Energetic components of the allosteric machinery in hemoglobin measured by hydrogen exchange

A hydrogen exchange (HX) functional labeling method was used to study allosterically active segments in human hemoglobin (Hb) at the α-chain N terminus and the β-chain C terminus. Allosterically important interactions that contact these segments were removed one or more at a time by mutation (Hbs Cowtown, Bunbury, Barcelona, Kariya), proteolysis (desArg141α, desHis146β), chemical modification (N-ethylsuccinimidyl-Cys93β), and the withdrawal of extrinsic effectors (phosphate groups, chloride). The effects of each modification on HX rate at the local and the remote position were measured in the deoxy Hb T-state and translated into change in structural free energy at each position.