On the Heat Stability of Amyloid-Based Biological Activity: Insights from Thermal Degradation of Insulin Fibrils

<div><p>Formation of amyloid fibrils in vivo has been linked to disorders such as Alzheimer’s disease and prion-associated transmissible spongiform encephalopathies. One of the characteristic features of amyloid fibrils is the high thermodynamic stability relative both to native and disordered states which is also thought to underlie the perplexingly remarkable heat resistance of prion infectivity. Here, we are comparing high-temperature degradation of native and fibrillar forms of human insulin. Decomposition of insulin amyloid has been studied under helium atmosphere and in the temperature range from ambient conditions to 750°C using thermogravimetry and differential scanning calorimetry coupled to mass spectrometry. While converting native insulin into amyloid does upshift onset of thermal decomposition by ca. 75°C, fibrils remain vulnerable to covalent degradation at temperatures below 300°C, as reflected by mass spectra of gases released upon heating of amyloid samples, as well as morphology and infrared spectra of fibrils subjected to incubation at 250°C. Mass spectra profiles of released gases indicate that degradation of fibrils is much more cooperative than degradation of native insulin. The data show no evidence of water of crystallization trapped within insulin fibrils. We have also compared untreated and heated amyloid samples in terms of capacity to seed daughter fibrils. Kinetic traces of seed-induced insulin fibrillation have shown that the seeding potency of amyloid samples decreases significantly already after exposure to 200°C, even though corresponding electron micrographs indicated persisting fibrillar morphology. Our results suggest that amyloid-based biological activity may not survive extremely high temperature treatments, at least in the absence of other stabilizing factors.</p></div

Temporal profiles of high temperature incubations of amyloid samples.

<p>Powder sample were heated to 100, 200, 250, 350°C with a linear ramp 10°C/min under helium atmosphere. After desired temperature was reached, isothermal conditions were held for 10 minutes, followed by gradual cooling of the samples down to room temperature.</p

Aggregation kinetics of human insulin seeded at 37°C with fibrils exposed to high temperatures, probed by ThT fluorescence.

<p>Error bars correspond to standard deviations of fluorescence intensity calculated for six microplate wells with identical samples.</p

TEM (left) and SEM (right) images of amyloid fibrils before and after heating.

<p>Scale bars are 200 nm.</p

Infrared absorption (A) and second derivative (B) spectra of amyloid fibrils before and after exposure to high temperatures.

<p>Inset in panel A shows normalized spectra in the amide I/II spectral region. Second derivative FT-IR spectra in panel B are shown only for the amide I/II region with omission of featureless 750°C spectrum.</p

Thermal stability of human insulin in the amyloid (A, C) and native (B, D) states.

<p>Panels A and B presents thermogravimetric curves (black lines) together with the corresponding heat flow profiles (red lines) obtained through heating of samples form 50 to 750°C with a linear ramp 10°C/min under helium atmosphere. Panels C and D show TGA-MS evolution profiles of gases released during thermal analysis at selected m/z channels and assigned to different molecules.</p