Prediction of the Feasibility of Using the âªGold Standardâ« Thioflavin T to Detect Amyloid Fibril in Acidic Media.
Anal Chem
; 96(5): 2158-2164, 2024 02 06.
Article
in En
| MEDLINE
| ID: mdl-38269442
ABSTRACT
Ordered protein aggregates, amyloid fibrils, form toxic plaques in the human body in amyloidosis and neurodegenerative diseases and provide adaptive benefits to pathogens and to reduce the nutritional value of legumes. To identify the amyloidogenic properties of proteins and study the processes of amyloid fibril formation and degradation, the cationic dye thioflavin T (ThT) is the most commonly used. However, its use in acidic environments that induce amyloid formation in vitro can sometimes lead to misinterpretation of experimental results due to electrostatic repulsion. In this work, we show that calculating the net charge per residue of amyloidogenic proteins or peptides is a simple and effective approach for predicting whether their fibrils will interact with ThT at acidic pH. In particular, it was shown that at pH 2, proteins and peptides with a net charge per residue > +0.18 are virtually unstained by this fluorescent probe. The applicability of the proposed approach was demonstrated by predicting and experimentally confirming the absence of ThT interaction with amyloids formed from green fluorescent (sfGFP) and odorant-binding (bOBP) proteins, whose fibrillogenesis was first carried out in an acidic environment. Correct experimental evidence that the inability to detect these fibrils under acidic conditions is precisely because of the lack of dye binding to amyloids (and not their specific structure or the low fluorescence quantum yield of the bound dye) and that the number of ThT molecules associated with fibrils increases with decreasing acidity of the medium was obtained by using the equilibrium microdialysis approach.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Benzothiazoles
/
Amyloid
Type of study:
Prognostic_studies
/
Risk_factors_studies
Limits:
Humans
Language:
En
Journal:
Anal Chem
Year:
2024
Type:
Article
Affiliation country:
RUSSIA