Role of ionic strength on hemoglobin interparticle interactions and subunit dissociation from light scattering

The average diffusion coefficients of hemoglobin (bovine CO-Hb) in dilute solutions (phi = 0.005), as obtained by cumulant analysis of the autocorrelation functions of the scattered light, are found to depend upon pH and ionic strength. A decrease of the diffusion coefficient is observed vs ionic strength for two salts, NaCl or CaCl2, in the range 10-300 mM, when the solution pH is away from that corresponding to the isolectric point of the protein. It is shown here that the results can be interpreted in terms of electrostatic effects with a model that should include both interparticle interactions and subunit dissociation of the protein. Global analyses of the first cumulant diffusion coefficient vs ionic strength were then applied in order to obtain accurate values of the hemoglobin charge vs pH and of its single particle diffusion coefficient. A quantitative estimate of the dissociation was also obtained, leading then to good descriptions both of the electrostatic and of the non-electrostatic contribution to the protein tetramer to dimer dissociation process.