Abstract
The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the α(A)-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the α(D)-globin gene). The α(D)-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O(2) affinity in the presence of allosteric effectors such as organic phosphates and Cl(-) ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O(2) affinity stems primarily from changes in the O(2) association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the α(D)-globin gene that is shared with the embryonic α-like globin gene.
Highlights
The functional significance of hemoglobin heterogeneity remains a mystery
The ␣D-globin gene originated via tandem duplication of an embryonic ␣-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function
Functional Properties of Avian HbA and HbD Isoforms—O2equilibrium measurements revealed consistent functional differences between the HbA and HbD isoforms, as HbD was generally characterized by a higher O2 affinity in the presence of IHP (2-fold molar excess over Hb) and in the presence of IHP ϩ 0.1 M ClϪ (Table 1)
Summary
The functional significance of hemoglobin heterogeneity remains a mystery. Results: In adult birds, the HbD isoform (related to embryonic hemoglobin) exhibits distinct oxygenation properties relative to the major HbA isoform. Because embryonic and adult-expressed Hbs exhibit a number of consistent functional differences [42], the scenarios depicted, D–F, suggest the possibility that HbA/D isoform differentiation may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function To investigate this possibility and to examine the functional evolution of the ␣-like globin genes, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Results of the comparative sequence analysis revealed that isoHb differentiation is attributable to roughly equal numbers of post-duplication amino acid substitutions that occurred in the ␣A- and ␣D-globin genes
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