1. Dr. Shumaila Asim Lecture # 4
HEMOGLOBIN
Dr. Shumaila Asim Lecture # 4

2. Introduction The main function of red blood cell
Transfer of O2 from lungs to tissue
Transfer of CO2 from tissue to lungs
To accomplish this function red cells has hemoglobin (Hb)
Each red cell has 640 million molecules of Hb

3. Introduction
Hemoglobin (Hb), protein constituting 1/3 of the red blood cells
Synthesis begins in proerythroblast
65% at erythroblast stage
35% at reticulocyte stage
Two parts
Heme
Globin

5. Synthesis of Hemoglobin (Hb)
Heme & globin produced at two different sites in the cells
Heme in mitochondria
Globin in polyribosomes
Well synchronized

6. Synthesis of Hemoglobin

7. Synthesis of Heme Protoporphyrin ring with an iron atom in centre
The main site is mitochondria as it contains ALAS
Mature red cell does not contain mitochondria

8. Structure of Heme

9. HEME-CONTAINING PROTEINS
 Hemoglobin
 Myoglobin
 Cytochromes
 Catalase
 Some peroxidases

10. Synthesis of globin

11. Synthesis of globin
Various types of globin combines with heme to from different hemoglobin
Eight functional globin chains, arranged in two clusters the
β- cluster (β, γ , δ and ɛ globin genes) on the short arm of chromosome 11
α- cluster (α and ζ globin genes) on the short arm of chromosome 16

12. Synthesis of globin Globin synthesis, starts at 3rd week of gestation
Embryonic
Haemoglobin Gower I ( ζ2ɛ2)
Haemoglobin Portland ( ζ2γ2)
Haemoglobin Gower II (α2ɛ2)
Fetal : HbF (α2γ2), HbA (α2β2)
Adult : HbA, HbA2 ( α2δ2), HbF.

14. Globin gene clusters

15. Globin chain switch

16. Alpha & beta chains

17. Chain interaction
The chains interact with each other via hydrophobic interactions
Therefore, hydrophobic amino acids are not only present in the interior of the protein chains, but also on the surface.
Electrostatic interactions (salt bridges) and hydrogen bonds also exist between the two different chains.

18. Types of Hb Normal: HbA (97%) HbA2 (2%) HbF (1%) HbA1c Abnormal:
Carboxy Hb
Met Hb
Sulf Hb

19. HbA structure
Oxygen binding to hemoglobin

20. Types of hemoglobin Fetal hemoglobin (HbF):
Major hemoglobin found in the fetus and newborn
Tetramer with two a and two g chains
Higher affinity for O2 than HbA
Transfers O2 from maternal to fetal circulation across placenta

21. Types of hemoglobin HbA2: Appears ~12 weeks after birth
Constitutes ~2% of total Hb
Composed of two α and two δ globin chains

22. Types of hemoglobin HbA1c: HbA undergoes non-enzymatic glycosylation
Glycosylation depends on plasma glucose levels
HbA1c levels are high in patients with diabetes mellitus

23. Abnormal Hbs Unable to transport O2 due to abnormal structure:
Carboxy-Hb: CO replaces O2 and binds 200X tighter than O2 (in smokers)
Met-Hb: Contains oxidized Fe3+ (~2%) that cannot carry O2
Sulf-HB: Forms due to high sulfur levels in blood (irreversible reaction)

24. Hemoglobin structure
Hemoglobin is tetrameric hemeprotein (fur protein chains known as globins with each bound to heme.
In adults, the four globin proteins are of two different types known as α and β, so a hemoglobin protein is an α2β2 globin protein.
The α and β chains contain multiple α-helices where α contains 7 α-helices and β contains 8 α-helices (similar to myoglobin).

25. Hemoglobin is allosteric
Hemoglobin is an allosteric protein (from Greek "allos" = "other", and "stereos" = "shape").
An allosteric protein: a protein where binding of a molecule (ligand) to one part of the protein affects binding of a similar or a different ligand to another part of the protein.
Hemoglobin exists in two forms, T-state and R-state
The T-state is also known as the "taut" or "tense" state and it has a low-binding affinity to oxygen.
The R-state is known as the "relaxed" state and it has 500 times higher affinity to oxygen than as the T conformation .
Binding of O2 causes conformational changes in hemoglobin, converting it from the low affinity T-state to the high affinity R-state .

26. How does the structure change?
When heme is free of oxygen, it has a domed structure and iron is outside the plane of the heme group.
When oxygen binds to an iron atom, heme adopts a planar structure and the iron moves into the plane of the heme pulling proximal histidine (F8) along with it.

27. How does the structure change?
This movement triggers
changes in tertiary structure of individual hemoglobin subunits and
breakage of the electrostatic bonds at the other oxygen-free hemoglobin chains.

28. The saturation curve is sigmoidal because…
Conformational changes lead to cooperativity among binding sites.
Binding of the first O2 breaks some salt bridges with the other chains increasing the affinity of the binding of a second molecule.
Binding of the second O2 molecule breaks more salt bridges increasing the affinity towards binding of a third O2 even more, and so on.

30. Derivatives of hemoglobin
Oxyhemoglobin (oxyHb) = Hb with O2
Deoxyhemoglobin (deoxyHb) = Hb without O2
Methemoglobin (metHb) contains Fe3+ instead of Fe2+ in heme groups
Carbonylhemoglobin (HbCO) – CO binds to Fe2+ in heme in case of CO poisoning or smoking. CO has 200x higher affinity to Fe2+ than O2.
Carbaminohemoglobin (HbCO2) - CO2 is non-covalently bound to globin chain of Hb. HbCO2 transports CO2 in blood (about 23%).
Glycohemoglobin (HbA1c) is formed spontaneously by nonenzymatic reaction with Glc. People with DM have more HbA1c than normal (› 7%). Measurement of blood HbA1c is useful to get info about long-term control of glycemia.

31. Another significance of distal histidine