nonpolar amino acid 6th position of  chain

  43. HbS - Defect Mechanism of Sickling : Long fibrous precipitates of deoxyHbS Condition : at low [O2]

  44. Clinical Manifestations of Sickle Cell Anemia Sickled RBC’s are fragile Cause hemolysis, Block capillaries Result : Poor blood supply to tissues This results in 1. Life long hemolytic anemia 2. Tissue damage and pain 3. Increased susceptibility to infections and 4. Premature death.

  45. Heterozygous Individuals are resistant to Plasmodium falciparum Malaria Malarial parasite (plasmodium falciparum) - part of its life cycle in RBCs. Sickled RBCs have shorter life span and the life cycle of the parasite is affected. Diagnosis : Sickle cell anemia is diagnosed by electrophoresis, where HbS migrates faster than HbA towards anode.

  46. Sickle cell anemia is diagnosed by electrophoresis in acidic medium HbS will moves faster than HbA Electrophoresis in alkaline medium - the HbS will moves slower and HbA will move faster

  47. Thalassemias • Thalassemiasare a group of hereditary hemolytic disorders characterized by impairment in the synthesis of globin chains. • Molecular Basis : Thalassemias are characterized by a defect in the production of either  or  - globin chain, but there is no abnormality in the primary structure of the individual chains.

  48. Thalassemias • Two types • Molecular basis : depending on the globin chains that are defective. • -Thalassemia : This is caused by decreased synthesis or complete absence of -globin chains. • -Thalassemias:Results due to decreased synthesis or total lack of- -globin chains. • In thalassemia, life span of RBCs is decreased leading to hemolytic anemia.

  49. Question Bank Hemoglobin –Structure, Function, Abnormal Hb • 1. Carbonic anhydrase. (3) • 2. Chloride bicarbonate shift (3) • 3. Chloride shift (3/4) • 4. Chloride bicarbonate shift (2) • 5. Name 3/4 Heme proteins and their functions (3/4) • 6. How is 2, 3 –Bisphosphoglycerate (2, 3- BPG) synthesized in erythrocytes? What is the importance of 2, 3 –BPG in erythrocytes? (3) • 7. Methemoglobin (3/4) • 8. Carboxy hemoglobin. (3) • 9. Explain the biochemical basis: carbon monoxide poisoning leads to hypoxia. (3) • 10. Discuss the manifestation molecular basis and laboratory diagnosis of Sickle cell disease. What is the biological advantage of Sickle cell trait? (3/4) • 11. Sickle cell hemoglobin (3/4) • 12. Explain the biochemical basis: sickling of RBCs in sickle cell anemia. (3) • 13. Hemoglobinopathies. (3/4)

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