A Biochemical–Biophysical Study of Hemoglobins from Woolly Mammoth, Asian Elephant, and Humans

Abstract

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.

Role of β/δ101Gln in Regulating the Effect of Temperature and Allosteric Effectors on Oxygen Affinity in Woolly Mammoth Hemoglobin

“…As reported previously (10), the Hill coefficients for rHb WM and rHb AE are lower than those of Hb A under all experimental conditions, suggesting a difference in the cooperativity of the oxygenation process for these Hbs (for details, see Supporting Information Figure 2S). The n 50 values for the mutants rHb WM (β/δQ101E), rHb WM (αN5K, β/δQ101E), and rHb WM (β/δQ101K), are maintained around the expected value of 1.8–2.5 in the range of pH 5.8–8.4 in 0.1 M MES or NaPi buffer, while the n 50 values for rHb WM (β/δQ101D) and rHb WM (αN5K, β/δA12T, β/δS86A) are found to be lower (1.0–2.0) under the same experimental conditions.…”

“…Experiments were conducted in the presence and absence of 0.5 mM inositol hexaphosphate (IHP), in the pH range 5.5 to 8.5. For these measurements, the rHbs were kept at 100–120 μM (in terms of heme) to minimize tetramer-dimer dissociation (10). Each sample was checked before and after measurements for met-hemoglobin (met-Hb) in a spectrophotometer.…”

“…Lower effect of temperature on the O 2 affinity of Hb has been observed previously from some arctic animals, such as Eskimo dog, reindeer, and musk ox (2–8), but the key amino acid residues for this property have not been confirmed. Recently, recombinant Hbs of woolly mammoth (rHb WM) and Asian elephant (rHb AE) have been successfully expressed in Escherichia coli and isolated for biochemical-biophysical studies (9, 10). The O 2 affinity of rHb WM is much less dependent on temperature than that of human normal adult Hb (Hb A) or rHb AE (9, 10).…”

“…Recently, recombinant Hbs of woolly mammoth (rHb WM) and Asian elephant (rHb AE) have been successfully expressed in Escherichia coli and isolated for biochemical-biophysical studies (9, 10). The O 2 affinity of rHb WM is much less dependent on temperature than that of human normal adult Hb (Hb A) or rHb AE (9, 10). This property could have an adaptive role for woolly mammoths in having a unique hemoglobin to deliver O 2 to their extremities.…”

Elephantid Genomes Reveal the Molecular Bases of Woolly Mammoth Adaptations to the Arctic

“…Thus, while the molecular bases of some phenotypic traits have been identified, these studies generally are limited to a few wellcharacterized genes and pathways with relatively simple and direct genotype-phenotype relationships (Chan et al, 2010;Hoekstra et al, 2006;Lang et al, 2012;Smith et al, 2013;Storz et al, 2009). Previous structural and functional studies, for example, have shown that amino acid polymorphisms in the woolly mammoth hemoglobin b/d fusion gene (HBB/HBD) reduce oxygen affinity (Campbell et al, 2010;Yuan et al, 2011Yuan et al, , 2013, whereas amino acid polymorphisms in both the woolly mammoth and Neandertal melanocortin 1 receptor (MC1R) genes were hypomorphic compared to the ancestral alleles Rö mpler et al, 2006). Most traits, however, have complex genotype-phenotype relationships with phenotypic divergence arising through the accumulation of numerous variants of small individual effects rather than one or a few mutations of large effect.…”