The first application of terephthalate fluorescence for highly selective detection of hydroxyl radicals in thylakoid membranes
Iva Šnyrychová A B C and Éva Hideg A C DA Institute of Plant Biology, Biological Research Center, Szeged, Hungary.
B Laboratory of Biophysics, Department of Experimental Physics, Faculty of Science, Palacký University, Olomouc, Czech Republic.
C These authors contributed equally to this article.
D Corresponding author. Email: ehideg@brc.hu
Functional Plant Biology 34(12) 1105-1111 https://doi.org/10.1071/FP07150
Submitted: 16 June 2007 Accepted: 20 September 2007 Published: 27 November 2007
Abstract
Possibilities and limitations of the detection of hydroxyl radicals via the conversion of terephthalate (TPA) into the strongly fluorescent hydroxyterephthalate were investigated in order to adapt this method for chlorophyll-containing samples. Using model chemical sources of various reactive oxygen species, we confirmed that TPA detects hydroxyl radicals very sensitively, but is not reactive to either hydrogen peroxide or superoxide radicals. As a new result, we showed that the conversion of TPA to hydroxyterephthalate cannot be induced by singlet oxygen, which may be produced in photosynthetic systems under stress. Until now, the TPA method has not been used in photosynthesis research, so necessary adaptations to minimise the effects of chlorophyll and buffering sugars on hydroxyl radical detection were also explored and optimal conditions for using the method in thylakoid preparations are suggested. Anticipating further plant physiology applications, usefulness of the TPA method was tested in a wider range of pH than reported earlier. To demonstrate that this simple and highly specific method can be used as an alternative approach for the detection of hydroxyl radicals in plant samples, we measured these radicals in isolated thylakoid membranes exposed to 312 nm ultraviolet radiation.
Acknowledgements
Iva Šnyrychová was supported by a Visegrád Post-Graduate Scholarship (VPSP, S-023–2006) from the International Visegrád Fund and by the grant of The Ministry of Education, Youth and Sports of the Czech Republic (MSM 6198959215). Terephthalic acid was a kind gift from Dr Tamás Kálai (Department of Medicinal and Organic Chemistry, University of Pécs, Hungary).
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