pH-dependent self-association of zinc-free insulin characterized by concentration-gradient static light scattering

Insulin self-association at pH 1.85, 1.95, 3.0, 7.2, 8.0 and 10 was studied via composition gradient light scattering (CG-SLS). At pH 1.95 in acetic acid, insulin was found to exist as a monomer, and in pH 1.85 HCl as a dimer. At pH values of 3.0-8.0, the dependence of scattering intensity upon total insulin concentration at concentrations of up to 1.5mg/mL may be quantitatively accounted for by a simple isodesmic association equilibrium scheme requiring only a single association constant for addition of monomer to monomer or any oligomer. At pH 10, the association constant for addition of monomer to monomer was found to be smaller than the association constant for addition of monomer to all higher oligomers by a factor of approximately five. The isodesmic association scheme was also found to quantitatively account for the concentration dependence of the weight-average molecular weight derived from previously published sedimentation equilibrium measurements made at pH 7.0, and the best-fit value of the stepwise equilibrium constant obtained therefrom was in excellent agreement with that obtained from analysis of the light scattering data obtained at pH 7.2.