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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.

Equilibrium self-association of Zn-insulin at pH 7.0 was characterized over the range 0.3-5mg/mL by simultaneous measurement of static and dynamic light scattering. Analysis of static light scattering yielded a concentration-dependent weight-average molecular weight, and analysis of dynamic light scattering yielded a concentration-dependent intensity-average diffusion coefficient. The concentration dependence of both quantities may be accounted for to within experimental precision by a simple model, according to which the basic structural unit of Zn-insulin at concentrations exceeding 0.3mg/mL is a hexamer H. With increasing total protein concentration, hexameric protomers may self-associate in accordance with an isodesmic scheme in which a protomer may add to any prexisting oligomer H(n) to form H(n+1) with an invariant stepwise equilibrium association constant.

A method for rapid detection and quantitative characterization of associations between two proteins in solution is presented, based upon continuous measurement of the Rayleigh light scattering and UV absorbance of a solution whose composition is varied with time in a controlled fashion. Collection and subsequent analysis of data are both rapid and semiautomatic. The results of validation experiments demonstrate that the technique can correctly identify complexes and reliably evaluate equilibrium constants for hetero-association.