Conditions Restricting Allosteric Transitions in Carp Hemoglobin

Below pH 5.6, in the presence of endogenous polyphosphates, carp hemoglobin has a very low ligand affinity, and ligand binding is noncooperative, the values of n in the Hill equation being 0.75 and 1.0 for oxygen and carbon monoxide, respectively (Tan, A. L., De Young, A., and Noble, R. W. (1972) J. Biol. Chem. 247, 2493–2498). It has been postulated that under these conditions the molecule remains in the low affinity deoxy conformation even when liganded. Raising the pH above 5.6 or removing the organic phosphates converts carp hemoglobin to a molecule that exhibits the usual cooperative ligand binding. Ligand affinity and the degree of cooperativity are phosphate- and pH-dependent. Above pH 8.2 in the presence of organic phosphates and above pH 7.5 in their absence, the ligand-binding properties approach those characteristic of a molecule that again remains in a single conformation, only this time with a high ligand affinity. Whenever ligand binding was cooperative, the rate of CO recombination with partially liganded hemoglobin produced by partial flash photolysis was faster than that with fully unliganded hemoglobin produced by full flash photolysis. Under conditions when the molecule was thought to remain in one structure, the rates of CO recombination upon full and partial flash photolysis were equal. The time course of the conformation change in carp hemoglobin relative to ligand binding has been examined by measuring the rate of release of a fluorescent polyphosphate analogue during CO binding. At low pH there was a marked lag between dye release and CO binding. This lag decreased and was finally reversed with increased pH in agreement with our other results. Kinetic studies of CO combination at wave lengths near the Soret isosbestic point revealed a difference in the rate of CO binding to α and β chains of carp hemoglobin. Although our results for the most part can be explained on the basis of a simple two-state model of hemoglobin, computer fits of the time course of the CO combination reaction could not be achieved with such a simplified scheme, but rather required intra-dimer interactions.