This study is aimed at investigating the molecular basis of environmental adaptation of woolly mammoth hemoglobin (Hb) to the harsh thermal conditions of the Pleistocene Ice-ages. To this end, we have carried out a comparative biochemical-biophysical characterization of the structural and functional properties of recombinant hemoglobins (rHb) from woolly mammoth (rHb WM) and Asian elephant (rHb AE) in relation to human hemoglobins Hb A and Hb A2 (a minor component of human Hb). We have obtained oxygen equilibrium curves and calculated O2 affinities, Bohr effects, and the apparent heat of oxygenation (ΔH) in the presence and absence of allosteric effectors [inorganic phosphate and inositol hexaphosphate (IHP)]. Here, we show that the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP, suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally, 1H-NMR spectroscopy data indicates that both α1(β/δ)1 and α1(β/δ)2 interfaces in rHb WM and rHb AE are perturbed, whereas only the α1δ1 interface in Hb A2 is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM presumably facilitated woolly mammoth survival in the Arctic environment.

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