Molecular and physicochemical characterization of hemoglobin from the high-altitude Taiwanese brown-toothed shrew (Episoriculus fumidus)

Abstract

Red-toothed shrews (subfamily Soricinae) exhibit the highest mass-specific rates of O₂ consumption recorded among eutherian mammals, though surprisingly no data appears to be available on the functional characteristics of their hemoglobin (Hb). As a first step in addressing this shortcoming, we investigated the O₂ binding characteristics of Taiwanese brown-toothed shrew (Episoriculus fumidus) Hb and its temperature and pH dependence in the absence and presence of anionic red blood cell effectors. Although comparative data regarding the intrinsic O₂ affinity of other shrew species are currently unavailable, our data suggest that the sensitivity of this high-elevation endemic species' Hb to allosteric effector molecules is similar to that of the two lowland species of white-toothed (crocidurine) shrews examined to date. The efficient exploitation of blood O₂ reserves by E. fumidus appears to be achieved via synergistic modulation of O₂ affinity by Cl⁻ and organic phosphates that moreover dramatically lowers the overall enthalpy of oxygenation of their Hb. Oxygen unloading is presumably further enhanced by a relatively high Bohr effect (ΔLog P₅₀/ΔpH = -0.69) and marked reduction in the titratable histidine content (predicted low proton buffering value) of the component globin chains relative to human HbA. Notably, however, the limited data available suggest these latter attributes may be widespread among shrews and hence likely are not adaptations to chronic altitudinal hypoxia per se.

Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different altitudinal ranges

“…For example, the loss of the β146His Bohr group may be partly compensated for by the α113Leu→His substitution in marmotine ground squirrels, which results in the gain of two titratable His residues per tetramer relative to human HbA. The same α113Leu→His substitution was also suggested to contribute to a large Bohr effect in mole and shrew Hb (Campbell et al, 2012). Detailed mutagenesis and crystallographic studies will be required to illuminate the structure-function relationships of golden-mantled ground squirrel Hb.…”

Altered hemoglobin co-factor sensitivity does not underlie the evolution of derived fossorial specializations in the family Talpidae

“…Interestingly, β4 Ser and β5 Gly are also found in the three species of shrews for which sequence data is available (Fig. 2), while Taiwanese brown-toothed shrew (Episoriculus fumidus) Hb-the only other eulipotyphlan species for which comparable data exists-exhibits a comparably depressed DPG sensitivity as moles (0.22; Campbell et al, 2012). Taken together these results strongly suggest that a blunted DPG sensitivity phenotype may in fact be an ancestral trait for Eulipotyphla (i.e.…”

“…Prior to conducting experiments, the major Hb component was isolated from thawed blood samples by preparative isoelectric focusing (see Fig. S1), concentrated by centrifugal ultrafiltration, and stored in the CO form at -80°C as previously described (Campbell et al, 2010;Campbell et al, 2012). These Hb samples were then diluted to create 0.1 M HEPES buffer solutions over a range of pH's and containing varying concentrations of KCl and DPG; final Hb4 concentrations for the Galemys trials was 0.05 mM, while those for the Uropsilus experiments ranged from 0.02 to 0.05 mM.…”

“…PCRs were purified with serapure magnetic beads and DNA was eluted in 20 μl of nuclease-free water. Table 1 for GenBank Accession numbers); note that the adult expressed βlike globin genes of shrews, moles, and hedgehogs is orthologous to the HBD genes of other eutherian mammals (Campbell et al, 2012;Gaudry et al, 2014), and thus the protein product is a δ-globin chain (as opposed to a β-globin chain). We followed the manufacturing protocol of Mybaits for hybridization using an Eppendorf Mastercycler Nexus Thermal Cycler (Thermo Fisher Scientific, Canada).…”

Enthalpic partitioning of the reduced temperature sensitivity of O2 binding in bovine hemoglobin

“…Given that the normal temperature sensitivity (i.e. heat uptake by Hb upon deoxygenation in the tissues, and heat liberation upon oxygenation at the respiratory surfaces) constitutes outward heat transport (that accounts for 9% of the heat produced by glucose metabolism), a reduction in temperature sensitivity will decrease Hb-mediated heat transfer, thus contributing to maintain warm tissues in regionally heterothermic animals (Weber and Wells, 1989;Campbell et al, 2012).…”