Physical studies of the hemocyanin of the marine gastropod, Kelletia Kelleti (forbes)

Abstract

1. 1. The hemocyanin of the Californian whelk, Kelletia kelleti, investigated at pH and ionic conditions close to physiological, has a molecular weight close to 9.0 × 10 6 and a sedimentation constant of 114S, characteristic of the di-decameric structure of molluscan hemocyanins. Light-scattering measurements at pH 8.0, 0.05M Mg 2+, 0.01 M Ca 2+ gave a molecular weight of 9.0 ± 0.6 × 10 6 , and scanning transmission electron microscopy produced nearly the same particle mass of 9.22 ± 0.50 × 10 6 daltons (Da). 2. 2. Light-scattering measurements on the fully dissociated monomers in the presence of 8.0 M urea and at pHs 10.6 and 11.0 gave molecular weights of 4.50 × 10 5–4.91 × 10 5 , that are close to one-twentieth of the mass of the parent di-decameric hemocyanin assembly. 3. 3. Changes in pH produced a bell-shaped molecular weight profile, with molecular weights close to 9.0 × 10 6 in the pH region of about 5.5–8.0, and progressive dissociation to 4.5 × 10 5 Da monomers in the region below pH. 40 and above pH 9.0 or 10, depending on the absence or presence of stabilizing Mg 2+ ions (0.01 M). 4. 4. In the absence of divalent ions some aggregation of hemocyanin was found at pHs close to 5.0, with observed molecular weights above 10 × 10 6 (investigated at a hemocyanin concentration of 0.10 g/l). The early studies of Condie and Langer ( Science 144, 1138–1140, 1965) had shown that Kelletia kelleti hemocynanin aggregates at acidic pHs close to the isoelectric point, forming linear polymers of the hemocyanin di-decamers. 5. 5. The influence of various ureas and salts on the subunit structure and the denaturation of the dissociated subunits, followed by light-scattering and optical rotation, indicate hydrophobic stabilization of the subunit contacts within the decameric sub-assemblies, as well as in the folding or tertiary structure of the constituent subunits.