We have measured hemoglobin oxygen uptake vs. the partial pressure of oxygen, with independently controlled activities of chloride and water. This control is effected by combining different concentrations of NaCl and sucrose in the bathing solution to achieve: (i) water activities were varied and Cl- activity was fixed, (ii) both water and Cl- activities were varied with a traditional NaCl titration, or (iii) Cl- activities were varied and water activity was fixed by adding compensating sucrose. Within this analysis, the Cl(-)-regulated loading of four oxygens can be described by the reaction Hb.Cl- + 4 O2 + 65 H2O in equilibrium with Hb.4O2.65H2O + Cl-. The dissociation of a neatly integral chloride, rather than the nonintegral 1.6 chlorides inferred earlier from simple salt titration, demonstrates the need to recognize the potentially large contribution from changes in water activity when titrating weakly binding solutes. The single-chloride result might simplify structural considerations of the action of Cl- in hemoglobin regulation
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