Trimethylammonium-diphenylhexatriene and propionic acid-diphenylhexatriene are sensitive fluorescence probes of...

Amyloids are fibril aggregates of proteins that are rich in β-sheet structure. Amyloid formation is usually detected using dyes that bind the fibril state, and the kinetics of amyloid formation are often followed by adding amyloid sensitive dyes to a solution of the protein or polypeptide of interest. We tested the ability of two diphenylhexatriene (DPH) based fluorescence probes to monitor amyloid formation using human islet amyloid polypeptide (hIAPP), also known as amylin, as a test case. Human islet amyloid polypeptide forms amyloid in type-2 diabetes and readily aggregates in vitro. DPH has been shown to detect amyloid formed by human islet amyloid polypeptide, but the poor solubility of the probe causes significant practical issues and DPH can bind to commonly used 96-well plastic plates employed with plate readers, thereby limiting its usefulness. Trimethylammonium-DPH (a cationic variant) and propionic acid-DPH (an anionic variant) are sensitive to hIAPP amyloid formation even at a ratio of peptide to compound of 64:1. Much weaker fluorescence enhancement is detected in the presence of freshly dissolved, non-amyloid, human islet amyloid polypeptide. The kinetics of hIAPP amyloid formation can be followed in situ using trimethylammonium-DPH. The two dyes have different excitation and emission maximum relative to thioflavin-T and thus can be useful for minimizing inner filter effects in certain assays. Both DPH derivatives are commercially available in high purity.