Studies of human embryonic haemoglobins

Abstract

In this study, the three human embryonic haemoglobins Gower I (ζ2ε2), Gower II (α2ε2) and Portland (ζ2γ2) have been investigated. Due to ethical and practical problems in obtaining these proteins from natural sources, a plasmid based yeast expression system has been used to express large quantities of each protein. Optimal growth conditions for the expression of the three embryonic proteins, involving a three step growth protocol, have been developed. Agitation of the cells with glass beads, followed by a three step purification protocol, yielded essentially pure haemoglobins. Physicochemical characterization showed them to be in the "native" state. Extensive growth of the cultures leads to the formation of an abnormal form of haemoglobin. This form sulphaemoglobin is produced within the cell and its occurrence could be shown to be time dependent. Further investigations indicated that the production of this abnormal form only starts some time after induction of the culture. Sulphaemoglobin free samples of each of the three human embryonic haemoglobins have then been used to investigate their function employing equilibrium and kinetic methods. Oxygen-binding studies showed, that under what are usually considered physiological conditions, each of the embryonic haemoglobins showed a high oxygen affinity, coupled to a high degree of co-operativity. It was also found, that the degree of co-operativity was unchanged under all conditions used in equilibrium studies.