Light-scattering investigation of the subunit dissociation of Homarus americanus hemocyanin. Effects of salts and ureas.
The subunit dissociation of the hemocyanin of the lobster, Homarus americanus, by the various salts of the Hofmeister series and the hydrophobic reagents of the urea-guanidinium chloride (GdmCl) class was investigated by laser light scattering molecular weight measurements. The dissociations of the hemocyanin dodecamers to hexamers by the various salts and the lower members of the urea series are found to be rapid and reversible, as predicted by the mass action law for monomer-dimer type of reactions. The salts are found to be very effective dissociating reagents with the unusual order of increasing effectiveness, Cl- less than Br- less than I- less than ClO4-, SCN-. The ureas and GdmCl are found to be relatively ineffective dissociating agents. In addition, the ureas show a decreasing order of effectiveness in going from urea to methyl-, ethyl-, and propylurea. This suggests that hydrophobic interactions are not the dominant stabilizing forces between the pairs of hexamers that form the dodecameric structure. Polar and ionic interactions appear to be the major stabilizing forces of the dodecameric structure. The use of equations derived for predicting the effects of dissociating reagents and salts on the structure of subunit proteins [Herskovits, T. T., & Ibanez, V. S. (1976) Biochemistry 15, 5715-5721] together with binding and Setschenow constants based on model amino acid data is found to give good account of the dissociation behavior observed with the salts, urea, and methylurea in the presence of calcium ion at both pH 7.8 and pH 9.5. The apparent number of amino acids at the contact areas of the hexamers, Napp, required to fit the dissociation data were found to be 24 +/- 8 at pH 7.8 and 23 +/- 4 at pH 9.5. However, because of the possible effects of molecular microheterogeneity, the estimates of amino acids at the contact areas must be viewed with caution, depending on further investigations.
D000818:Animals; D000838:Anions; D006433:Hemocyanins; D008027:Light; D046911:Macromolecular Substances; D008433:Mathematics; D008970:Molecular Weight; D008121:Nephropidae; D012492:Salts; D012542:Scattering, Radiation; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014508:Urea
D000838:Anions; D046911:Macromolecular Substances; D012492:Salts; D014508:Urea; D006433:Hemocyanins