Expression, purification, and characterization of human hemoglobins Gower-1 (ζ2ε2), Gower-2 (α2ε2), and Portland-2 (ζ2β2) assembled in complex transgenic–knockout mice

Embryonic ζ- and ε-globin subunits assemble with each other and with adult α- and β-globin subunits into hemoglobin heterotetramers in both primitive and definitive erythrocytes. The properties of these hemoglobins—Hbs Gower-1 (ζ2ε2), Gower-2 (α2ε2), and Portland-2 (ζ2β2)—have been incompletely described as they are difficult to obtain in quantity from either primary human tissue or conventional expression systems. The generation of complex transgenic–knockout mice that express these hemoglobins at levels between 24% and 70% is described, as are efficient methods for their purification from mouse hemolysates. Key physiological characteristics—including P50, Hill coefficient, Bohr effect, and affinity for 2,3-BPG—were established for each of the 3 human hemoglobins. The stability of each hemoglobin in the face of mechanical, thermal, and chemical stresses was also determined. Analyses indicate that the ζ-for-α exchange distinguishing Hb Portland-2 and Hb A alters hemoglobin O2-transport capacity by increasing its P50 and decreasing its Bohr effect. By comparison, the ε-for-β exchange distinguishing Hb Gower-2 and Hb A has little impact on these same functional parameters. Hb Gower-1, assembled entirely from embryonic subunits, displays an elevated P50 level, a reduced Bohr effect, and increased 2,3-BPG binding compared to Hb A. The data support the hypothesis that Hb Gower-2, assembled from reactivated ε globin in individuals with defined hemoglobinopathies and thalassemias, would serve as a physiologically acceptable substitute for deficient or dysfunctional Hb A. In addition, the unexpected properties of Hb Gower-1 call into question a common hypothesis for its primary role in embryonic development.