Experimental resolution of cooperative free energies for the ten ligation states of human hemoglobin.

Abstract

Tetrameric human Hb can assume 10 molecular forms that differ in the number and configuration of ligands bound at the 4 heme sites. For each of these species determined the cooperative free energy, i.e., the deviation in free energy of ligation from that which would obtain for the same sites binding as independent .alpha. and .beta. subunits. These cooperative free energies were resolved from measurements on the dissociation into dimers of tetramers in which each subunit is either unligated (Fe2+ deoxy) or ligated by conversion into the cyanomet form (Fe3+ CN). Each Hb tetramer apparently acts as a 3-level molecular switch. During the course of ligation, the total cooperative free energy (6 kcal/mol over all 4 binding steps) is expended in 2 transitions that are synchronized with particular ligation steps. Whether a cooperative energy transition occurs or not depends upon how the ligation step changes both the number and configuration of ligated subunits. The Hb tetramer is thus a combinatorial switch. The finding of 3 distinct free energy levels for the 10 ligation states suggests the existence of 3 major structural forms of the Hb tetramer.