Expression, purification, and characterization of human hemoglobins Gower-1 (zeta(2)epsilon(2)), Gower-2 (alpha(2)epsilon(2)), and Portland-2 (zeta(2)beta(2)) assembled in complex transgenic-knockout mice

Embryonic zeta- and epsilon -globin subunits assemble with each other and w ith adult alpha- and beta -globin subunits into hemoglobin heterotetramers in both primitive and definitive erythrocytes, The properties of these hemo globins-Hbs Gower-1 (zeta (2)epsilon (2)), Gower-2 (alpha (2)epsilon (2)), and Portland-2 (zeta (2)epsilon (2)), have been incompletely described as t hey are difficult to obtain in quantity from either primary human tissue or conventional expression systems. The generation of complex transgenic-knoc kout mice that express these hemoglobins at levels between 24% and 70% is d escribed, as are efficient methods for their purification from mouse hemoly sates. Key physiological characteristics-including P-50, Hill coefficient, Bohr effect, and affinity for 2,3-BPG-were established for each of the 3 hu man hemoglobins. The stability of each hemoglobin in the face of mechanical , thermal, and chemical stresses was also determined. Analyses indicate tha t the zeta -for-alpha exchange distinguishing Hb Portland-2 and Hb A alters hemoglobin O-2-transport capacity by increasing its P-50 and decreasing it s Bohr effect. By comparison, the epsilon -for-beta exchange distinguishing Hb Gower-2 and Hb A has little impact on these same functional parameters. Hb Gower-1, assembled entirely from embryonic sub-units, displays an eleva ted P-50 level, a reduced Bohr effect, and increased 2,3-BPG binding compar ed to Hb A. The data support the hypothesis that Hb Gower-2, assembled from reactivated epsilon globin in individuals with defined hemoglobinopathies and thalassemias, would serve as a physiologically acceptable substitute fo r deficient or dysfunctional Hb A. In addition, the unexpected properties o f Hb Gower-1 call into question a common hypothesis for its primary role in embryonic development.