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

Abstract

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.

Figure 1

Mean values (open circles) of R/K_opt obtained from 14 detectors, pooled from replicate runs, are plotted as a function of log₁₀ w_tot of insulin concentration (mg/ml). The plots in each panel represent the different pH at which the experiments were performed: [a] pH 1.95 acetic acid); [b] pH 1.85 (HCl); [c] pH 3.0; [d] pH 7.2; [e] pH 8.0; and [f] pH 10.0. Best fit obtained from isodesmic self-association model is shown in panels [a] to [e] as a solid line. The best fit line shown in panel [f] was obtained from enhanced isodesmic self association model.

Figure 2

Normalized M_w distribution to the monomeric insulin molecular weight (5400 dalton) is plotted as a function of concentration (log₁₀ w_tot (mg/ml)). Each drawn curve ( red, pH 7.2; blue, pH 8.0; cyan pH 3.0; solid black, pH 1.95, 20% acetic acid; dotted black, pH 1.85, 0.025 M HCl; and green, pH 10) represent the M_W distribution as obtained from corresponding best fit parameters values for isodesmic self-association model (enhanced isodesmic for pH 10) calculated for each set of experimental conditions.

Figure 3

[a] Dependence of weight-average molecular weight of insulin upon protein concentration at pH 7.0, obtained from sedimentation equilibrium experiments (data points) and calculated according to best-fit of the isodesmic model as described in text with the best-fit parameters given in Table 2 (curve). [b] Calculated dependence of the weight-average molecular weight of insulin upon protein concentration according to best-fit of the isodesmic model to data of Pekar and Frank (green) and calculated according to best-fit of the isodesmic model to CG-SLS data obtained in the present study at pH 7.2 (Figure 1, panel d.)