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Roles of Fe-Histidine bonds in stability of hemoglobin: Recognition of protein flexibility by Q Sepharose

Shigenori Nagatomo, Teizo Kitagawa, Masako Nagai
Published: 1 June 2021

Abstract: Using various mutants, we investigated to date the roles of the Fe-histidine (F8) bonds in cooperative O2 binding of human hemoglobin (Hb) and differences in roles between α and β subunits in the α2β2 tetramer. An Hb variant with a mutation in the heme cavity exhibited an unexpected feature. When the β mutant rHb (βH92G), in which the proximal histidine (His F8) of the β subunit is replaced by glycine (Gly), was subjected to ion exchange chromatography (Q Sepharose column) and eluted with an NaCl concentration gradient in the presence of imidazole, yielded two large peaks, while the corresponding α mutant, rHb (αH87G), gave a single peak similar to Hb A. The β mutant rHb proteins under each peak had identical isoelectric points (pI) according to isoelectric focusing electrophoresis. Proteins under each peak were further characterized by Sephadex G-75 gel filtration, far-UV CD, 1H NMR, and resonance Raman spectroscopy. We found that rHb (βH92G) exists as a mixture of αβ dimers and α2β2 tetramers, and that hemes are released from β subunits in a fraction of the dimers. An approximate amount of released hemes were estimated to be as large as 30% with Raman relative intensities. It is stressed that Q Sepharose columns can distinguish differences in structural flexibility of proteins having identical pIs by altering the exit rates from the porous beads. Thus, the role of Fe-His (F8) bonds in stabilizing the Hb tetramer first described by Barrick et al. was confirmed in the present study. In addition, it was found in this study that a specific Fe-His bond in the β subunit minimizes globin structural flexibility.
Keywords: Mutant hemoglobin / ion exchange chromatography / molecular recognition / Q Sepharose / iron-histidine bond / tetramer-dimer equilibrium

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