HEMOGLOBIN DERIVATIVES

DERIVATIVES OF HEMOGLOBIN

1. Haemoglobin Derivatives Gandham . Rajeev

2. Hemoglobin derivatives are formed by the combination of different ligands with the heme part, or change in the oxidation state of iron .

3. Carboxy-Hemoglobin ( CO- Hb ) Hemoglobin binds with carbon monoxide ( CO) to form carboxy-Hb . The affinity of CO to Hb is 200 times more than that of oxygen. It is then unsuitable for oxygen transport .

4. When one molecule of CO binds to one monomer of the hemoglobin molecule, it increases the affinity of others to O2; so that the O2 bound to these monomers are not released . This would further decrease the availability of oxygen to the tissues.

5. Carbon Monoxide Poisoning CO is a colorless , odorless , tasteless gas generated by incomplete combustion. CO poisoning is a major occupational hazard for workers in mines. Breathing the automobile exhaust in closed space is the commonest cause for CO poisoning

6. The carboxy-Hb level in normal people is 0.16%. An average smoker has an additional 4% of CO- Hb . One cigarette liberates 10–20 ml carbon monoxide into the lungs .

7. Clinical Manifestations Clinical symptoms manifest when carboxy-Hb levels exceed 20%. Breathlessness , headache , nausea , vomiting, & chest pain. At 40-60 % saturation , death can result. Administration of O2 is the treatment.

8. Methemoglobin (Met- Hb ) When the ferrous ( Fe 2+ ) iron is oxidized to ferric (Fe 3+ ) state, met- Hb is formed. Small quantities of met- Hb formed in the RBCs are readily reduced back to the ferrous state by met- Hb reductase enzyme systems. About 75 % of the reducing activity is due to enzyme system using NADH & cytochrome b5

9. Methemoglobinemias Normal blood has only less than 1% of methemoglobin . It has markedly decreased capacity for oxygen binding and transport . An increase in methemoglobin in blood, ( methemoglobinemia ) is manifested as cyanosis . Causes may be congenital or acquired .

10. Congenital Methemoglobinemia Presence of Hb variants like HbM can cause congenital methemoglobinemia . Cytochrome b5 reductase deficiency is characterized by cyanosis from birth. 10-15 % of hemoglobin may exist as methemoglobin . Oral administration of methylene blue , 100-300 mg/day or ascorbic acid 200-500 mg/day decreases met- Hb level to 5-10% and reverses the cyanosis .

11. Acquired or Toxic Methemoglobinemia Met- hemoglobinemia may develop by intake of water containing nitrates or due to absorption of aniline dyes. Drugs which produce met- hemoglobinemia - acetaminophen , phenacetin , sulphanilamide, amyl nitrite, & sodium nitroprusside .

12. Sulf-hemoglobinemia When hydrogen sulfide acts on oxy- Hb , sulf-hemoglobin is produced. It occur in people taking drugs like sulphonamides, phenacetin , acetanilide , dapsone , etc. It cannot be converted back to oxy- hemoglobin .

13. Hemoglobinopathies Abnormal hemoglobins are the resultant of mutations in the genes that code for α or β chains of globin As many as 400 mutant hemoglobins are known. About 95% of them are due to alteration in single amino acid of globin

14. Types of abnormal Hb Two types: If the mutation affects structural gene, it results in replacement of a single amino acid in Hb by some other amino acid resulting into abnormal Hb . E.g : Hb -S, Hb -M, Hb -C, Hb -D & others.

15. If the mutation affects the regulator gene, which affects the rate of synthesis of peptide chains, the amino acid sequence remains unaffected. E.g : Thalassaemias

16. G lobin synthesis The globin genes are organised into two gene families or clusters α -Gene family: There are 2 genes coding for α -globin chain present on each one of chromosome 1 6. The ζ (zeta)-gene , other member of a-gene cluster is also found on chromosome 16 & is active during the embryonic development

17. β -Gene family: The synthesis of β-globin occurs from a single gene located on each one of chromosome 11. This chromosome also contains four other genes . One ε -gene expressed in the early stages of embryonic development.

18. Two γ -genes ( Gγ & Aγ ) synthesize γ-globin chains of fetal hemoglobin ( HbF ). One δ -gene producing δ -globin chain found in adults to a minor extent (HbA2).

19. Sickle-cell anemia ( HbS ) Sickle-cell anemia ( HbS ) is the most common form of abnormal hemoglobins . Erythrocytes of these patients adopt a sickle shape (crescent like) at low oxygen concentration It primarily occurs in the black population .

20. Molecular basis of HbS The glutamic acid in the 6 th position of β chain of HbA is changed to valine in HbS . This single amino acid substitution leads to polymerization of hemoglobin molecules inside RBCs . This causes a distortion of cell into sickle shape

21. Normal & HbS

22. The substitution of hydrophilic glutamic acid by hydrophobic valine causes a localized stickiness on the surface of the molecule The deoxygenated HbS may be depicted with a protrusion on one side and a cavity on the other side, so that many molecules can adhere and polymerize

23. The sickled cells form small plugs in capillaries. Occlusion of major vessels can lead to infarction in organs like spleen. Death usually occurs in the second decade of life .

24. Homozygous and heterozygous HbS Sickle cell anemia is said to be homozygous , if caused by inheritance of two mutant genes (one from each parent) that code for β -chains . In case of heterozygous HbS , only one gene (of β -chain) is affected while the other is normal

25. The erythrocytes of heterozygotes contain both HbS & HbA & the disease is referred to as sickle cell trait. The individuals of sickle-cell trait lead a normal life, & do not usually show clinical symptoms .

26. Abnormalities associated with HbS Life-long hemolytic anemia : The sickled erythrocytes are fragile & their continuous breakdown leads to life-long anemia . Tissue damage and pain: The sickled cells block the capillaries resulting in poor blood supply to tissues. This leads to extensive damage & inflammation of certain tissues causing pain.

27. Increased susceptibility to infection : Hemolysis & tissue damage are accompanied by increased susceptibility to infection & diseases. Prematured eath : Homozygous individuals of sickle-cell anemia die before they reach adulthood (< 20 years)

28. Mechanism of sickling in sickle-cell anemia G lutamate is a polar amino acid & it is replaced by a non-polar valine in sickle-cell hemoglobin . This causes a marked decrease in the solubility of HbS in deoxygenated form S olubility of oxygenated HbS is unaffected

29. Sticky patches & formation of deoxyhemoglobin fibres The substitution of valine for glutamate results in a sticky patch on the outer surface of β -chains . It is present on oxy- & deoxyhemoglobin S but absent on HbA . There is a site or receptor complementary to sticky patch on deoxyHbS .

30. The sticky patch of one deoxyHbS binds with the receptor of another deoxyHbS & this process continuous resulting in the formation of long aggregate molecules of deoxyHbS T he polymerization of deoxy-HbS molecules leads to long fibrous precipitates.

31. These stiff fibres distort the erythrocytes into a sickle or crescent shape The sickled erythrocytes are highly vulnerable to lysis . ln case of oxyHbS , the complementary receptor is masked, although the sticky patch is present.

32. HbS gives protection against malaria HbS affords protection against Plasmodium falciparum infection Hence the abnormal gene was found to offer a biologic advantage.

33. Sickle cell trait protects from malaria

34. D iagnosis of sickle cell anemia Sickling test: A simple microscopic examination of blood smear prepared by adding reducing agents such as sodium dithionite. Sickled erythrocytes can be detected under the microscope

35. Electrophoresis Electrophoresis at alkaline pH shows a slower moving band than HbA . At pH 8.6, carboxyl group of glutamic acid is negatively charged. Lack of this charge on HbS makes it less negatively charged , & decreases the electrophoretic mobility At acidic pH, HbS moves faster than HbA . In sickle cell trait , both the bands of HbA and HbS can be noticed

36. Electrophoresis at pH 8.6

37. Management of sickle cell disease Administration of sodium cyanate inhibits sickling of erythrocytes Cyanate increases the affinity of O2 to HbS & lowers the formation of deoxyHbS It causes certain side effects like peripheral nerve damage In severe anemia , repeated blood transfusion is required. It result in iron overload & cirrhosis of liver

38. Hemoglobin C disease Cooley's hemoglobinemia ( HbC ) is characterized by substitution of glutamate by lysine in the sixth position of β -chain . Due to the presence of lysine, HbC moves more slowly on electrophoresis compared to HbA and HbS . HbC disease occurs only in blacks . Both homozygous & heterozygous individuals of HbC disease are known . It is characterized by mild hemolytic anemia . No specific therapy is recommended .

39. Hemoglobin D Caused by the substitution of glutamine in place of glutamate in the 121 st position of β -chain . Several variants of HbD are identified from different places indicated by the suffix. For instance, HbD ( Punjab) HbD , on electrophoresis moves along with HbS .

40. Hemoglobin E Most common abnormal hemoglobin after HbS . lt is estimated that about 10% of the population in South-East Asia ( Bangladesh, Thailand, Myanmar) suffer from HbE disease . In India , it is prevalent in West Bengal. HbE is characterized by replacement of glutamate by lysine at 26 th position of β -chain . The individuals of HbE ( either homozygous or heterozygous ) have no clinical manifestations

41. Thalassemias Thalassemias are a group of hereditary hemolytic disorders characterized by impairment/imbalance in the synthesis of globin chains of Hb Thalassemias (Greek: thalassa -sea) mostly occur in the regions surrounding the Mediterranean sea, hence the name. Also prevalent in Central Africa , India.

42. Molecular basis of thalassemias Hemoglobin contains 2 α & 2 β globin chains . The synthesis of individual chains is so coordinated that each α -chain has a β -chain partner & they combine to finally give hemoglobin ( α 2 β 2). Thalassemias are characterized by a defect in the production of α -or β -globin chain

43. Thalassemias occur due to a variety of molecular defects Gene deletion or substitution, Underproduction or instability of mRNA, Defect in the initiation of chain synthesis, Premature chain termination .

44. α - Thalassemiasas α - Thalassemias are caused by a decreased synthesis or total absence of α -globin chain of Hb . There are four copies of α -globin g ene , two on each one of the chromosome 16. Four types of α - thalassemias occur which depend on the number of missing α -globin genes

45. Salient features of different α - thalassemias Silent carrier state is due to loss of one of the four α - globin genes with no physical manifestations . α -Thalassemia trait caused by loss of two genes (both from the same gene pair or one from each gene pair). Minor anemia is observed

46. Hemoglobin H disease, due to missing of three genes, is associated with moderate anemia Hydrops fetalis is the most severe form of α - thalassemias due to lack of all the four genes. The fetus usually survives until birth & then dies.

47. β - thalassemias Decreased synthesis or total lack of the formation of β -globin chain causes β - thalassemias . The production of α -globin chain continues to be normal, leading to the formation of a globin tetramer ( α 4 ) that precipitate. This causes premature death of erythrocytes. There are mainly two types of β - thalassemias

48. β -Thalassemia minor This is an heterozygous state with a defect in only one of the two β -globin gene pairs on chromosome 11. Also known as β - thalassemia trait , is usually asymptomatic, since the individuals can make some amount of β -globin from the affected gene

49. β -Thalassemia major This is a homozygous state with a defect in both the genes responsible for β -globin synthesis. The infants born with β -thalassemia major are healthy at birth since β -globin is not synthesized during the fetal development

50. They become severely anemic and die within 1-2 years. Frequent blood transfusion is required for these children. This is associated with iron overload which in turn may lead to death within 15-20 years

51. References Text book of Biochemistry – U Satyanarayana Text book of Biochemistry – DM Vasudevan Text book of Biochemistry – MN Chatterjea

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