Limulus hemocyanin is a 48-subunit complex that is composed of eight immunochemically distinct subunits. Conditions were established that allowed for comparison of the structural and functional properties of the native molecule with those of its 24-subunit and 12-subunit dissociation products by analytical ultracentrifugation, functional analysis, stopped-flow light scattering, and circular dichroism spectroscopy. The 48-subunit complex was found to be specifically stabilized by calcium ions. When 48-subunit molecules at pH 7 were rapidly mixed with a pH 9 buffer containing a calcium chelator, the dissociation to monomers was complete within minutes. The rate is dependent upon the concentration and Ca2+ affinity of the chelator, consistent with the formation of a transient chelator-calcium ion-protein complex during the dissociation process. Dissociation to the level of 24-subunit molecules occurs much more slowly than does the dissociation of 24-subunit molecules to monomers. The dissociation to monomers, from 48-, 24-, or 12-subunit aggregates, is a highly cooperative process, and the corresponding reassembly reactions show appreciable hysteresis. In contrast, the reversible dimerization of dodecamers appears to be a rapidly equilibrating system. The varied aggregation states of Limulus hemocyanin are characterized by different circular dichroism spectra. These are, however, not unambiguously associated with the aggregation-state changes in that the CD spectra were found to be sensitive to pH under conditions where the aggregation state was unaltered. The 24-subunit complex of Limulus hemocyanin was found to be similar in its functional properties to the native 48-subunit aggregate. No unique functional properties thus appear to be associated with the 48-subunit ensemble that is found only in horseshoe crabs. Upon dissociation to the level of dodecamers, the reverse Bohr effect, cooperatively, and modulation of oxygen affinity by NaCl are still present although diminished in magnitude. Taken together, these studies clearly indicate that the interactions that stabilize dodecamers are different in pH and ion sensitivity from the interactions that are involved in formation of the 24-subunit complex from dodecamers and that these in turn differ from the interactions that stabilize the 48-subunit complex. These results are consistent with other lines of evidence that indicate that interactions at these different levels are dominated by structurally and functionally distinct types of subunits.
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