Title: Hemocyanin of the Molluscan Concholepas concholepas Exhibits an Unusual Heterodecameric Array of Subunits
Abstract: We describe here the structure of the hemocyanin from the Chilean gastropod <i>Concholepas concholepas</i> (CCH), emphasizing some attributes that make it interesting among molluscan hemocyanins. CCH exhibits a predominant didecameric structure as revealed by electron microscopy and a size of 8 MDa by gel filtration, and, in contrast with other mollusc hemocyanins, its stabilization does not require additional Ca<sup>2+</sup> and/or Mg<sup>2+</sup> in the medium. Polyacrylamide gel electrophoresis studies, analyses by a MonoQ FPLC column, and Western blots with specific monoclonal antibodies showed that CCH is made by two subunits noncovalently linked, named CCH-A and CCH-B, with molecular masses of 405 and 350 kDa, respectively. Interestingly, one of the subunits undergoes changes within the macromolecule; we demonstrated that CCH-A has an autocleavage site that under reducing conditions is cleaved to yield two polypeptides, CCH-A1 (300 kDa) and CCH-A2 (108 kDa), whereas CCH-B remains unchanged. The CCH-A nick occurs at 4 °C, increases at 37 °C, and is not inhibited by the addition of protease inhibitors and/or divalent cations. Since the CCH structure is a heterodimer, we investigated whether subunits would be either intermingled, forming heterodecamers, or assembled as two homogeneous decamers. Light scattering and electron microscope studies of the <i>in vitro</i> reassociation of purified CCH subunits demonstrated that the sole addition of Mg<sup>2+</sup> is needed for its reassembly into the native decameric molecule; no homodecamer reorganization was found with either CCH-A or CCH-B subunits alone. Our evidence showed that <i>C. concholepas</i> hemocyanin is an unusual example of heterodecameric organization.