Oxygen equilibrium curves have been measured to determine the binding constant at each oxygenation step (Ki) for various cross-linked hemoglobins, (aj8)A(a/3)cXL, (a+CN|8)A(a/3)cXL, (ajfT^)A(a/J)cXL, (a+CN/^CN)A(a/3)cXL, and (a|8+CN)A(a+CN|8)CXL, where the subscripts A and C denote that the aB dimer is derived from human normal adult hemoglobin and mutant hemoglobin C (B6Glu — Lys), respectively, and XL denotes cross-linking between the lysyl residues at position 82 in the two B chains by bis(3,5-dibromosalicyl) fumarate as described by Miura and Ho [Miura, S., & Ho, C. (1982) Biochemistry 21, 6280–6287]. The oxygen equilibrium data indicate that the oxygen affinity increases with the number of cyanomet hemes carried by the cross-linked mixed valency hybrid hemoglobins. The oxygen binding property depends not only on the number of the subunits carrying cyanomet hemes but also on the distribution of cyanomet hemes among the four subunits. A striking effect is observed in singly cyanomet valency hybrid hemoglobins; namely, (a+CNB)A(aB)cXL exhibits lower oxygen affinity and higher cooperativity than (aB+CN)A(aB)cXL. The magnitude of the Adair constants and their pH dependency of (a+CNB)A(aB)cXL (Ki, i = 1–3) are analogous to those of the Adair constants of (aB)A(aB)cXL (Ki,i = 2–4), whereas such an analogy is not observed between (aB+CN)A(aB)cXL and (aB)A(aB)cXL. The doubly cyanomet mixed valency hybrid cross-linked hemoglobins exhibit high oxygen affinity and reduced cooperativity, and their Adair constants are not analogous to K3 and K4of (aB)A(aB)cXL. The present results on the oxygen binding properties of cross-linked mixed valency hybrid hemoglobins provide additional support to the conclusion based on a previous proton nuclear magnetic resonance investigation of these hybrid hemoglobins that there are at least three functional and energetically important structures of hemoglobin in going from the deoxy to the ligated state. Thus, the cooperative oxygenation process of human normal adult hemoglobin cannot be simply described by two-structure allosteric models.
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