Amyloidogenic Growth Observation of Stem Bromelain via Atomic Force Microscopy

In this paper, we report on the amyloidogenic fibril formation of stem bromelain (SB) by using atomic force microscopy (AFM). Stem bromelain (SB), a proteolytic enzyme, is widely used in industries and medicine, making it essential to understand the factors affecting aggregation. Amyloid formation entails the assembly of proteins into highly ordered, β-sheet-rich fibrillar structures; yet while heating is a recognized trigger for SB fibrillation, the extent of continued fibril growth at room temperature incubation and its nanoscopic morphological observation remain unexplored. Here, SB was heated in pH 10.8 borate buffer at 65 °C for 10 h, then incubated at room temperature for 1, 3, and 7 days, respectively. A time-course imaging directly visualized the morphological progression from small, dispersed protofibrils on day 1 to increasingly pronounced fibrillar bundles on day 3 and dense, interconnected amyloid networks by day 7. Quantitative analysis of AFM images revealed a progressive increase in alignment in the orientation distribution, which shows directional growth of fibril on mica substrate. Moreover, there is a clear upward trend in fibril coverage area over time, with day 7 showing significantly higher coverage, which implies structural organization. We also introduce a technique that provides an accessible, high-resolution approach for real-time morphological studies of SB protofibril elongation and provides new insights into the kinetics and organizational dynamics of amyloid fibril formation.