Light-scattering investigation of the dissociation behavior of Lunatia heros and Littorina littorea hemocyanins

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Abstract
The subunit structure and dissociation of the hemocyanins of 2 marine snails, L. heros and L. littorea, were investigated by light-scattering MW methods. The hemocyanins of both species of snails are readily dissociated to fragments of 1/10 and 1/20 of the parent proteins of close to 9 .times. 106 daltons by either increasing the pH or using dissociating reagents of the hydrophobic urea series or some of the Hofmeister salts. The lower members of the latter group of reagents, NaCl, and to some extent also NaBr were found to have only marginal effects on the observed MW transitions, suggesting that the 2 hemocyanins investigated possess .beta.-type subunits, which are known to be resistant to NaCl dissociation. The MW profiles obtained with the various dissociating reagents were single inverted sigmoidal-shaped curves for both Lunatia and Littorina hemocyanins, suggesting overlapping transitions. The ultracentrifugation patterns and the species-distribution plots based on the urea dissociation data of Littorina hemocyanin suggest the presence of whole, half, and 1/10 MW species in the dissociation transition region. Fitting of the urea dissociation data of Littorina hemocyanin obtained at both pH 5.7 and pH 8.0, assuming a sequential 2-step dissociation scheme used in previous studies, was found to be consistent with a model of a few hydrophobic binding sites at the contact areas of the half-molecules and a much larger apparent number of binding sites (Napp) at the side to side contacts of the 1/10 molecules. Model calculations also showed that with much larger Napp values for the first dissociation step, approaching that of the 2nd step of the dissociation reaction (Napp = 50-60), biphasic curves would be produced. This suggests that the stabilizing interactions between the basic decameric units of the marine hemocyanins are largely nonhydrophobic in origin, and therefore must be due primarily to polar and ionic interactions. The much larger Napp estimates characterizing the 2nd step of the dissociation reaction are comparable to those obtained with the .alpha. component of Helix pomatia hemocyanin. Hydrophobic stabilization of each half-molecule through side to side contact of the dimeric subunits is suggested by both the latter observations and also finding that the dissociation of Littorina, as well as Lunatia, hemocyanin by the urea series follows the expected order of increasing effectiveness with increasing hydrophobicity of the dissociating reagent.