Light-scattering investigation of the subunit structure and dissociation of Octopoda hemocyanins

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Abstract
The molecular weights, subunit dissociation, and conformation in solution of the hemocyanins of three species of octopi were investigated by light-scattering, ultracentrifugation, absorbance, and circular dichroism methods. The molecular weights of the hemocyanins of Octopus bimaculoides, Octopus bimculatus, and Octopus rubescens obtained by light scattering were 3.3 .times. 106, 3.4 .times. 106, and 3.5 (.+-.0.3) .times. 106, respectively. The average molecular weights of the fully dissociated hemocyanins of the same octopi, investigated at alkaline pH and in the presence of 8 M urea and 6 M guanidinium chloride (GdmCl), were found to be close to one-tenth of those of the parent proteins, with average molecular masses of 3.4 .times. 105, 3.3 .times. 105, and 3.3 (.+-.0.3) .times. 105. These findings confirm the earlier observations of van Holde and co-workers with other cephalopod hemocyanins that the basic cylindrical assembly of molluscan hemocyanins consists of 10 subunits. Circular dichroism and absorbance measurements suggest that the dissociated subunits at alkaline pH and in concentrated urea solutions retain their native, multidomain folding. Fairly concentrated GdmCl above 3-4 M is necessary to unfold fully the dissociated hemocyanin chains. Molecular weight measurements studied as a function of reagent concentration with the urea and Hofmeister salt series as dissociating agents show that the ureas are very effective dissociating agents, while the salts are ineffective to moderately effective reagents for octopus hemocyanin. With the ureas the increasing order of effectiveness with greater hydrophobicity of the reagent, in going from urea to methyl-, ethyl-, propyl-, and butylurea, suggests that the basic decameric unit of molluscan hemocyanins in solution is hydrophobically stabilized. The more quantitative interpretation of the dissociation behavior of octopi hemocyanins is made simpler by the absence of stabilizing interdecamer contacts encountered with the land and marine snail hemocyanins, which are largely nonhydrophobic in character [Herskovits, T. T., Mazzella, L. J., and Villanueva, G. B. (1985) Biochemistry 24, 3862-3870]. The fit of the urea dissociation data obtained with O. bimaculoides hemocyanin could be best accommodated with a model of interacting dimers with about 40-50 apparent amino acid residues (Napp) at the contact areas of each dimer forming the parent decamer, with each dimer stabilized by a larger number of amino acid contacts represented by Napp values of 110-150 amino acid groups per monomer.