Effects of Ions on Insulin Aggregation Status and Formation of Insulin Fibrils

Yi Xie，Chen Lv，Huilin Tan，Weiqun Tian，＊

1Biology Base Class2，College of Life Sciences at Wuhan University，Wuhan，430072，China；

2 Department of Biomedical Engineering，Wuhan University School of Basic Medical Sciences，Wuhan，430071，China；＊Corresponding authors

It is crucial that maintain the insulin in solution in the monomer state to guarantee its physiological activity.Therefore，it is necessary to find the related ions which are able to promote depolymerization of insulin hexamer，while inhibition of the formation of insulinβ-amyloid fibrils.The research on these ions can be a clue to help to improve the bioavailability of insulin formulations and be references for the diagnosis，therapeutic intervention and inspiration of other protein misfolding diseases.In order to separate the insulin polymers with the effect of zinc ions，gradient PAGE electrophoresis method has been used in this study.As a result，the insulin trimmer，tetramer，pentimer，hexamer and heptimer have been separated.Moreover，this study explored and stabilized the experimental program of insulin amyloid fibrils culture protocol and system.The pictures of insulin fiber under inverted light microscope and scanning electron microscope have been taken to analysis the morphologyof the fibrils.At the same time，the formation process of insulin fiber has been obtained.Two-stage continuous formation of visual images of filaments and mature fibers has been recalled and examined.Finally，the ThT staining fluorescence intensity and the A330UV absorbance analysis have been chosen to get the formation of insulinβ-amyloid fibers in the four different aqueous medium within 216hours.The preliminary draw of total insulin fibers is the highest in phosphate buffer（PB，pH 7.4），followed by 150mM Tris-Cl buffer（pH 7.4）and 150mM phosphate buffer solution（PBS，pH 7.4），and in saline solution（0.9%NaCl，pH 7.4）is the least.