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10/12/2014 Hb Structure & Functions Blood chemistry Hb Structure & Functions Dr. Vishnu Kumar Awasthi Dr. Vishnu Kumar Awasthi Assistant Professor – In.

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Presentation on theme: "10/12/2014 Hb Structure & Functions Blood chemistry Hb Structure & Functions Dr. Vishnu Kumar Awasthi Dr. Vishnu Kumar Awasthi Assistant Professor – In."— Presentation transcript:

1 10/12/2014 Hb Structure & Functions Blood chemistry Hb Structure & Functions Dr. Vishnu Kumar Awasthi Dr. Vishnu Kumar Awasthi Assistant Professor – In – Charge HLS, Department of Biochemistry Assistant Professor – In – Charge HLS, Department of Biochemistry

2 10/12/2014 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 CO 2

3 10/12/2014 Haemoglobinstructure 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.

4 10/12/2014 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% HbA 2 and about 1% HbF.

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6 CHHC NH C N CH HC NHNH C HCHC CC N HCHC CH HN CC CH C HC C CHCH CHCH Pyrrole Porphyrin What is Porphyrin ? N

7 Heme Heme = Porphyrin + iron

8 10/12/2014  Hemoglobin is a O 2 and CO 2 transport protein found in the RBCs  Hemoglobin is an oligomeric protein made up of 2 α β dimers, a total of 4 polypeptide chains: α 1 β 1 α 2 β 2.  Total Mr of hemoglobin is 64,500.  The α (141 aa) and β (146 aa) subunits have < 50 % identity.

9 10/12/2014  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. 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.

12 10/12/2014  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

13 10/12/2014  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.

14 10/12/2014  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

15 10/12/2014  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.

16 10/12/2014  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

17 10/12/2014  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.

18 10/12/2014  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.

19 10/12/2014 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.

20 10/12/2014 Oxygen dissociation curve  i. The ability of Hb to load and unload oxygen at physiological pO 2 is shown by oxygen dissociation curve (ODC)

21 10/12/2014 A Percentage saturation pO2 in mm of Hg

22 10/12/2014 B Percentage saturation pO2 in mm of Hg

23 10/12/2014 C Percentage saturation pO2 in mm of Hg

24 10/12/2014 D Percentage saturation pO2 in mm of Hg

25 10/12/2014  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 42 0 C.

26 10/12/2014  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%.

27 10/12/2014 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) Hill equation (A V Hill, nobel prize,1922)

28 10/12/2014 B. Positive co operativity Hb HbO 2 HbO 4 HbO 6 HbO 8 Homotropic interaction

29 10/12/2014 c. Each successive addition of O 2, increase the affinity of Hb to O 2 synergistically.  D. Similarly, binding of 2, 3 – BPG at a site other than the oxygen binding site, lowers the affinity for oxygen (heterotropic interaction).

30 10/12/2014 Alteration of structure Diagrammatic representation of subunit interaction in Hemoglobin

31 10/12/2014  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 α 1 - β 2 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.

32 10/12/2014  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) (2x alpha)+(2x beta)→2x(alpha-beta) (Deoxy-Hb) (oxy-Hb)

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34 3. The Bohr Effect i.The influence of pH and pCO 2 to facilitate oxygenation of Hb in the lungs and deoxygenation at the tissues is known as the Bohr effect (1904). ii.Binding of CO 2 forces the release of O 2 iii.When the pCO 2 high, CO 2 diffuses into the RBCs CO 2 + H 2 O → H 2 CO 3 → H + + HCO 3 - CO 2 + H 2 O → H 2 CO 3 → H + + HCO 3 - Carbonic Carbonic Anhydrase Anhydrase Iv. When carbonic acid is ionizes, the intracellular pH falls. The affinity of Hb for oxygen is decreased and oxygen is unloaded

35 10/12/ The chloride shift CO 2 H2O + CO2 H2CO3 Carbonic anhydrase HCO3- H+H+ HHb +O2 Cl- Cl shift (in tissues) Cl- HCO3- O2- To cells  When CO2 is taken up ---- HCO3- ↑ NN HbO 2 Chloride enters into RBC

36 10/12/2014  When the blood reaches the lungs, reverse reaction takes place

37 10/12/ The chloride shift CO 2 H2O + CO2 H2CO3 Carbonic anhydrase HCO3- H+H+ HHb +O2 Cl- Cl shift (in lungs) Cl- HCO3- O2- Air  When O2 is taken up ---- NN HbO 2 Chloride comes out of RBC Air

38 10/12/ Effect of temperature  p50 = the pO 2 at which Hb is half saturated  p50 of normal Hb = 26 mmHg (at 37 o C)  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.

39 10/12/ 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

40 10/12/2014 Transport of CO 2  At rest, about 200 ml of CO 2 is produced /minute in tissues. The CO 2 is carried by the following 3 ways:- 1. Dissolved form: 1. Dissolved form: CO 2 + H 2 O H 2 CO 3 HCO H + CO 2 + H 2 O H 2 CO 3 HCO H +

41 10/12/ Isohydric transport of CO 2 1.Haldane effect: The H+ ions are buffered by the deoxyhemoglobin. 2.In tissue 3.Oxy-Hb is more – (negatively) charged than deoxy-Hb 4. In the Lungs 4. In the Lungs H - Hb + 4O 2 Hb (O 2 )4 + H + H - Hb + 4O 2 Hb (O 2 )4 + H +

42 10/12/ The proton released in the RBC combine with HCO 3 - forming H 2 CO 3 which would dissociate to CO 2, that is expelled through pulmonary capillaries. 6. As the HCO 3 - level inside the erythrocytes falls, more and HCO 3 - gets into the RBC, and chloride diffuse out.

43 10/12/ Carriage as carbamino- Hb R-NH2 + CO2 R-NH-COOH 4. Clinical Applications. 1- Hypoxic states,O 2 affinity decreased. 1- Hypoxic states,O 2 affinity decreased. - ODC shift right. - ODC shift right. - increased in 2,3-BPG increased in RBC. - 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.

44 10/12/ The Red cell 2,3-BPG level is decreased in acidosis and increased in alkalosis, ODC shift to right. 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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