Effect of point mutation on structure-function correlation of hemoglobin variants, HbE and HbD Punjab.

ABSTRACT

Hemoglobinopathies are examples of autosomal recessive disorders of human hemoglobin. Hemoglobin E (HbE) and Hemoglobin D Punjab (HbD Punjab) are two of the most common hemoglobin variants geographically spread across Asian continent. These two variants differ from normal human hemoglobin (HbA) at a single amino acid residue caused by the point mutation of ß globin gene. The presence of the mutated amino acid residue causes perturbation in the function of both variants. However, the structure-function correlation of these variants has not been established yet. In the present study, we analyzed the conformational changes associated with oxygenation of hemoglobin variants using hydrogen/deuterium exchange-based mass spectrometry of backbone amide hydrogens of α and ß globin chains in the tetrameric hemoglobin molecule. We also performed the functional assay of these variants using oxygen dissociation equilibrium curve. Compared to HbA, both variants showed reduced oxygen affinity, as reported earlier. The functional perturbations exhibited by these variants were correlated well with their structural alterations with respect to the reported changes in the residue level interactions upon oxygenation of normal hemoglobin, monitored through the hydrogen/deuterium exchange kinetics of several peptic peptides originated from the isotopically exchanged oxy and deoxy forms of HbE and HbD Punjab.