1. Blood chemistry Hb Structure & Functions
Dr. Vishnu Kumar Awasthi
Assistant Professor – In – Charge HLS, Department of Biochemistry
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2. Objectives Structure of hemoglobin Transport of oxygen by Hb
Oxygen dissociation curve (ODC)
Factors affecting ODC
Heme-heme interaction and co-operativity
Effect of pH and pCO2
The Bohr Effect
The chloride shift
Effect of temperature
Effect of 2,3-BPG
Transport of CO2
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3. Haemoglobin structure
Haemoglobin (Hb) is the most abundant porphyrin – containing compound.
It is a tetramer made up of four subunits.
Each subunit contains a heme group and a polypeptide chain.
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4. Haemoglobin structure
Normal level of Hb in blood of males is 14 – 16 g/dl and in females, 13 – 15 g/dl.
Normal adult blood contains 97% HbA, about 2% HbA2 and about 1% HbF.
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6. What is Porphyrin ? HC HC HC CH HC CH HC C C CH NH N HC C C CH NH HN
Pyrrole HC HC HC CH HC CH HC C C CH NH N HC C C CH NH HN HC C C CH N N HC C C CH CH CH

7. Heme
Heme = Porphyrin + iron

8. Hemoglobin is a O2 and CO2 transport protein found in the RBCs
Hemoglobin is an oligomeric protein made up of 2 α β dimers, a total of 4 polypeptide chains: α1 β1 α2 β2.
Total Mr of hemoglobin is 64,500.
The α (141 aa) and β (146 aa) subunits have < 50 % identity.
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9. similar; all three are members of the globin family.
• The 3D- structures of α (141 aa) and β (146 aa) subunits of hemoglobin and the single polypeptide of myoglobin are very
similar; all three are members of the globin family.
• Each subunit has a haem-binding pocket
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11. The polypeptide chains are of five types viz. α, β, γ, δ and ε
The α chain is made up of 141 amino acids.
The β , γ, δ and ε chains are made up of 146 amino acid residues each.
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12. A small amount of HbA2 is also found in adults which is α2 δ2.
Normal adult haemoglobin (HbA) is made up of four haem groups, two α chains and two β chains, and is represented as α2 β2.
A small amount of HbA2 is also found in adults which is α2 δ2.
Foetal haemoglobin (HbF) is α2 γ2
Embryonic haemoglobin is α2 ε2
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13. The histidine residues linked to iron are present at positions 58 and 87 in α chains and at positions 63 and 92 in other chains.
The bond between iron and the distal histidine residue (His87 or His92) is unstable.
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14. This results in the formation of an iron-oxygen bond
The distal iron-histidine bond is broken when haemoglobin is exposed to high oxygen tension
This results in the formation of an iron-oxygen bond
The binding of oxygen to haemoglobin changes the conformation of haemoglobin
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15. Two conformations have been described, T (taut) and R (relaxed).
Deoxygenated Hb exists in T form which is stabilised by 2,3-bisphosphoglycerate (2,3-BPG) which is formed from 1, 3-BPG (an intermediate in glycolytic pathway) when there is a deficiency of oxygen in the tissues.
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16. T and R states of Hemoglobin
• Hemoglobin exists in two major conformational
states: Relaxed (R ) and Taut or Tense (T)
• R state has a higher affinity for O2.
• In the absence of O2, T state is more stable; when O2
binds, R state is more stable, so hemoglobin
undergoes a conformational change to the R state.
• The structural change involves readjustment of interactions between subunits
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17. 2,3-BPG enters this cavity and cross links the two β chains.
There is a central cavity in the haemoglobin molecule surrounded by the four polypeptide chains.
2,3-BPG enters this cavity and cross links the two β chains.
When oxygen tension increases, 2,3-BPG is displaced and the T form changes into R form.
During this transition, one pair of α and β subunits rotates by 15° relative to the other pair.
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18. Each subunit of haemoglobin can bind one oxygen molecule.
Since there are four subunits in a molecule of haemoglobin, one molecule can bind four oxygen molecules.
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19. Transport of oxygen by Hb
Hb has all the requirements of an ideal
respiratory pigment:
It can transport large quantities of oxygen
It has great solubility
It can take up an release oxygen at appropriate partial pressure
It is powerful buffer.
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20. Oxygen dissociation curve
i. The ability of Hb to load and unload oxygen at physiological pO2 is shown by oxygen dissociation curve (ODC)
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21. Percentage saturation
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pO2 in mm of Hg

22. Percentage saturationB
Percentage saturation
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pO2 in mm of Hg

23. Percentage saturation
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pO2 in mm of Hg

24. Percentage saturationD
Percentage saturation
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pO2 in mm of Hg

25. A. Theoretical curve as per mass action.
B. Sigmoid curve, due to heme-heme interaction (hill effect).
C. Further shift to right due to carbon dioxide (Bohr effect) and BPG. This curve represents the pattern under normal conditions.
D. further shift to right when temp is increased to 420C.
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26. ii. At the oxygen tension in the pulmonary alveoli, the Hb is 97% saturated with oxygen. Normal blood with 15 gm/dl of Hb can carry 20 ml of oxygen /dl of blood.
iii. In the tissue capillaries, where the pO2 is only 40 mmHg, theoretically Hb saturation is 75%. Thus under STP conditions, blood can release only 22%.
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27. Factors affecting ODC 1. Heme-heme interaction and co-operativity:-
A. the sigmoid shape of ODC – due to allosteric effect, or co-operativity.
equilibrium of Hb=O2
Hill equation (A V Hill, nobel prize,1922)
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28. B. Positive co operativityHb
HbO2
HbO4
HbO6
HbO8
Homotropic interaction
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29. c. Each successive addition of O2, increase the affinity of Hb to O2 synergistically.
D. Similarly, binding of 2, 3 – BPG at a site other than the oxygen binding site, lowers the affinity for oxygen (heterotropic interaction).
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30. Alteration of structure
Diagrammatic representation of subunit interaction in Hemoglobin
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31. The Hb subunits are moved relative to one another.
During oxygen uptake, the T form to the R form with disruption of the salt bridges .
The Hb subunits are moved relative to one another.
During oxygenation, the α1 - β2 interface shows movement.
The two subunits slip over each other.
The quaternary structure of oxy Hb is described as R form; and that of de-oxy Hb is T form.
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32. (2x alpha)+(2x beta)→2x(alpha-beta) (Deoxy-Hb) (oxy-Hb)
When oxygenation occurs the salt bonds are broken successively. Thus on oxygenation, the Hb molecule can form two similar dimers.
(2x alpha)+(2x beta)→2x(alpha-beta)
(Deoxy-Hb) (oxy-Hb)
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34. 3. The Bohr Effect
The influence of pH and pCO2 to facilitate oxygenation of Hb in the lungs and deoxygenation at the tissues is known as the Bohr effect (1904).
Binding of CO2 forces the release of O2
When the pCO2 high, CO2 diffuses into the RBCs
CO2 + H2O → H2CO3 → H+ + HCO3-
Carbonic
Anhydrase
Iv. When carbonic acid is ionizes, the intracellular pH falls. The affinity of Hb for oxygen is decreased and oxygen is unloaded
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35. 4. The chloride shift Cl shift (in tissues) HHb +O2
When CO2 is taken up ----HCO3- ↑
Cl shift (in tissues)
CO2
H2O + CO2
Carbonic anhydrase
H2CO3
HbO2 H+ N N
HCO3-
HHb +O2
HCO3-
Cl-
O2-
To cells
Cl-
Chloride enters into RBC
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36. When the blood reaches the lungs, reverse reaction takes place
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37. 4. The chloride shift Cl shift (in lungs) HHb +O2
When O2 is taken up ----
Cl shift (in lungs)
H2O + CO2
CO2
Air
Carbonic anhydrase
H2CO3
HbO2 H+ N N
HCO3-
HHb +O2
HCO3-
Cl-
O2-
Air
Cl-
Chloride comes out of RBC
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38. 5. Effect of temperature p50 = the pO2 at which Hb is half saturated
p50 of normal Hb = 26 mmHg (at 37oC)
Elevation of temp. causes 88 % increase in p50
ODC shifts to left at low temp.
Under febrile conditions , increased needs of oxygen met by a shift in ODC to right.
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39. 6. Effect of 2,3-BPG Normal 2,3-BPG level=15 ± 1.5 mg/g Hb.
2,3-BPG == high in children
2,3-BPG is produced from 1,3-BPG, an intermediate of glycolytic pathway.
2,3-BPG, preferentially binds to deoxyHb and stabilizes T form
When T form reverts to R, 2, 3-BPG ejected
During oxygenation, BPG released
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40. Transport of CO2
At rest, about 200 ml of CO2 is produced /minute in tissues.
The CO2 is carried by the following 3 ways:-
1. Dissolved form:
CO2 + H2O H2CO HCO3- + H+
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41. 2. Isohydric transport of CO2
Haldane effect: The H+ ions are buffered by the deoxyhemoglobin.
In tissue
Oxy-Hb is more – (negatively) charged than deoxy-Hb
4. In the Lungs
H - Hb + 4O Hb (O2)4 + H+
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42. 5. The proton released in the RBC combine with HCO3- forming H2CO3 which would dissociate to CO2, that is expelled through pulmonary capillaries.
6. As the HCO3- level inside the erythrocytes falls, more and HCO3- gets into the RBC, and chloride diffuse out.
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43. 3. Carriage as carbamino- Hb
R-NH2 + CO R-NH-COOH
4. Clinical Applications.
1- Hypoxic states,O2 affinity decreased.
- ODC shift right.
- increased in 2,3-BPG increased in RBC.
2. In anemia, increased oxygen unloading will ensure proper oxygenation of tissues.
3. 2,3-BPG level varies as Hb conc.
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44. 4. The Red cell 2,3-BPG level is decreased in acidosis and increased in alkalosis, ODC
shift to right.
5. Transfusion of large vol. of stored blood which has a low level of 2,3-BPG can lead sudden hypoxia and a left shifted ODC.
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