Oxygen binding by Limulus polyphemus hemocyanin: allosteric modulation by chloride ions

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Abstract
60S L. polyphemus hemocyanin contains 48 O2-binding sites. The effect of Cl- on the O2-binding properties of this huge molecule was examined by equilibrium and rapid-mixing techniques. Ultracentrifugation verified that the sedimentation coefficient of the hemocyanin was 60S in all of the conditions examined. Cl- lowered the O2 affinity of Limulus hemocyanin at all pH values, from pH 7.0-9.5, with maximum effectiveness at pH 8.5. Kinetically this decrease in affinity was reflected in a pH-dependent increase of the O2 dissociation rate. Cl- shifted the deoxy asymptotes of the Hill plots (the low affinity, n = 1, portion) and ultimately stabilized the 60S structure in a noncooperative low-affinity state. Based on the shift of the deoxy asymptotes, equilibrium association constants for the reactions: Hcy + O2 .tautm. Hcy-O2 (K1), Hcy + Cl .tautm. Hcy-Cl (K2), Hcy-O2 + Cl .tautm. Hcy-Cl-O2 (K3), and Hcy-Cl + O2 .tautm. Hcy-Cl-O2 (K4), were calculated where Hcy represented hemocyanin in the initial portion of the binding curve where O2 binding was noncooperative. The effect of Cl- on O2-binding by Limulus hemocyanin was satisfactorily accounted for within a concerted 2-state model (Monod, et al, (1965)) with the modification that this ligand not only changed the allosteric equilibrium constant, but also changed the nonexclusive binding coefficient by decreasing the O2 affinity of the unliganded state (K1 > K4). This interpretation, which implied the presence of more than 1 apparent T state, was supported by kinetic data. Curve-fitting within the context of the modified MWC model suggested that the hexamer was the allosteric unit in the 60S molecule.