The structure of partially oxygenated hemoglobin. A highly reactive intermediate toward a sulfhydryl titrant.

It is known that Cys beta 93 in hemoglobin A reacts with sulfhydryl reagents more rapidly in the oxy than in the deoxy form. In this study, the reaction of the residues toward 4,4'-dithiodipyridine was measured at various degrees of saturation with O2. Analysis of initial rates of the reaction revealed that an intermediate, which reacts even more rapidly than the oxy form, occurs in the course of O2 binding to hemoglobin A, especially at higher degrees of saturation. Its occurrence was independent of the overall O2 affinity of hemoglobin. Time courses of the reaction were measured at pH 8.0, where the change in O2 saturation of hemoglobin by the 4,4'-dithiodipyridine reaction is negligible, and they were analyzed by the curve-fitting procedure. It was found that the rapidly reacting species appears in parallel with the Adair intermediate carrying three O2 molecules, and their close relationship was suggested. Furthermore, the analysis of the time course indicated the existence of another molecular species with an intermediate reactivity between those of oxy and deoxy forms. It was concluded that the ligand-linked structural changes in hemoglobin take place through several steps.