Measuring Assembly and Binding in Human Embryonic Hemoglobins

Abstract

Publisher Summary Although the adult human hemoglobin is the most thoroughly studied protein and arguably one of the best understood, in terms of its function, the methods and models developed to investigate the detailed actions of this protein have not been widely applied to other hemoglobin systems. This chapter seeks to explore the methods and results obtained from the application of a range of techniques used to measure the assembly and binding processes that occur in the human embryonic hemoglobins. The human embryonic hemoglobins consist of a group of three hemoglobins synthesized in yolk sac-derived megaloblasts from approximately week 3 to week 16 of gestation. This chapter serves to illustrate the fact that, in order to assign the biological activity of a novel hemoglobin system, it is necessary to apply a wide range of methodologies. Only then, a proper understanding of this complex protein that shows both ligand and redox sensitivity within a multisubunit allosteric context can be obtained. Specifically, the methodologies described in this chapter have been applied successfully to the study of the three human embryonic hemoglobins obtained from recombinant yeast expression systems. Each of these hemoglobin expression systems consists of Saccharomyces cerevisiae cells containing plasmids based on the naturally occurring yeast 2μ plasmid. The plasmids have been engineered to contain each of the appropriate globin genes, linked in tandem to artificial galactose promoters. Nevertheless, the approaches described here are, in principle, applicable to any hemoglobin system.