Question

Compare the binding isotherm of normal hemoglobin (at pH 7.4, with 7.8 mM 2,3 BPG) with that of the following variations. For each variant, clearly explain why or why not the binding isotherm changes the way it does.

Explain Please!

a) Hemoglobin at pH 6.9, 7.8 mM 2,3 BPG

b) Hemoglobin in which a glutamate residue (at a position normally exposed to the solvent) has been substituted for a valine residue (at pH 7.4, with 7.8 mM 2,3 BPG)

c) Hemoglobin at pH 7.4 with 9.4 mM 2,3 BPG

d) Hemoglobin with a mutation in the beta chain position 94 (position FG1) (at pH 7.4, with 7.8 mM 2,3 BPG). In this mutation Asp94 ->His.

Answer 1

The binding of O₂ to hemoglobin is affected by the concentration of H⁺ ions and CO2 in the surrounding tissue, the Bohr effect.

a) Hemoglobin at pH 6.9, 7.8mM BPG

In actively metabolizing tissue, such as muscle, the concentrations of these two substances are relatively high i.e. at low pH and at hydrogen ion concentration. This effectively causes a shift of the O₂ dissociation curve for hemoglobin to the right, promoting the release of O₂. This comes about because there are H⁺binding sites, primarily His146 in the b-chain., which have a higher affinity for binding H⁺deoxyhemoglobin than in oxyhemoglobin. An increase in CO₂ causes an increase in H⁺ ions due to the action of the enzyme carbonic anhydrase which catalyses the reaction:

CO₂ + H₂O ------------> HCO₃^- + H⁺

b) Hemoglobin in which glutamate residue is substitued with valine residue

Several hundred abnormal hemoglobins have been characterised, giving rise to the so-called hemoglobinopathies. Probably the best characeterized hemoglobinopathy is sickle-cell anemia (HbS). This disease is characterised by the patient's erythrocytes having a characteristic sickle or crescent shape. The molecular basis of this disease is the change of a glutamic acid residue for a valine at position 6 of the -chain, resulting in the substitution of a polar residue by a hydrophobic one.

This nonconservative substitution of valine for glutamate give HbS a sticky hydrophobic patch on the outside chain of its -chains. In the corner between helices E and F of the -chain of deoxy HbS is a hydrophobic site that is complementary to the sticky patch. Thus, the complementary site on one deoxy HbS molecule can bind to the sticky path on another deoxy-HbS molecule, resulting in the formation of long fibres of hemoglobin molecules that distort the erythrocytes. In oxy-HbS the complementary site is masked, so the formation of the long fibres occurs only when there is a high concentration of the deoxygenated form of HbS.

c) Hemoglobin at high level of 2,3 BPG

Increase in the level of BPG decreases oxygen affinity of hemoglobin and hence mediates oxygen release, which occurs at the cells.

2,3 biphosphoglycerate is a highly anionic organic phosphate molecules which is present in erythrocytes along with the hemoglobin. This molecule promotes the release of O₂ from hemoglobin by lowering the affinity of the protein for O₂. 2,3-BPG binds in the small cavity inthe center of the four subunits. In oxyhemoglobin, this cavity is too small fo it whereas in deoxyhemoglobin it is large enough to accommodate a single molecule of BPG. On binding, it forms ionic bonds wiht the positively charged amino acid side-chains in the -subunits, stabilizing the quaternary structure . H⁺, CO₂ and BPG are all allosteric effectors as they favour the conformation of deoxyhemoglobin and therefore promote the release of O₂.