Dissociation and reassembly of Limulus polyphemus hemocyanin.

Limulus polyphemus hemocyanin is a 3.3 x 10(6)-Mr protein containing 48 subunits in an assemblage of eight hexamers. The molecule can be dissociated into monomers and dimers at pH 8.9 in the presence of 0.01 M EDTA. These subunits are heterogeneous and can be separated into five zones (I--V) by DEAE-Sephadex chromatography. Reassembly experiments were carried out with varied subunit mixtures, based on different combinations of the five chromatographic zones, in order to study the structural role of the diverse subunits in the eight-hexamer molecule. The reassembly products were analysed by electron microscopy and ultracentrifugation. No structural role for zone I could be found. Zone V and possibly zone II are needed to form structures larger than hexamers. Absence of zone III causes irregular aggregation of hexamers. Zone IV and perhaps zone II are needed to make eight-hexamer molecules from four-hexamer molecules. From these results we conclude that there is a high degree of subunit specificity in the inter-subunit contacts in the native Limulus hemocyanin molecule.