Abstract
Abstract 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.
Highlights
The equilibrium curves are hyperbolic, indicating the absence of homotropic cooperative effects in the ligand-binding process. It was these properties that led Noble et al [1] and Tan et al [2] to postulate that at low pH, carp hemoglobin is frozen in the low affinity structure usually associated with deoxygenated hemoglobin and that the transition to the high affinity form, which results in cooperative effects in functionally normal hemoglobin, does not occur
Since the effect of phosphate removal on ligand affinity mimics the effect of increasing pH, it was of interest to determine if the effects of these two variables on the cooperativity of ligand binding would be similar. For this reason we have studied the pH dependence of the binding of oxygen and carbon monoxide to carp hemoglobin stripped of organic phosphates
PH dependence of carbon monoxide binding to stripped carp hemoglobin at 20” was studied
Summary
PH 5.6, in the presence of endogenous polyphosphates, carp hemoglobin has a very low ligand aflinity, 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 The equilibrium curves are hyperbolic, indicating the absence of homotropic cooperative effects in the ligand-binding process It was these properties that led Noble et al [1] and Tan et al [2] to postulate that at low pH, carp hemoglobin is frozen in the low affinity structure usually associated with deoxygenated hemoglobin and that the transition to the high affinity form, which results in cooperative effects in functionally normal hemoglobin, does not occur. If a hemoglobin is frozen in a single conformation, whether liganded or not, these two rates should be equal We have used this method to study carp hemoglobin in the presence and absence of organic phosphates over a wide pH range. These findings have been evaluated by computer simulation giving an approximation to the kinetic behavior of the two types of chains in carbon monoxide binding
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