The kinetics and equilibria of squirrel-fish hemoglobin. A Root effect hemoglobin complicated by large subunit heterogeneity.

The functional properties of squirrel-fish hemoglobin have been measured by studying ligand binding equilibria and kinetics. The results show that squirrel-fish hemoglobin has a Root effect with a corresponding stabilization of the low affinity state. The properties of this state are pH dependent even in the absence of cooperativity. The effect of ATP shifts the overall ligant affinity towards the low affinity state and is characteristic of the allosteric effect caused by organic phosphates. Under pH and ATP conditions favoring the low affinity conformational state, a 10-fold difference in the binding kinetics of carbon monoxide to the alpha and beta subunits is observed.

Kinetic studies on the binding affinity of human hemoglobin for the 4th carbon monoxide molecule, L4.

L4, the affinity of hemoglobin for the 4th CO molecule, has been determined for human adult hemoglobin (HbA) as a function of pH and the presence of organic phosphates by measuring the kinetic parameters for the reaction. l'4, the rate of combination of CO with the triliganded molecule, was measured by flash photolysis while l4, the rate of CO dissociation for the ligand-saturated molecule, was measured by ligand replacement. L4 is pH-dependent and affected by 2,3-diphosphoglycerate. Additionally, this pH dependence of the high affinity state is largely eliminated by carboxypeptidase A digestion. L4 for human fetal hemoglobin (HbF) in phosphate buffers was also determined and found to be pH-dependent. These results cannot be reconciled within the framework of the two-state allosteric model. Additional structures in the conformational equilibrium due to either intermediates in the T to R transition or two or more R states must exist.

Structural states and transitions of carp hemoglobin.

The wide ligand affinity range previously observed for carp hemoglobin is bounded at both extremes by regions of constant affinity. Within these regions, pH, organic phosphates, and the extent of ligand binding have no effect on the measured affinity and the cooperativity of ligand binding is greatly reduced or absent. The rates of CO recombination to fully and partially unliganded carp hemoglobin, under various organic phosphate and pH conditions, are shown to reflect this behavior. Constant kinetic rates are seen to directly correspond to the regions of constant affinity. Therefore, these are taken to be single protein conformations, one of high and one of low ligand affinity. In the simplest view, these conformations represent the R and T states of a two-state model, and most of the properties of carp hemoglobin are explained quite well within this framework. Increases in either hydrogen or phosphate ion concentrations favor the stabilization of the low affinity structure of even fully liganded carp hemoglobin. We have studied the structural transition from high to low affinity by monitoring the absorption spectra of carp hemoglobins at constant pH as a function of organic phosphate concentration. We find that different spectra are induced in both carp methemoglobin and cyanomethemoglobin by inositol hexaphosphate addition. Furthermore, the dependence of the magnitude of the spectral changes on pH and organic phosphate concentration is the close agreement with that predicted from studies of the ligand binding properties of the molecule.

Ligand binding properties of hemoglobin 3 of the trout, Salmo gairdneri. The occurrence of an acid Bohr effect in the absence of heme-heme interaction.

The four components of hemoglobin from the rainbow trout (Salmo gairdneri) have been isolated. The oxygen affinities of the first two components eluted from the DEAE-cellulose column have much smaller pH dependencies than the last two components. These components have very low O2 affinities at low pH. The effect of pH on the equilibrium and kinetics of ligand binding to the third fraction, the pH-dependent component present in greatest amounts, has been studied. Measurements of ligand binding equilibria demonstrate the presence of both an alkaline and an acid Bohr effect. In the region of the alkaline Bohr effect the value of n in the Hill equation is a function of ligand affinity. For CO binding n decreases as the pH is decreased until at pH 6, the minimum ligand affinity is reached. At this pH there is also a complete loss of cooperative ligand binding. Decreasing the pH further results in an increase of ligand affinity, but this acid Bohr effect is not associated with a reappearance of cooperativity. This suggests that Fraction 3 of S. gairdneri is frozen in the low affinity, deoxygenated conformation at low pH and that the quaternary structure does not change even when fully liganded. However, the properties of the low affinity conformation of this hemoglobin are pH-dependent.