Direct and Indirect Pathways of Functional Coupling in Human Hemoglobin Are Revealed by Quantitative Low-Temperature Isoelectric Focusing of Mutant Hybrids
Functional energetic coupling within human hemoglobin has been explored by using quantitative analysis of asymmetric mutant hybrid equilibria. Previous work showed that the free energy of cooperativity is largely attributable to alterations in free energy that accompany changing interactions at the interface between α1β1 and α2β2 dimers [Pettigrew et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1849]. However, the issue of how cooperativity-linked sites in the molecule are energetically coupled in manifesting cooperative ligation is still not well delineated. In this paper we address the questions of what types of functional coupling pathways are operational in hemoglobin, what some of their characteristics are, and how they are related to one another. By constructing asymmetric mutant hybrid hemoglobins, we can assay how two structurally identical, symmetrically equivalent sites are energetically coupled in manifesting subunit assembly and/or cooperative ligation. Asymmetric hybrid hemoglobins, i.e., those containing a single modified site, cannot be isolated and must be studied in equilibrium with their symmetric parent molecules. In order to study these asymmetric hybrid equilibria, we have developed new theory and quantitation techniques to augment the low-temperature quenching and isoelectric focusing procedures of Perrella et al. [(1978) Anal. Biochem. 88, 212]. Studies of these mutant hybrid hemoglobins have provided evidence for three distinct types of energetic coupling within the hemoglobin tetramer. All α1β2 interface sites examined are involved in coooperativity-linked indirect coupling. Within the context of this indirect “pathway” there exist two different types of direct long-range coupling. One of these classes of direct long-range pathways is linked to cooperative ligand binding while the other class is not. © 1990, American Chemical Society. All rights reserved.
Publication Source (Journal or Book title)
LiCata, V., Speros, P., Rovida, E., & Ackers, G. (1990). Direct and Indirect Pathways of Functional Coupling in Human Hemoglobin Are Revealed by Quantitative Low-Temperature Isoelectric Focusing of Mutant Hybrids. Biochemistry, 29 (42), 9771-9783. https://doi.org/10.1021/bi00494a003