title: Evaluation of an Ultrafast Molecular Rotor, Auramine O, as a Fluorescent Amyloid Marker.
abstract: Recently, Auramine O (AuO) has been projected as a fluorescent fibril sensor, and it has been claimed that AuO has an advantage over the most extensively utilized fibril marker, Thioflavin-T (ThT), owing to the presence of an additional large red-shifted emission band for AuO, which was observed exclusively for AuO in the presence of fibrillar media and not in protein or buffer media. As fibrils are very rich in β-sheet structure, a fibril sensor should be more specific toward the β-sheet structure so as to produce a large contrast between the fibril form and native protein form, for efficient detection and in vitro mechanistic studies of fibrillation. However, in this report, we show that AuO interacts significantly with the native form of bovine serum albumin (BSA), which is an all-α-helical protein and lacks the β-sheet structure, which are the hallmarks of a fibrillar structure. This strong interaction of AuO with the native form of BSA leads to a large emission enhancement of AuO for the native protein itself, and leads to a low contrast between the BSA protein and its fibrils. More importantly, the large red-shifted emission band of AuO, reported in the presence of human insulin fibrils, and which was projected as its major advantage over ThT, is not observed in the presence of BSA fibrils as well as fibrils from other proteins, such as lysozyme, human serum albumin, and β-lactoglobulin. Thus, our results provide information on the universal applicability of the distinctive and claimed-to-be-advantageous photophysical features reported for AuO in human insulin fibrils towards fibrils from other proteins. Time-resolved fluorescence measurements also support the proposition of a strong interaction of AuO with native BSA. Additionally, tryptophan emission of the protein has been explored to further elucidate the binding mechanism of AuO with native BSA. Evaluation of thermodynamic parameters revealed that the binding of AuO with native BSA involved positive enthalpy and entropy changes, suggesting dominant contributions from hydrophobic and electrostatic interactions toward the association of AuO with native BSA. Molecular docking calculations have been performed to identify the principal binding location of AuO in native BSA.
authors.affiliation: Radiation & Photochemistry Division, ‡Radiation Safety Systems Division, Bhabha Atomic Research Centre , Mumbai 400085, India.
authors.forename: Niyati H
authors.initials: NH
authors.lastname: Mudliar
authors.affiliation: Radiation & Photochemistry Division, ‡Radiation Safety Systems Division, Bhabha Atomic Research Centre , Mumbai 400085, India.
authors.forename: Biswajit
authors.initials: B
authors.lastname: Sadhu
authors.affiliation: Radiation & Photochemistry Division, ‡Radiation Safety Systems Division, Bhabha Atomic Research Centre , Mumbai 400085, India.
authors.forename: Aafrin M
authors.initials: AM
authors.lastname: Pettiwala
authors.affiliation: Radiation & Photochemistry Division, ‡Radiation Safety Systems Division, Bhabha Atomic Research Centre , Mumbai 400085, India.
authors.forename: Prabhat K
authors.initials: PK
authors.lastname: Singh
mesh_terms: D000818:Animals; D001576:Benzophenoneidum / Q000737:chemistry / Q000378:metabolism*; D052160:Benzothiazoles; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007328:Insulin / Q000737:chemistry / Q000378:metabolism; D062105:Molecular Docking Simulation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D007328:Insulin; D013844:Thiazoles; C009462:thioflavin T; D001576:Benzophenoneidum; D012710:Serum Albumin, Bovine