Hemocyanin of the chiton Acanthopleura granulata

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Abstract
The subunit structure and solution conformation of the hemocyanin of the chiton Acanthopleura granulata were investigated by light-scattering, ultracentrifugation, viscosity, absorbance, and circular dichroism methods. The molecular weight, determined by light scattering at pH 7.4 in the presence of 0.05 M Mg2+ and 0.01 M Ca2+, was (4.2 .THETA. 0.3) .times. 106, while those of dissociated subunits in the presence of 8.0 M urea (at pH 7.4) and at pH 10.7 were found to be 4.57 .times. 105 and 4.58 .times. 105, respectively. Circular dichroism and absorbance measurements at 222 and 346 nm indicate only minor changes in the conformation of the folded domains of the hemocyanin subunits in these dissociating solvents. As with the hemocyanins of the snails Busycon canaliculatum, Lunatia heros, and Littorina littorea, exposure to 4.0-6.0 M guanidinium chloride (GdmCl) is found to produce unfolding of the domains, resulting in much more pronounced spectral changes and a further drop in molecular weight. A Mw of 3.2 .times. 105 was obtained with Acanthopleura hemocyanin in 6.0 M GdmCl, suggesting hidden breaks in the polypeptide chains analogous to those observed with the gastropodan hemocyanins. Both urea and pH dissociation showed gradual declines in the molecular weights, consistent with a decamer-dimer-monomer scheme of subunit dissociation. The bell-shaped molecular weight profiles obtained in the pH region from 5 to 11 can be accounted for by assuming two proton-linked groups per dimer, characterized by apparent pK values of 5.5 and 9.5, and the further involvement of five to eight acidic and five to eight basic groups per monomer, having apparent pK values of 5.0 and 10.2. The urea dissociation implicates a significantly larger number of interacting groups per hemocyanin dimer and monomer and suggests hydrophobic stabilization of the subunits. A good account of the urea dissociation data, studied at pH 8.5 and a protein concentration of 0.1 g .cntdot. L-1, was obtained with an apparent estimate of 30 amino acid groups (Napp) at the areas of contacts of each dimer forming the basic decameric assembly and an additional 100 groups per monomer forming each dimer.