Multimerization and Aggregation of Native-State Insulin: Effect of Zinc

Abstract

The aggregation of insulin is complicated by the coexistence of various multimers, especially in the presence of Zn(2+). Most investigations of insulin multimerization tend to overlook aggregation kinetics, while studies of insulin aggregation generally pay little attention to multimerization. A clear understanding of the starting multimer state of insulin is necessary for the elucidation of its aggregation mechanism. In this work, the native-state aggregation of insulin as either the Zn-insulin hexamer or the Zn-free dimer was studied by turbidimetry and dynamic light scattering, at low ionic strength and pH near pI. The two states were achieved by varying the Zn(2+) content of insulin at low concentrations, in accordance with size-exclusion chromatography results and literature findings (Tantipolphan, R.; Romeijn, S.; Engelsman, J. d.; Torosantucci, R.; Rasmussen, T.; Jiskoot, W. J. Pharm. Biomed. 2010, 52, 195). The much greater aggregation rate and limiting turbidity (τ(∞)) for the Zn-insulin hexamer relative to the Zn-free dimer was explained by their different aggregation mechanisms. Sequential first-order kinetic regimes and the concentration dependence of τ(∞) for the Zn-insulin hexamer indicate a nucleation and growth mechanism, as proposed by Wang and Kurganov (Wang, K.; Kurganov, B. I. Biophys. Chem. 2003, 106, 97). The pure second-order process for the Zn-free dimer suggests isodesmic aggregation, consistent with the literature. The aggregation behavior at an intermediate Zn(2+) concentration appears to be the sum of the two processes.

Uniform mPEG‐b‐PMETAC enables pH‐responsive delivery of insulin

“…Insulin is prone to aggregate in neutral solution, and forms monomers or dimers at pH 2 or lower, and at pH 9 or higher. 31,32 Therefore, in the pH range of 3.0-7.0, insulin molecules prefer to form hexamers which favor the stability of insulin. Figure 5(b) shows the CD spectra of insulin in the presence of mPEG-b-PMETAC.…”

Comparison of NMR and Dynamic Light Scattering for Measuring Diffusion Coefficients of Formulated Insulin: Implications for Particle Size Distribution Measurements in Drug Products

“…Formulations used for this study contain protein at sub-mM concentration and excipients such as Zn 2+ , m -cresol and/or phenol. These formulation conditions stabilize insulin dimer, hexamer and also higher-order structure formation (19,24,25). Hexameric or higher-order structures have also been observed for insulin analogues under these conditions (18,26–28).…”

The Diabetes Susceptibility Gene SLC30A8 that Encodes the Zinc Transporter ZnT8 is a Pseudogene in Guinea Pigs Potentially Contributing to Low Guinea Pig Islet Zinc Content

“…High islet zinc levels have been reported to be important for multiple aspects of pancreatic islet beta cell function including paracrine signaling from beta to alpha cells (Hardy et al 2011; Robertson et al 2011), proinsulin processing (Dunn 2005), crystallization of insulin hexamers (Chausmer 1998; Dunn 2005) and promotion of insulin aggregation (Xu et al 2012), which may be protective against proteolysis (Zimmerman and Yip 1974). However, the importance of islet zinc appears at odds with the observation that in some mammalian species, for example Guinea pigs ( Cavia porcellus ), islet zinc content is very low (Havu et al 1977; Zimmerman and Yip 1974).…”