Energetic Differences at the Subunit Interfaces of Normal Human Hemoglobins Correlate with Their Developmental Profile

Abstract

A previously unrecognized function of normal human hemoglobins occurring during protein assembly is described - - self-regulation of subunit pairings and their durations arising from the variable strengths of their subunit interactions. Although it is known that many mutant human hemoglobins have altered subunit interface strengths, those of the normal embryonic, fetal, and adult human hemoglobins have not been considered to differ significantly. However, in a comprehensive study of both types of subunit interfaces of seven of the eight normal oxy human hemoglobins, we found that the strength, i.e. the free energies of the tetramer-dimer interfaces, contrary to previous reports, differ by 3-orders of magnitude and display an undulating profile similar to the transitions (“switches”) of various globin subunit types over time. The dimer interface strengths are also variable and correlate linearly with their developmental profile; embryonic hemoglobins are the weakest, fetal hemoglobin is of intermediate strength, and adult hemoglobins are the strongest. The pattern also correlates generally with their different O2 affinities and responses to allosteric regulatory molecules. Acetylation of fetal hemoglobin weakens its unusually strong subunit interactions and occurs progressively as its expression diminishes and adult hemoglobin A formations begins; a causal relationship is suggested. The relative contributions of globin gene order and competition among subunits due to differences in their interface strengths were found to be complementary and establish a connection between genetics, thermodynamics, and development.

Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces

“…2(C), blue curve] consistent with its rapid onset at 6 weeks 16,17 due to its very low-free energy as calculated from its dissociation constant. 13 Subsequently, slow posttranslational acetylation at the N-terminus of its c-subunit (HbF 1 , a 2 c In addition, the c-subunits of HbF undergo unproductive c 2 -homodimer formation, which limits the amount of functional ac heterodimers. 20 The other adult Hb (HbA 2 , a 2 d 2 ) displays an abnormal equilibrium between dimers and monomers as shown by its wide peak [ Fig.…”

“…The preferential assembly of a 2 b 2 over a 2 c 2 has been demonstrated by in vitro studies involving mixing of isolated Hbs. 12,13,22 Thus, when the weak embryonic Hbs f 2 b 2 , f 2 c 2 , and a 2 e 2 are mixed, they slowly dissociate and reassemble to form a 2 b 2 (HbA), the strongest tetramer, through subunit exchange, that is,…”

“…The same conclusion regarding the preferential assembly of a 2 b 2 over a 2 c 2 has been reached by in vivo studies using a yeast expression system in which a, b, and c genes in different orders were allowed to compete; HbA (a 2 b 2 ) was consistently formed in greater amounts than (a 2 c 2 ) HbF, regardless of gene order. 13 Hence, a major driving force in the developmental formation of Hbs may be competition among subunits to form more favorable pairs (and eventually tetramers) with progressively stronger subunit interfaces. However, even though we have demonstrated subunit competition/exchange, we have no evidence that such a mechanism is physiologically operative.…”

“…12,13 In this article, these equilibria have been studied together in a single measurement as linked events. Information on individual equilibria enabled us to choose a Hb concentration where differences in their subunit interface bonding strengths would be manifested; if too high a Hb concentration had been studied, the equilibria of all the Hbs would have been shifted toward the tetrameric state thus obscuring any differences between them.…”

Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding

“…We reported earlier (4, 10) that the T/D equilibria for the fetal and embryonic hemoglobins differ significantly from that of adult hemoglobin (13–15) and each equilibrium was found to be completely reversible. Of particular interest, however, was the finding that the embryonic hemoglobins deviate from the adult hemoglobin paradigm regarding their ( D/M ) interfaces, which are relatively weak in comparison to those of adult and fetal hemoglobins (10, 17–19) as shown in Fig. 2.…”