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Intermediate states of assembly in the dissociation of gastropod hemocyanin by hydrostatic pressure.

Under physiological conditions, the oxygen-transport protein from the gastropod Megalobulimulus ovatus, an extracellular hemocyanin, is composed of 20 identical subunits organized into a cylindrical structure (M(r) 9 x 10(6); 100 S). It dissociates in the pressure range of 0.4-2.5 kbar, as observed by spectroscopic methods (light scattering and intrinsic fluorescence) and gel filtration. In contrast to what is seen with smaller proteins, especially dimers, the pressure-dissociation curves for hemocyanin show little dependence on concentration, suggesting that native hemocyanin exists as a population of molecules with different free energies of association. The pressure-induced dissociation results from an equilibrium in which each aggregate responds to pressure independently of the others and, at any given pressure, is in one of two states, whole or dissociated, which persists for long times when compared with the duration of the experiment. The subunit-subunit affinity of dissociated hemocyanin is much lower than that of associated subunits, suggesting that a conformational drift of monomers occurs. When hemocyanin undergoes dissociation in the absence of calcium and at high pH (> 7.2), a large fraction of the dissociated products changes to a conformation that generates stable intermediate states of assembly, lacking the ability to fully reassemble into decamers and didecamers. These intermediates consist primarily of dimers (M(r) 900,000), and they bind oxygen reversibly with a higher affinity than the native hemocyanin. The binding of calcium or protons changes the conformation back to the "associable" state, which finally generates the assembled structure. The dissociation process is highly reversible at low pH (6.8-6.0) or in the presence of millimolar concentrations of calcium. At pH 5.7, dissociation is negligible at pressures up to 2.5 kbar. A decrease in pH from 7.6 to 6.6 increases the half-dissociation pressure (p 1/2) by 1.3 kbar, corresponding to a stabilization of 1.35 kcal per mole of subunit. The effects of Ca2+ and H+ may mean that, in vivo, special ionic conditions or other factors are required to be present at the assembly sites of oligomeric proteins such as hemocyanin.