| Summary: | A mass spectrometry-based method was developed for studying hemoglobin assembly/dissociation. The dynamics of bovine hemoglobin assembly were investigated by monitoring monomers/oligomers equilibria in solution with electrospray ionization mass spectrometry and circular dichroism spectroscopy. The mass spectral data confirm that bovine hemoglobin dissociation involves a step where heme is first lost from the β-chain of the α*β*-dimer to form a heme-deficient dimeric species or semi-hemoglobin dimer (α*β*). The experimental data provide strong evidence that binding of a partially unstructured apo-β-chain to a tightly folded holo-α-chain to form the semi-hemoglobin dimer is the initial step of hemoglobin assembly. Such binding locks the β-chain in a highly ordered conformation, which allows for an efficient heme acquisition. This step is followed by the docking of the two hemoglobin dimers to form a tetrameric form of the protein. Also, investigation of the individual apo- and heme-reconstituted globin chains show that although apo-α- and apo-β-chains are very flexible, dynamic, and have similar protein structures (the globin fold), reconstituted α-chains are monomeric and exhibit conformational structures very similar to holo-α-chains in the complex Hb equilibrium mixture. Reconstituted β-chains oligomerize to form the tetrameric homo-β-globin species HbH found in alpha thalassemic disorder. This suggests that dynamics play an important, perhaps the most important, role in the hemoglobin assembly process. We hypothesize that the intrinsic protein disorder exhibited by the bovine hemoglobin β-chains and the asymmetry of globin interaction reported in this work developed due to evolutionary pressure to provide a vital safeguard that may inhibit random oligomerization and aggregation in the crowded environment of the red blood cell, thus directing the assembly process along the correct pathway. The data support this hypothesis as hemoglobins analyzed from all organisms that evolved after the emergence of separate α- and β-chains (bovine, porcine, human, and modern fish hemoglobins) exhibit the expected asymmetry in the roles played by their globin chains in assembly. In contrast, hemoglobin from the mollusk Scapharca, which parallels the emergence of separate α- and β-chains does not exhibit asymmetry. In the analysis of the modern fish hemoglobin from Gadus morhua, the Atlantic cod, it was noticed that the measured molecular weights for the α- and β-chains did not correspond to masses calculated from their published sequences. Using mass spectrometry-based methods 65% sequence coverage was achieved, and a number of mutations were identified in both globin chains. A feasibility study for the use of mass spectrometry to study the interaction between hemoglobin and its scavenger protein haptoglobin is also presented.
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