Analysis of the Quaternary Structure of Hemoglobin Beckman Variant and Molecular Interpretation of Its Functional Abnormality: A Mass-Spectrometry-Based Approach.

Abstract

Electrostatic attraction between α and β globin chains holds the subunits together in a tetrameric human hemoglobin molecule (α2 β2 ). Compared to normal globin chains, the affinity of a mutant chain to its partner globin might be different in genetic variants of hemoglobin. This leads to an unequal abundance of normal and variant hemoglobin in heterozygous samples, even though the rates of synthesis of both the normal and variant chains are the same. The aforementioned affinities across various globin chains might be assessed by quantification of the different forms of the tetramers present in a variant hemoglobin sample. In the present study, by exploiting mass differences between globin chains, differently populated hemoglobin tetramers present in hemoglobin (Hb) Beckman, a β variant (βA135D), were structurally characterized. The relative populations of dissymmetric tetramers (α2 β2 , α2 ββV , and α2 βV2 ) indicated that both β and βV have different affinities towards the α globin chain. Conformational dynamics analyzed from hydrogen/deuterium exchange kinetics of the three peptide fragments of Hb Beckman in its oxy state displayed molecular insight into its functional abnormality. However, in comparison to normal hemoglobin (α2 β2 ), the point mutation did not show any change in the collision cross-sections of the functionally active conformers of the variant hemoglobin molecules (α2 ββV and α2 βV2 ).