Quantifying the Self-Association of Insulin at Neutral pH Using Composition-gradient Multi-Angle Light Scattering

The self-association of macromolecules in solution, including insulin^1,2, can be quantified using composition-gradient multi-angle light scattering (CG-MALS). CG-MALS enables rapid, reproducible, label-free quantification of biomolecular self-association.

This technique measures the change in weight-average molar mass as a function of concentration to determine the absolute stoichiometry and affinity of these interactions. While a semi-quantitative study of insulin self-association may be carried out by coupling MALS instrumentation to size-exclusion chromatography³, this article describes the use of CG-MALS to quantify the self-association of insulin at neutral pH in the absence of zinc, identifying a progressive, isodesmic self-assembly process.  

Sample Preparation and Instrumentation

Human insulin samples were prepared in a buffer composed of 20mM sodium phosphate pH 7.2, 0.1M NaCl, 1mM EDTA and measured by means of an extinction coefficient of 1.05 AU/(g/L*cm) at 276nm. Insulin solutions and buffers were filtered using Anotop syringe-tip filters with 0.02µm pore size by centrifugation at 2500g for 15 minutes. Experiments were carried out at 25°C in duplicate using two stock concentrations of either 3.2mg/ml or 0.3mg/ml.

CG-MALS experiments were performed using a Calypso II to produce concentration gradients in line with a Shimadzu SPD-6AV UV/Vis spectrometer and a mini-DAWN TREOS three-angle light scattering photometer (Figure 1). The CALYPSO software was used to fit the light scattering and concentration data to different self-association models to identify the association scheme that best delineated the data.

Figure 1. Calypso Hardware Set-up.

Experimental Results

As expected, the measured light scattering and concentration data for insulin are characteristic of a self-associating molecule (Figure 2). Although the maximum measured M_w under these conditions (38kDa) is that of a hexamer, the increase in molar mass with concentration cannot be described by a simple model of monomer-hexamer equilibrium 6I⇄I₆ with an equilibrium association constant, K_A=[I₆]/[I]^₆. As illustrated in Figure 2, this type of assembly underestimates the measured Mw by as much as 30% for concentrations below ~0.2mg/mL (34 µM) and overestimates the Mw by as much as 25% for concentrations between 0.2 and 2mg/mL (34-340 µM).

Figure 2. The measured LS signal as a function of concentration (blue diamonds) is significantly greater than the expected LS signal for a non-interacting monomer with M_w = 6kDa (gray line).

Rather, the light scattering and concentration data are best fit by an isodesmic self-assemble model. According to this model, each insulin monomer couples to a growing insulin cluster with constant affinity as follows:

The equilibrium association constant K2 is related to the affinity per binding site, K_D=1/ K₂. For the data shown in Figures 2 and 3, the best fit is isodesmic self-assembly with affinity K_D = 52µM.

Figure 3. The increase in M_w as a function of concentration corresponds to an isodesmic self-association (solid line) and is not characteristic of a simple model of monomer-hexamer equilibrium (dashed line).

The self- association model can be used to calculate the equilibrium distribution of oligomers, as shown in Figure 4. As the total concentration reaches the isodesmic binding site affinity (~50µM or 0.3mg/mL), insulin molecules self-associate, and the monomer fraction in solution rapidly decreases, indicating the self-association of insulin molecules. At concentrations above ~0.6mg/mL (~100µM), even the dimer fraction decreases due to the formation of higher order oligomers.

Figure 4. Equilibrium distribution of species.

At the maximum concentration measured (~500µM), monomers represent only 27% mol/mol and dimers 20% mol/mol. Figure 4 provides the molar compositions up to 15-mers, which represent 0.3% mol/mol of the stock solution.

Conclusion

The results clearly demonstrate the ability of CG-MALS to quantify the isodesmic self-assembly of human insulin in the absence of zinc. Even though the overall Mw only increased by ~6-fold over the concentrations studied (~0.3-3mg/mL, ~5-500µM), the Mw change as a function of concentration is not described well by simple monomer-hexamer equilibrium. Instead, a true description of the complexes present at equilibrium needs to consider higher order insulin oligomerization, including species containing >10-mer under these conditions.

References

Adapted from "Self-Association of Insulin Quantified by CG-MALS" white paper by Wyatt Technology. Graphs and illustrations reprinted with permission from Wyatt Technology.

1. Arun K. Attri, Cristina Fernández, Allen P. Minton., (2010). pH-dependent self-association of zinc-free insulin characterized by concentration-gradient static light scattering. Biophys. Chem., Vol. 148, 28-33.
2. Arun K. Attri, Cristina Fernández, Allen P. Minton., (2010) Self-association of Zn-insulin at neutral pH: Investigation by concentration gradient-static and dynamic light scattering. Biophysical J., Vol. 148, 23-27.
3. Application of Column Chromatography (SEC/GPC) and MALS for Characterizing Monomers, Dimers, Hexamers and Aggregates of Insulin

This information has been sourced, reviewed and adapted from materials provided by Wyatt Technology.

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