A Biophysical-Biochemical Comparison Of Hemoglobins From Mammoth, Asian Elephant, And Human

This study is aimed at investigating the molecular basis of environmental adaptation of hemoglobin from tropical climate to low temperature in the Arctic region. We have carried out a biochemical-biophysical characterization of the structural and functional properties of hemoglobins from woolly mammoth (Hb WM) and Asian elephant (Hb AE) and compared those to human hemoglobins (Hb A and Hb A2) in 0.1 M phosphate buffer. Hb A consists of two α and two β subunits. Hb AE was found to contain two α subunits and two β/δ fusion subunits. Hb WM was expressed by inserting Asian elephant α-like and β/δ-like cDNA into our E. coli Hb plasmid (pHE2), and then introducing the mammoth-specific residue differences (αK5N, β/δT12A, β/δA86S, and β/δG101Q) into the Asian elephant plasmid. Since Hb AE and Hb WM contain β/δ fusion chains, we have also compared them to Hb A2, which contains δ chains instead of the β subunits present in Hb A. Oxygen affinity, Bohr effect, and cooperativity of oxygenation were measured at different temperatures and pH and 1H-NMR spectra were obtained for structural comparisons for each Hb. Our results show: (i) Hb AE has the higher O2 affinity as compared to Hb WM, Hb A2, and Hb A; (ii) the effect of an allosteric effector, inositol hexaphosphate (IHP), is the most prominent on Hb A2 as compared to Hb A, Hb AE, and Hb WM. 1H NMR results indicates that the α1β/δ1 and α1β/δ2 interfaces are perturbed in both Hb AE and Hb WM, whereas only the α1δ1 interface is perturbed in Hb A2 compared to Hb A. Hb AE and Hb WM have structural features that are very different from that of Hb A2 and Hb A, consistent with the altered functional properties.