Epi-fluorescence Imaging at the Air-Water Interface of Fibril Formation in Bovine Serum Albumin and Human Insulin

Amyloid proteins undergo fibrillation at high temperatures or low pH. This transition from α-helices to β-sheets is characteristic of many neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, diabetes, and mad cow disease. Previously, fibrillation has been imaged under conditions that do not mimic in-vivo conditions using transmission electron microscopy (TEM), atomic force microscopy (AFM), and fluorescence. In the present study, the fibrillation processes of bovine serum albumin (BSA) and human insulin were imaged with epi-fluorescence under conditions more similar to in-vivo. It was hypothesized that while individual fibrils would not be seen with epi-fluorescence, the change in tertiary structure would alter the nature of monolayer at the air-water interface and a change in the characteristics of the images from small, circular domains to more stagnant string-like structures would be seen. Images were taken at the air-water interface with (5-octadecanoylamino)-fluorescein (ODFL) using a Langmuir monolayer technique. BSA fibrils were formed at physiological pH, a 50mM electrolyte concentration, and elevated temperatures. The imaging done at the air-water interface and at physiological pH more closely imitate in-vivo conditions. Transmission electron microscopy (TEM), surface-pressure isotherms, and circular dichroism (CD) were also used to monitor fibril formation. Preliminary images for BSA support the hypothesis that a change in the images will be seen; however experiments are ongoing to provide further support for this conclusion.