Interference of low-molecular substances with the thioflavin-T fluorescence assay of amyloid fibrils

• Published in: Journal of peptide science Vol. 18; no. 1; pp. 59 - 64
• Main Authors: Noormägi, Andra, Primar, Kateryna, Tõugu, Vello, Palumaa, Peep
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.01.2012
• Subjects: aggregation; amyloid; Amyloid - analysis; Amyloid - metabolism; Amyloid beta-Peptides - analysis; Amyloid beta-Peptides - metabolism; Azure Stains - adverse effects; Azure Stains - metabolism; Biological Assay; Diabetes Mellitus, Type 2 - diagnosis; Diabetes Mellitus, Type 2 - metabolism; False Positive Reactions; fibrillization; fluorescence; Fluorescent Dyes - analysis; Fluorescent Dyes - metabolism; Humans; insulin; Insulin - analysis; Insulin - metabolism; Kinetics; Neurodegenerative Diseases - diagnosis; Neurodegenerative Diseases - metabolism; Protein Structure, Secondary; Spectrometry, Fluorescence; Tannins - adverse effects; Tannins - metabolism; Thiazoles - analysis; Thiazoles - antagonists & inhibitors; Thiazoles - metabolism
• ISSN: 1075-2617; 1099-1387
• DOI: 10.1002/psc.1416

ABSTRACT Abnormal fibrillization of amyloidogenic peptides/proteins has been linked to various neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as with type‐II diabetes mellitus. The kinetics of protein fibrillization is commonly studied by using a fluorescent dye Thioflavin T (ThT) that binds to protein fibrils and exerts increased fluorescence intensity in bound state. Recently, it has been demonstrated that several low‐molecular weight compounds like Basic Blue 41, Basic Blue 12, Azure C, and Tannic acid interfere with the fluorescence of ThT bound to Alzheimers' amyloid‐β fibrils and cause false positive results during the screening of fibrillization inhibitors. In the current study, we demonstrated that the same selected substances also decrease the fluorescence signal of ThT bound to insulin fibrils already at submicromolar or micromolar concentrations. Kinetic experiments show that unlike to true inhibitors, these compounds did neither decrease the fibrillization rate nor increase the lag‐period. Absence of soluble insulin in the end of the experiment confirmed that these compounds do not disaggregate the insulin fibrils and, thus, are not fibrillization inhibitors at concentrations studied. Our results show that interference with ThT test is a general phenomenon and more attention has to be paid to interpretation of kinetic results of protein fibrillization obtained by using fluorescent dyes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. Basic Blue 41, Basic Blue 12, Azure C and Tannic acid decrease fluorescence intensity of ThT bound to insulin fibrils. In insulin fibrillization assay monitored by ThT fluorescence, these compounds show apparent inhibition of fibrillization. Unlike to true inhibitors, these compounds did not decreased the fibrillization rate or increased the lag‐period of insulin fibrillization.