Title
Fluorescence “off” and “on” signalling of esculetin in presence of copper and thiol: A possible implication in cellular thiol sensing
Author
Rupali G Shinde, Ayesha A. Khan, Amit Kunwar, V. S. Tripathi, Atanu Barik
Year
2018
Journal
Photochemical & Photobiological Sciences
Abstract
The interaction of cupric ion with esculetin, a dihydroxy coumarin derivative, was studied by absorption and fluorescence spectroscopic methods in aqueous medium. Esculetin formed a complex in presence of cupric ion which was characterised by the shift in its absorption band from 350 nm to 389 nm and the quenching of its fluorescence intensity at 466 nm. From Job’s plot and fluorescence quenching studies, the stoichiometry of copper ion and esculetin in the complex was estimated to be 1:2 respectively. Interestingly incubation of Cu(II)-esculetin complex with a thiol peptide, glutathione (GSH) showed restoration of fluorescence intensity as well as absorption maxima to that of the pure esculetin. Incubation with other common thiol and non- sulphur amino acids did not show similar restoration of photophysical properties of the complex except in the case of cysteine. Mechanistically it was evident that two molecules of GSH were consumed in reducing Cu(II)-esculetin complex, which subsequently split into copper ion and esculetin. In this process GSH was converted to oxidised GSH (GSSG) as evident from the mass spectroscopy and HPLC studies. The above florescence regeneration behaviour of copper esculetin system in presence of GSH was also observed in the cellular system using Chinese Hamster Ovary (CHO) as model cells. In conclusion, these studies may find application in developing sensor for detecting the cellular thiol level.
Instrument
FP-8500
Keywords
Fluorescence, Chemical stability, Ligand binding, Kinetics, Quenching, Sensors, Materials