Anion–Induced Amyloid Fibrillation of Human Insulin In vitro

The physiologically relevant anions like iodide, acetate, and nitrate affect the structure of insulin through interaction and charge neutralization of the protein. For this reason, monomeric insulin shows the formation of amyloid–like oligomeric aggregates in the presence of these anions under physiological concentration. Multi–spectroscopic and imaging techniques are used to investigate and understand the mechanism of the salt effect on insulin.

Abstract

In the present study, we have focused on the effect of three biologically important salt anions on insulin aggregation. The result of the present study reveals that salts differing in their anions (NaI, NaOAc, and NaNO₃) induce the self–assembly formation of insulin even at low physiological (in the micromolar range) salt concentration with efficacy that follows the order I⁻>CH₃COO⁻>NO₃ ⁻. Here, the anion–driven aggregation of insulin does not follow either the Hofmeister series or electroselectivity series at very low salt concentrations; instead, the binding of anions at low pH to the positively charged residues of insulin is determined by a mechanism where the salt anions promote the fibrillation of insulin and modify the morphology of the monomeric precursor molecule. Both the nucleation and fibril elongation are controlled by the electrostatic forces and hydrophobic interactions. Aggregation mechanism and aggregate morphology were investigated employing a combination of Thioflavin T (ThT) and ANS fluorescence, circular dichroism (CD), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Overall, the present data will enhance the idea to rationalize the anion effects on the aggregation of amyloid-prone protein and to understand the influence of charged biomolecules in cellular compartments.

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