A kinetic analysis of the binding of oxygen and carbon monoxide to lamprey hemoglobin. Petromyzon marinus and Petromyzon fluviatilis.

We have studied the ligand-binding reactions of lamprey (Petromyzon marinus and Petromyzon fluviatitis) hemoglobin with O2 and CO in the pH range 9.0 to 5.6 by means of both rapid reaction and equilibrium techniques. All the data can be accurately described by a model in which it is assumed that there are two forms of hemoglobin, a high affinity monomer and a lower affinity dimer, and that the formation of the dimer is pH dependent and requires the protonation on the protein of one specific site which has a pK of 6.0. For the deoxyhemoglobin monomer-dimer reactions, the dissociation equilibrium constant, K2.1, is estimated as 0.5 pM at pH 5.6; the association rate constant as 2 X lo6 M-lsec-l; and the dissociation rate constant as 1 per sec. The binding rates for the reaction of CO with the monomer and the dimer are 0.9 X lo6 M-’ set-l and 0.12 X lo6 M-' set-l, respectively, and for the reaction of 02, 10 X lo6 M-’ set-l and 0.25 X lo6 M-' set-I. The dissociation rates of CO and O2 from the monomer are 0.09 and 80 per set, respectively, and for the monomer the partition coefficient, M, is 89. In the proposed mechanism the deoxyhemoglobin dimer itself is supposed to be noncooperative, and cooperativity, as seen at acid pH, results from the oxygen-linked dissociation of the liganded dimer which has a dissociation rate constant of 150 per sec. This dissociation gives rise to a high affinity species, and thus produces the observed cooperativity. The rate constants that gave the best fits to the kinetic data were used to simulate equilibrium 09-binding curves. The model accurately predicts the equilibrium behavior of this protein without considering aggregation above the level of the dimer. Physiologically, higher aggregates may not have functional importance.