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Fluorescence “off” and “on” signalling of esculetin in the presence of copper and thiol: a possible implication in cellular thiol sensing

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Abstract

The interaction of the cupric ion with esculetin, a dihydroxy coumarin derivative, was studied by absorption and fluorescence spectroscopic methods in aqueous medium. Esculetin formed a complex in the presence of the 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 the copper ion and esculetin in the complex was estimated to be 1: 2 respectively. Interestingly, the incubation of the Cu(ii)–esculetin complex with a thiol peptide, glutathione (GSH), showed restoration of the fluorescence intensity as well as absorption maxima to that of pure esculetin. Incubation with other common thiol and non-sulphur amino acids did not show a similar restoration of the photophysical properties of the complex except in the case of cysteine. Mechanistically, it was evident that two molecules of GSH were consumed in reducing the Cu(ii)–esculetin complex, which subsequently split into the copper ion and esculetin. In this process GSH was converted into oxidised GSH (GSSG) as evident from the mass spectroscopy and HPLC studies. The above florescence regeneration behaviour of the copper–esculetin system in the 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 sensors for detecting the cellular thiol level.

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Acknowledgments

RGS thanks the Department of Atomic Energy, Government of India for providing a fellowship under the BARC-SPPU collaborative research programme. The authors are thankful to Ms Vishwa V. Gandhi for helping in the cell culture experiments. The authors are also thankful to Head, RPCD, BARC and Head, Chemistry Department, SPPU for their constant encouragement and support.

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  1. Department of Chemistry, Savitribai Phule Pune University, Pune, 411007, India

    Rupali G. Shinde & Ayesha A. Khan

  2. Radiation and Photochemistry Division, Bhabha Atomic Research Centre, Mumbai, 400085, India

    Rupali G. Shinde, Amit Kunwar, V. S. Tripathi & Atanu Barik

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Shinde, R.G., Khan, A.A., Kunwar, A. et al. Fluorescence “off” and “on” signalling of esculetin in the presence of copper and thiol: a possible implication in cellular thiol sensing. Photochem Photobiol Sci 17, 1197–1205 (2018). https://doi.org/10.1039/c8pp00157j

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