Insulin and Polylysine as Model Polypeptides for FTIR Studies of the Pressure-effect on Protein Aggregation

Abstract

High pressure second derivative FTIR spectroscopy has been employed in order to investigate subtle differences between insulin aggregates obtained after 1, 2, 3, 4, and 5 hours of incubation at 70°C and pD = 2. This study shows that, although the prolonged heating of insulin does result in the increasing content of pressure-insensitive amyloid fibrils, the pressure-sensitive fraction is still present even after long heating of the sample. This suggests that in the inter-fibrillar spaces, small dissociation-prone aggregates are entrapped. High pressure up to approx. 600 MPa acted as if it were capable of partial reversing of the very late stages of the insulin fibrilization. Interestingly, for all the samples examined, pressure increasing up to 1 GPa would not result in further spectral changes. On the other hand, even pressure as low as 30 MPa has been shown to prevent completely aggregation of insulin. Studying heat- and pressure-induced spectral shifts of the amide I’ band of polylysine in antiparallel β-sheet conformation, the causative role of changing hydration has been implicated. Likewise, a pressure-induced hydration of the amyloid may explain the spectral changes occurring upon pressurization of the insulin fibrils.