Investigation of the Amyloid Properties of Winter Flounder Antifreeze Protein (AFP6) and the Application of Aqueous Curcumin in the Detection of this Protein

Abstract

A Pseudopleuronectes americanus antifreeze protein, AFP6, has previously been shown to form amyloid upon specific interaction with an ice surface. In this work, an in situ real-time assay for amyloid formation by amyloid beta was implemented in order to evaluate potential cross-seeding of amyloid formation by synthetic AFP6. However, insufficient AFP6 amyloid was produced to continue with the assay. Through this work, AFP6 was found to be easily separated from other proteins due to its solubility in acetone. Furthermore, although AFP6 is undetectable using common positive protein stains, aqueous curcumin allowed fluorescence-based detection of AFP6. The absorbance and fluorescence spectra of curcumin are both altered in the presence of AFP6, with more marked changes following 70 °C incubation. However, the melting point and secondary structure of AFP6 determined by circular dichroism were unchanged by curcumin. Thus, single-reagent methods for purifying and detecting this unusual protein were found to be effective.