In vitro retardation and modulation of human insulin amyloid fibrillation by Fe3+ and Cu2+ ions

Metal ions of the later part of the first-row transition series (Fe, Co, Ni, Cu, and Zn) can form bonds through the carboxylate, hydroxyl, thiol, and imidazole side chains of proteins and those bonds are significantly more stable than those formed by non-transition metals. Their adventitious binding to protein surfaces provides a great platform for maintaining the native structure of many biologically significant proteins and thus inhibiting the self-aggregation phenomenon of proteins. Aggregation followed by deposition of proteins used for therapeutic purposes either at the site of administration in vivo or during storage conditions in vitro is a major concern. In this work, human insulin is induced to form 'amyloid fibrils' at acidic pH and elevated temperature and then the inhibitory activities of two transition metal ions Fe3+ and Cu2+ towards insulin amyloid fibrillation were investigated. The results show that the formation of beta-sheet-rich fibrillar structures was inhibited more with Fe3+ than Cu2+-treated insulin. Dynamic light scattering and circular dichroism proved that Cu2+ was effective at lowering the interaction ratios with insulin, yet Fe3+ was effective in maintaining the size as well as the alpha-helical conformation of monomeric insulin. This metal complex (insulin-Fe3+ or insulin-Cu2+) delayed the nucleation step of aggregation and Fe3+ is far better than Cu2+ in this endeavor. However, both of the metal ions proved their effectiveness in preventing amyloid fibrillation in vitro and can modulate the morphology of the aggregates as revealed by TEM studies. Overall the two transition metal ions Fe3+ and Cu2+ employed in this investigation are essential micronutrients too and are required in minute concentrations. The present study will encourage the applications of transition metal ions Fe3+ and Cu2+ as effective interrupters of amyloidogenesis in aggregation-prone proteins showing pathogenic amyloid deposition.