Light-scattering investigation of the subunit dissociation of Homarus americanus hemocyanin. Effects of salts and ureas

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Abstract
The subunit dissociation of the hemocyanin of the lobster, H. 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 MW measurements. The dissociations of the hemocyanin dodecamers to hexamers by the various salts and the lower members of the urea series are rapid and reversible, as predicted by the mass action law for monomer-dimer type of reactions. The salts are very effective dissociating agents with the usual order of increasing effectiveness,Cl- < Br- < I- < ClO4-, SCN-. The ureas and GdmCl are relatively ineffective dissociating reagents. The ureas show a decreasing order of effectiveness in going from urea to methyl-, ethyl and propylurea. Hydrophobic interactions probably 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 together with binding and Setschenow constants based on model amino acid data gives a good account of the dissociation behavior observed with the salts, urea and methylurea in the presence of Ca 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 24 .+-. 8 at pH 7.8 and 23 .+-. 4 at pH 9.5. 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.