MULTIPLE LINKAGE IN PANULIRUS-INTERRUPTUS HEMOCYANIN

The linkage between various ligands of Panulirus interruptus hemocyanin, namely, oxygen, proton, and calcium and sodium ion, has been investigated. The binding of calcium, followed by means of a calcium ion selective electrode, revealed that the number of binding sites is strongly pH dependent. The overall dissociation constant of calcium is ~0.05 mM. No difference between binding to oxygenated and deoxygenated protein could be detected. Binding of calcium or sodium ions is associated with a release of protons. There is a difference of approximately 10-fold in apparent affinity between calcium and sodium. A smaller number of protons are released from deoxygenated as compared to oxygenated hemocyanin. Moreover, competition between the two cations for the same sites is suggested. Bohr effect measurements show a strong dependence of the release of oxygen-linked protons on the calcium and sodium concentrations. Measurements of oxygen equilibrium as a function of calcium, sodium, and hydrogen ions reveal specific effects of these cations on the biological function of the protein. At pH 7.6 there are at least two types of oxygen-linked calcium-binding sites, with dissociation constants of approximately 0.1 and 20 mM. These sites affect preferentially either the lower (T state) asymptote or the upper (R state) asymptote. Under the same conditions, sodium affects only the upper asymptote. At pH 6.4 the set of low-affinity oxygen-linked calcium-binding sites seems to be inactive. The observed phenomena are analyzed in the context of general linkage theory, and two possible linkage maps are designed to illustrate the various reciprocal effects of these allosteric ligands. Panulirus hemocyanin may be taken as an example of an extended system with different types of binding sites which may interact in order to control its specific biological function. © 1979, American Chemical Society. All rights reserved.