The Effects Of Sonication And Metal Ions On The Assembly Of Glucagon

Amyloid fibrils are filamentous assemblies of proteins that were initially observed in connectionwith clinical disorders such as Alzheimer’s diseases. The primary study showed that the aggregationprogress is not only dependent on the sequences of amino acids, but also closely related to theenvironmental factors such as metal ions, pH, sonication, temperature and so on. Our study involved intwo aspects:(1) we studied the ultrasound effect on the assembly of glucagon fibrils. In this work, weuse Atomic Force Microscopy (AFM) to investigate the morphology of protein aggregation. Moreover,thioflavin-T fluorescence was used to detect kinetics of the amyloid fibrillation of glucagon.Experimental results showed that ultrasound can promote the formation of glucagon fibrils initially andthus drastically reduce the “lag time”. The growth rate of fibrils as well as their morphologies wasremarkably dependent on an applied ultrasonic power or ultrasonic frequency. The secondary structureof assembled protein was characterized by micro Fourier transform infrared spectroscopy (Micro-FTIR). Infrared spectroscopy shift showed that the process of protein structure from α-helix to β-sheet.(2) The investigation of metal ions regulates aggregation of glucagon. we not only observed theglucagon fibrillation with Fe(Ⅲ), Cu(Ⅱ) and Fe(Ⅱ), but also used Micro-FTIR to study thesecondary-structure of those fibers. The attained results reveal that: when added with Fe(Ⅲ), the lagtime of glucagon fibrillation was delayed, morphology and secondary-structure of glucagon waschanged greatly; when added with Cu(Ⅱ), the speed of this fibrillation was accelerated, and themorphology was different, but its secondary-structure was unchanged; and another situation is addedwith Fe(Ⅱ),The speed of fibrotic process did not change, but the second structure has changed.Furthermore, we studied the glucagon fibrillation with Fe(Ⅲ), Cu(Ⅱ) bycircular dichroism (CD) andThT fluorescence, on its conclusion change in secondary structure was with the same FTIR,fluorescence intensity curve shows that the protein aggregation process with Fe (Ⅲ) was inhibited,while glucagon fibrosis rate with Cu(Ⅱ) was increased to some extent.