Oxygen and CO ligand binding kinetics have been studied for the hybrid hemoglobin (Hb) a(human):β(carp), hybrid II. Valency and half-saturated hybrids were used to aid in the assignment of the conformations of both chains. In hybrid II, an intermediate S state occurs, in which one chain has R- and the other T-state properties. In HbCO at pH 6 (plus 1 mM inositol hexaphosphate), the human α-chain is R state and the carp β-chain is T state. We have no evidence at this pH that the carp β-chain ever assumes the R conformation. At pH 6, the human α-chain shows human Hb R-state kinetics at low fractional photolysis and T-state rates for CO ligation by stopped flow. At pH 7, the human-chain R-state rate slows toward a carp hemoglobin rate. The carp β-chains, on the other hand, react 50% more rapidly in the liganded conformation than in carp hemoglobin, and while the human α-chains are in the R state, the two β-chains appear to function as a cooperative dimer. In this hemoglobin, the chains appear to be somewhat decoupled near pH 7, allowing a sequential conformational change from the R state in which the β-chains first assume T-state properties, followed by the α-chains. The rate of the R-T conformational change for the carp β-chains is at least 300 times greater than that for the human α-chains. At pH 9, the R → T conformational transition rate is at least 200 times slower than that for human hemoglobin. The carp β-chain R state at this pH reacts twice as rapidly with CO as in carp hemoglobin. Diagrams are presented showing all conformations supported by kinetic evidence. The ligand kinetics in the hybrid suggest that in carp hemoglobin, the β-chains prevent the carp /3-chains from assuming full R-state properties and are themselves biased toward the T conformation. The marked tendency of the carp /3-chains to assume the T state lends strong support to the model that assigns a central role to the /3-chain in the Root effect [Perutz, M. F., & Brunori, M. (1982) Nature (London) 299, 421-426], From the data at hand, however, it does not appear possible to assign a definite number of Bohr protons to the four individual chains in carp and human hemoglobins.
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