Hemoglobin

I AM MD TRANSFUSION MEDICINE PG @ SCTIMST, TRIVANDRUM

1. HEMOGLOBIN Dr.Gayathri.A.M . PRESENTATION-4

2. Haemoglobin (C 712 H 1130 O 245 N 214 S 2 Fe) 4

3. INTRODUCTION Oxygen binding globular protein of red blood cell with quaternary structure Tetramer metalloprotein that binds to oxygen, transports O2 to the tissues and returns CO2 and protons to the lungs Consists of four polypeptide subunits; 2 alpha chains(141AAs) and two beta chains(146AAs) Molecular wt. : 64,450 D

4. HEME + GLOBULIN + =

5. MAX PERUTZ 3D structure of hemoglobin was solved using X-ray crystallography in 1959 by Max Perutz (Cambridge)

6. HEME Haeme is Iron – porphyrin complex called IRON-PROTOPORPHYRIN IX Cyclic tetra- pyrrole consisting of four molecules of pyrrole linked by a- methylene bridges This planar network of conjugated double bonds absorbs visible light and colors heme deep red Substituents at the b -carbons of pyrrole ring are methyl (M), vinyl (V), and propionate (Pr) groups arranged in the order M, V, M, V, M, Pr, Pr, M

7. 4 PYRROLE RINGS + 4 METHYLENE BRIDGES + Fe 2+ = HEME

8. One atom of ferrous iron (Fe2+) resides at the center of the planar tetrapyrrole Oxidation of the Fe2+ of Hb to Fe3+ destroys their biologic activity M V M M Pr V Pr M

9. GLOBIN Globin in HbA is made up of 4 polypeptide chains: 2 a chains – 141 amino acids & 2 b chains – 146 amino acids Individual globin units are insoluble 1 units of Heme attached to 1 unit of Globin hence 1 Haemoglobin molecules contains 4 Iron Atoms which carry 4 molecules of oxygen Exterior surface of Hb is polar (hydrophilic) & Inner surface non polar (hydrophobic) which encloses the heme moiety

10. GLYCINE + SUCCINYL Co.A ALA SYNTHASE + VIT. B 6 AMINOLEVULINIC ACID (ALA) PORPHOBILINOGEN (PBG) ALA DEHYDROGENASE HYDROXYMETHYLBILANE (HMB) UROPORPHYRINOGEN III (UPG) COPROPORPHYRINOGEN III (CPG) PBG DEAMINASE UPG SYNTHASE UPG DECARBOXYLASE FEEDBACK INHIBITION BY HEME & STIMULATED BY CYP INDUCERS BLOCKED BY LEAD CONGENITAL DISORDER DUE TO DEFICIENCY- AIP CEP PCT HEME SYNTESIS

11. COPROPORPHYRINOGEN III (CPG ) PROTOPORPHYRINOGEN III (PPG) PROTOPORPHYRIN IX (PP) + Fe 2+ HEME FERROCHELATASE BLOCKED BY LEAD

12. HEME SYNTHESIS- MITOCHONDRIA GLOBIN SYNTHESIS- RIBOSOMES

13. Various types of globin combines with heme to form different haemoglobin Eight functional globin chains, arranged in two clusters: b - cluster (b, g , d and e globin genes) on the short arm of Chromosome 11 a - cluster ( a and z globin genes) on the short arm of Chromosome 16 GLOBIN SYNTHESIS

14. Globin synthesis, starts at 3rd week of gestation Embryonic Haemoglobin Gower I ( z 2 e 2 ) Haemoglobin Portland ( z 2 g 2 ) Haemoglobin Gower II ( a 2 e 2 ) Fetal : HbF ( a 2 g 2 ), HbA ( a 2 b 2 ) Adult : HbA (96-98 %), HbA2( a 2 d 2 )[1.5-3.2%], HbF (0.5-0.8%)

15. GLOBIN CHAIN SWITCH

16. HEME + POLYPEPTIDE CHAINS HEMOGLOBIN #SOURCE OF PROTEIN: FOOD OF ANIMAL ORIGIN, MUSCLES, etc. #SOURCE OF IRON: DIETARY IRON & FROM DEGRADATION OF RBCs.

17. IRON ABSORPTION IRON METABOLISM

18. ROLE OF OTHER METALS & VITAMINS Copper – Promotes Absorption, Mobilization & Utilization of Iron Cobalt – Increases production of Erythropoietin Calcium – Conserve Iron & subsequent utilization VIT B12, Folic acid - Help in synthesis of nucleic acid VIT C - Helps in absorption of iron from gut (Fe3+ to Fe2+) Role of bile salts - Important for proper absorption of copper & nickel

19. CONFORMATIONAL CHANGES FOLLOWING O 2 BINDING Hemoglobin bind four molecules of O 2 per tetramer, one per heme COOPERATIVE BINDING: A molecule of O 2 binds to a hemoglobin tetramer more readily if other O 2 molecules are already bound This maximise both quantity & quality of O 2 loaded & released at various PO 2

20. Binding of the first O 2 molecule to deoxyHb shifts the heme iron towards the plane of the heme ring from a position about 0.04 nm beyond it This motion is transmitted to the proximal (F8) histidine and to the residues attached to it, which in turn causes the rupture of salt bridges between the carboxyl terminal residues of all four subunits One pair of a or b subunits rotates 15 degrees with respect to the other, compacting the tetramer

21. The iron atom moves into the plane of the heme on oxygenation Histidine F8 and its associated residues are pulled along with the iron atom

22. AFTER RELEASING OXYGEN AT TISSUES, Hb TRANSPORT CO 2 & PROTONS INTO LUNGS CO 2 + Hb – NH 3 + 2H + + Hb—NH– COO — Carbamates change the charge on amino terminals from positive to negative, favoring salt bridge formation between the a and b chains Hb carbamates : about 15% of the CO 2 in venous blood Remaining CO 2 is carried as bicarbonate, which is formed in RBC by the hydration of CO 2 to carbonic acid (H 2 CO 3 ), a process catalyzed by carbonic anhydrase CO 2 + H 2 0 H 2 CO 3 HCO 3 -- + H + DeoxyHb binds one proton for every two O 2 molecules released, contributing significantly to the buffering capacity of blood

23. Lower pH & carbamation , stabilizes the T state at peripheral tissue level enhances the delivery of O 2 In lungs, when O 2 binds to deoxyHb , protons are released & combine with bicarbonate to form carbonic acid & its dehydration catalyzed by carbonic anhydrase forms CO 2 , which is exhaled This reciprocal coupling of proton and O2 binding is termed the Bohr effect The Bohr effect is dependent upon cooperative interactions between the hemes of the Hb tetramer

24. BOHR EFFECT

25. PROTONS ARISE FROM RUPTURE OF SALT BRIDGES WHEN O2 BINDS Protons responsible for the Bohr effect arise from rupture of salt bridges during the binding of O 2 to T state Hb Conversion to the oxygenated R state breaks salt bridges involving chain residue His 146 which leads to dissociation of protons& it inturn drives the conversion of bicarbonate to carbonic acid Upon the release of O 2 , the T structure and its salt bridges re-form & this conformational change increases the p Ka (acid dissociation constant) of the chain His 146 residues, which bind protons By facilitating the re-formation of salt bridges, an increase in proton concentration enhances the release of O2 from oxygenated (R state) Hb Conversely, an increase in PO 2 promotes proton release.

26. 2,3-BPG & HEMOGLOBIN A low PO 2 in peripheral tissues promotes the synthesis of 2,3-BPG in erythrocytes from the glycolytic intermediate 1,3-BPG The hemoglobin tetramer binds one molecule of BPG in the central cavity formed by its four subunits However, the space between the H helices of the b chains lining the cavity is sufficiently wide to accommodate BPG only when Hb is in the T state( deoxy Hb) BPG forms salt bridges with the terminal amino groups of both b chains BPG therefore stabilizes deoxy (T state) Hb by forming additional salt bridges that must be broken prior to conversion to the R state

27. Residue H21 of the b subunit of HbF is Ser rather than His Since Ser cannot form a salt bridge, BPG binds more weakly to HbF than to HbA The lower stabilization afforded to the T state by BPG accounts for HbF having a higher affinity for O 2 than HbA .

28. OXY & DEOXY Hb

29. OXYGEN DISSOCIATION CURVE

30. ADAPTION TO HIGH ALTITUDES Physiologic changes include an increase in the number of erythrocytes and in their concentrations of hemoglobin and of BPG Elevated BPG lowers the affinity of HbA for O 2 (increases P50 ), which enhances release of O 2 at the tissues

31. BIOMEDICAL SIGNIFICANCES ANEMIA HEMOGLOBINOPATHIES(THALASSEMIAS, HbM , MET HB, HBS ) PORPHYRIA

32. ANEMIA Reduction in the number of red blood cells or of hemoglobin in the blood due to impaired synthesis of Hb ( eg , in iron deficiency) or impaired production of erythrocytes ( eg , in folic acid or vitamin B12 deficiency) Diagnosis : with spectroscopic measurement of blood hemoglobin levels

33. HEMOGLOBINOPATHIES It is estimated that more than 7% of the globe's population are carriers for Hb disorders Mutations in the genes that encode the a or b subunits of Hb potentially can affect its biologic function When a mutation does compromise biologic function, the condition is termed a hemoglobinopathy

34. THALASSEMIAS Genetic defects resulting in the partial or total absence of one or more a or b chains of Hb. Over 750 different mutations have been identified, but two are very common. Either the a chain (alpha thalassemias ) or b chain (beta thalassemias ) can be affected. A superscript indicates whether a subunit is completely absent ( a or b ) or whether its synthesis is reduced ( a + or b + ). Apart from marrow transplantation, treatment is symptomatic

35. HEMOGLOBIN F H EMOGLOBIN F ( HbF , α 2 γ 2 ) accounts for up to 90% of the circulating hemoglobin at birth Its synthesis starts to decline during the third trimester, and over the first year of life it is gradually replaced by adult hemoglobin, HbA (α 2 β 2 ) Normal adults have <1% of HbF , apparently confined to a subset of red blood cells called F cells which constitute about 3% of RBCs Most of the genetic disorders associated with persistent HbF production involve alterations of the structure of the β globin cluster The highest adult levels of HbF are seen in β and δβ thalassemia , or hereditary persistence of fetal hemoglobin (HPFH), in which HbF can constitute up to 100% of the hemoglobin; & sickle cell disease( HbF 5% and 20% of the total Hb)

36. METHEMOGLOBIN & HEMOGLOBIN M METHEMOGLOBINEMIA : Heme iron is ferric rather than ferrous, which can neither bind nor transport O 2 Methemoglobin can arise by oxidation of Fe2+ to Fe3+ as a side effect of agents such as sulfonamides , from hereditary hemoglobin M, or consequent to reduced activity of the enzyme methemoglobin reductase (reduce Fe 3+ of MetHb to Fe 2+ ) HEMOGLOBIN M : Histidine F8 (His F8) has been replaced by tyrosine Iron of HbM forms a tight ionic complex with the phenolate anion of tyrosine that stabilizes the Fe3+ form

37. In a -chain hemoglobin M variants , the R-T equilibrium favours the T state . Oxygen affinity is reduced, and the Bohr effect is absent. b -Chain HbM variants exhibit R-T switching , and the Bohr effect is therefore present. Mutations that favour the R state ( e.g., Hb Chesapeake ) increase O 2 affinity, thereby fail to deliver adequate O 2 to peripheral tissues resulting in tissue hypoxia that leads to polycythemia, an increased concentration of erythrocytes

38. HEMOGLOBIN S In HbS , the nonpolar amino acid valine has replaced the polar surface residue Glu6 of the b subunit, generating a hydrophobic "sticky patch" on the surface of the b subunit of both oxyHbS and deoxyHbS Both HbA and HbS contain a complementary sticky patch on their surfaces that is exposed only in the deoxygenated T state,thus at low PO 2 , deoxyHbS can polymerize to form long, insoluble fibers Binding of deoxyHbA terminates fiber polymerization, since HbA lacks the second sticky patch necessary to bind another Hb molecule

39. Representation of the sticky patch on hemoglobin S and its " receptor"on deoxyHb A and deoxyHbS . The complementary surfaces allow deoxyHbS to polymerize into a fibrous structure, but the presence of deoxyHb A will terminate the polymerization by failing to provide sticky patches

40. These twisted helical fibers distort the erythrocyte into a characteristic sickle shape, rendering it vulnerable to lysis in the interstices of the splenic sinusoids. They also cause multiple secondary clinical effects. A low PO 2 such as that at high altitudes exacerbates the tendency to polymerize. Emerging treatments for sickle cell disease include inducing HbF expression to inhibit the polymerization of HbS , stem cell transplantation, and, in the future, gene therapy

41. GLYCATED HEMOGLOBIN When blood glucose enters the RBCs, it glycates the e -amino group of lysine residues and the amino terminals of Hb. The fraction of hemoglobin glycated , normally about 5%, is proportionate to blood glucose concentration. Since the half-life of an erythrocyte is typically 60 days, the level of glycated hemoglobin (HbA1c ) reflects the mean blood glucose concentration over the preceding 6–8 weeks. Measurement of HbA1c therefore provides valuable information for management of diabetes mellitus

42. HEMOGLOBIN IN TRANSFUSION MEDICINE Criteria for donor selection: > 12.5g/dl ( > 38%) for allogenic transfusion & > 11g/dl ( > 33%) for autologous transfusion The methods to measure hemoglobin : Copper sulfate density method (semi-quantitative method) Spectrophotometric portable devices (quantitative method): HemoCue Donor Hb Checker, DiaSpect Hemoglobin Hematology analysers (quantitative method)

43. RED CELL CONCENTRATE TRANSFUSION CRITERIA FOR ANAEMIC PATIENTS The only indication for the transfusion of RCC is to correct or prevent tissue hypoxia; thus, the parameter “of choice” for making decisions should be intracellular PO 2 This parameter is not, however, usable for clinical purpose, instead Hb and the haematocrit ( Htc ) values are assessed Transfusion is recommended if Hb level is <6g/dl In hemolytic anaemias , there is no need for transfusion even though if Hb is <5g/dl unless hypoxic symptoms are present CRITERIA FOR TREATMENT OF ANEMIA

44. PORPHYRIAS   Group of disorders that result from increased production of porphyrin in the body mainly affecting skin & nervous system Occurs due to mutation in genes responsible for heme synthesis leading to specific enzyme deficiencies Symptoms of acute  porphyria  include abdominal pain, chest pain, vomiting, confusion, constipation, fever, and seizures Condition mimicking porphyria: lead poisoning, alcoholic liver disease.

45. Contn …… Triggers: Stress, drugs like sulfonamides , sulfonylureas , barbiturates, antifungals , progestrone,certain anticonvulsants & antibiotics, etc. Types : Autosomal dominant : Acute intermittent porphyria, Porphyria cutanea tarda , Hereditary coproporphyria , Porphyria variegata & Erythropoietic protoporphyria Autosomal recessive : Congential erythropoietic porphyria

46. REFERENCES Harper's Illustrated Biochemistry, 28 th Edition Harrison Manual of Medicine,19 th Edition AABB Technical Manual, 17 th Edition

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