title: 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.
abstract: Embryonic zeta- and epsilon-globin subunits assemble with each other and with adult alpha- and beta-globin subunits into hemoglobin heterotetramers in both primitive and definitive erythrocytes. The properties of these hemoglobins-Hbs Gower-1 (zeta(2)epsilon(2)), Gower-2 (alpha(2)epsilon(2)), and Portland-2 (zeta(2)beta(2))-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 P(50), 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 zeta-for-alpha exchange distinguishing Hb Portland-2 and Hb A alters hemoglobin O(2)-transport capacity by increasing its P(50) and decreasing its 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 subunits, displays an elevated P(50) 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 epsilon 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.
authors.affiliation: Department of Medicine and Pediatrics, University of Pennsylvania School of Medicine and The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: He
authors.forename: J E
authors.initials: JE
authors.lastname: Russell
mesh_terms: D019794:2,3-Diphosphoglycerate / Q000378:metabolism; D000818:Animals; D004622:Embryo, Mammalian / Q000378:metabolism; D005312:Fetal Blood / Q000737:chemistry; D015870:Gene Expression; D006418:Heme / Q000378:metabolism; D006455:Hemoglobins, Abnormal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013045:Species Specificity; D013329:Structure-Activity Relationship
chemical_list: D006455:Hemoglobins, Abnormal; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D019794:2,3-Diphosphoglycerate; D006418:Heme; C030447:hemoglobin Portland; C030446:hemoglobin Gower; D010100:Oxygen