Hemocyanin from Tachypleus gigas. I. Oxygen-binding properties

Hemocyanin was prepared from an Asian horseshoe crab, Tachypleus gigas. The hemocyanin was found to be similar to Limulus hemocyanin in the size of native molecules (48-mer) and dissociation under nonphysiological conditions. It also showed the reverse Bohr effect. The O2 affinity of the dissociated monomer was higher than that of the native molecule. Equilibrium O2 binding to T. gigas hemocyanin was studied with special attention to the effect of inorganic ions. Neutral salts decreased the O2 affinity of the associated hemocyanin. In the presence of CaCl2 the strength of the effect was in the order of Na+ greater than Cs+ not equal to K+ for the series of chlorides, and Br- not equal to Cl- greater than SO4(2-) for the series of Na+ salts. A high concentration of CaCl2 (50-500 mM) considerably increased the Hill coefficient. The O2 binding data obtained under various ionic conditions were analyzed by model fitting. The two-state concerted model could be fitted to the data, if the ligand affinity of the states was allowed to vary. Statistical tests of the fitting showed that the hexameric structure can be regarded as the functional unit under physiological conditions.