Roles of Amino Acid Residues in Woolly Mammoth Hemoglobin on the Temperature Effect of Oxygen Binding

Abstract

The O2 affinity of hemoglobin (Hb) is affected by allosteric effectors (e.g. H+, chloride, and organic phosphate) and temperature. Hb oxygenation is exothermic, i.e., the oxygen-binding affinity increases significantly with decreasing temperature. This makes it difficult for the Hb molecule in blood to deliver oxygen to the tissues under conditions of hypothermia during major surgical operations. We constructed plasmids to express recombinant woolly mammoth Hb (rHb WM) and Asian elephant Hb (rHb AE). Our biochemical-biophysical studies show that the apparent heat of oxygenation (ΔH) of rHb WM is less negative than that of rHb AE and human normal adult Hb (Hb A), suggesting that the O2 affinity of rHb WM is much less dependent on temperature. In order to investigate the key residues of the Hb molecule responsible for the temperature effect on O2 affinity, mutants with β/δ101 substitutions (β/δ101Gln→Glu, Lys, and Asp) in rHb WM have been expressed.Compared to rHb WM,these mutants exhibit a higher affinity for oxygen, and a more negative ΔH value under various conditions of pH, temperature, and salt concentration, with and without organic phosphates. Titrations for the O2 affinity of those mutant rHbs as a function of chloride concentrations indicate a lower heterotropic effect of this anion due to the replacement of β/δ101Gln, suggesting that the β/δ101Gln residue in rHb WM is important for its stronger response to chloride ions, and also responsible for its lower temperature effect of O2 affinity. NMR measurements for rHb WM and its mutants have been used to correlate their structural and functional properties. These findings could provide new insights into designing hemoglobin-based oxygen carriers (HBOCs) for treating patients undergoing therapeutic hypothermia (e.g. cardiac arrest, traumatic brain injury, stroke, etc.).