The Effects Of Pulsed Electric Fields On Insulin Amyloidosis

Under conditions that destabilize the native state, proteins can self-aggregate into fibrillar assemblies. In the form of amyloid fibrils or fibril precursors, the proteins not only lack their original biological function but also may be harmful to organisms. A series of human diseases are caused by the pathogenic deposition of proteins in the form of amyloid-like fibrils. As a model for disease-associated amyloids, insulin fibrillation is proposed to occur via partial unfolding of a monomeric intermediate. Stimulated by an intense electric field, the secondary structures of in-vitro insulin can be disturbed and transfer to unfolding aggregations or other conformations, which may inhibit the fibrillation process.This project is focusing on in-vitro studies employing two types of pulsed electric fields (PEFs), i.e.50 Hz and 33 Hz PEF, to see if there is possible causal connection between insulin fibrillation and the PEF exposure. Thioflavin T (ThT)-fluorescence, transmission electron microscopy (TEM) techniques, circular dichroism(CD) and Raman spectroscopy were employed simultaneously to follow the emperature-induced formation of amyloid fibrils by bovine insulin under amyloidogenic conditions (pH 2.0 and 60℃) regarding the effects of waveform, exposure duration and field intensity of PEF on the fibrillation mechanism of insulin.The Raman spectra and CD data confirm that the PEF exposed insulin molecules may primitively have a slight change in its native structure, causing aggregation. On heating to 60℃, partial unfolding occurs and results initially in nucleation centers, but aggregates in the PEF exposed insulin solution are difficult to dissolve to facilitate the unfolding of insulin molecules. Following this step, changes in the CD spectra and ThT indicate the extensive conversion of the molecular conformation from a-helical to β-sheet structure and the fibrillation velocity in the PEF exposed insulin is accelerated by the PEF exposure. At later stages, ThT kinetics curves indicate less mature fibrils in the PEF exposed insulin and TEM shows the morphology change of fibrils from long twisted fibrils to shorter and less matured fibrils and the CD spectra shows that the secondary structures of mature fibrils are also different. All the effects enhance when the exposure and electric intensity increase. The investigated evidences suggest that the PEF can inhibit insulin amyloidosis.