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:
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
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
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.
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.
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
Heme Heme = Porphyrin + iron
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.
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
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.
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
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.
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
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.
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
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.
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.
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.
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)
10/12/2014 A Percentage saturation pO2 in mm of Hg
10/12/2014 B Percentage saturation pO2 in mm of Hg
10/12/2014 C Percentage saturation pO2 in mm of Hg
10/12/2014 D Percentage saturation pO2 in mm of Hg
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.
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%.
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)
10/12/2014 B. Positive co operativity Hb HbO 2 HbO 4 HbO 6 HbO 8 Homotropic interaction
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).
10/12/2014 Alteration of structure Diagrammatic representation of subunit interaction in Hemoglobin
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.
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)
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
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
10/12/2014 When the blood reaches the lungs, reverse reaction takes place
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
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.
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
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 +
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 +
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.
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.
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.