LIGHT-SCATTERING INVESTIGATIONS OF THE SUBUNIT DISSOCIATION OF HUMAN AND HORSE HEMOGLOBINS
The subunit dissociation of human and horse hemoglobins has been studied by light-scattering molecular weight methods. Dissociation to (alpha)(beta) dimers of both proteins is effected by several reagents including urea, alkylureas, guanidinium chloride and some neutral salts of the Hofmeister series. Urea, methylurea and ethylurea have been found to effect increasingly greater dissociation of both proteins as the length of the alkyl sidechain of the urea is increased. More pronounced effects have been found in the case of human hemoglobin than in horse. These results have suggested that hydrophobic interactions are important in the stabilization of the native tetrameric form of both proteins, although not as important in the case of horse hemoglobin. The effects of the neutral salts of the Hofmeister series have been found to be dominated by the anion of these salts and the qualitative order of dissociating strength of these salts has been found with both proteins to follow the usual Hofmeister order, namely SCN('-), I('-(, ))> C10(,4) > Br('-(, ))> Cl('-) > F('-). The more effective salts of the series have been found to be strong dissociating agents regardless of the presence of hydrophobic interactions in the stabilization of the subunit protein. Equations derived to account for the dissociating effects of these various reagents on the subunit structure of proteins in terms of the binding or interaction constant of the reagent and the average amino acid of the protein are presented. Binding constants for the urea class of reagents have been evaluated independently based on denaturation and free energy of transfer data available in the literature. In the absence of such data for the neutral salts, the related Setschenow constants derived from solubility studies of model compounds in salt solutions available in the literature have been used to evaluate the binding constants for these reagents and have been found to give good account of the dissociating effects of these salts. Analysis of dissociation data based on these equations and binding constants have given estimates of the number of amino acid residues present at the areas of contact between the human and horse hemoglobin dimers. The numbers thus obtained of about 18-19 for human hemoglobin and about 15-16 for horse are consistent with the X-ray crystallographic structures of these proteins which suggest that contact areas in horse hemoglobin are smaller than the human ones. However, these structures also suggest that 27 and 18 amino acids are present at the areas of subunit contact of human and horse hemoglobins. It is shown that low estimates of the number of contact area amino acids are expected from dissociation studies due to the fact that the change in accessibility to solvent of the contact area amino acids that takes place as the protein dissociates is less than one. Analysis of the dissociation data obtained with urea and Gdm-Cl in terms of the free energy of transfer of the contact area amino acids from water to solution containing these reagents has yielded estimates of this average fractional change in accessibility, (alpha) of the contact area amino acids of human and horse hemoglobins. (alpha) values of 0.23 and 0.28 have thus been obtained from the human and horse proteins respectively which are very similar to those obtained based on accessible surface area calculations. Comparison of these values with those for amino acids considered to be fully accessible to solvent suggest that the contact area amino acids of human and horse hemoglobins are characterized by average fractional exposures of 0.63 and 0.78 respectively. These results have suggested that the actual number of contact area amino acids of human and horse hemoglobins are 30 and 21 respectively in reasonably good agreement with the X-ray structures of these proteins. These results thus extend the utility of the methods described for the study of other subunit proteins.
SAN GEORGE, RICHARD CHARLES, "LIGHT-SCATTERING INVESTIGATIONS OF THE SUBUNIT DISSOCIATION OF HUMAN AND HORSE HEMOGLOBINS" (1980). ETD Collection for Fordham University. AAI8020083.