Novel allosteric conformation of human HB revealed by the hydration and anion effects on O-2 binding

The effect of anions on the stability of different functional conformations of Hb is examined through the determination of the dependence of O-2 affin ity on water activity (a(w)). The control of a(w) is effected by varying th e sucrose osmolal concentration in the bathing solution according to the "o smotic stress" method. Thus, the hydration change following Hb oxygenation is determined as a function of Cl- and of DPG concentration. We find that o nly similar to 25 additional water molecules bind to human Hb during the de oxy-to-oxy conformation transition in the absence of anions, in contrast wi th similar to 72 that bind in the presence of more than 50 mM Cl- or more t han 15 mu M DPG. We demonstrate that the increase in the hydration change l inked with oxygenation is coupled with anion binding to the deoxy-Hb. Hence , we propose that the deoxy-Hb coexists in two allosteric conformations whi ch depend on whether anion is bound or not: the tense T-state, with low oxy gen affinity and anion bound, or a new allosteric P-state, with intermediat e oxygen affinity and free of bound anions. The intrinsic oxygen affinity o f this unforeseen P-state and the differential binding of Cl-, DPG, and H2O between states P and T and P and R are characteristics which are consisten t with those expected for a putative intermediate allosteric state of Hb. T hese findings represent a new opportunity to explore the structure-function relationships of hemoglobin regulation.