Download Novel agents for the in-situ detection of cysteine oxidation states

P042
Novel agents for the in-situ detection of cysteine
oxidation states
Shibani N. Ratnayake, Eric Lattmann
and Helen R. Griffiths
Aston University, Birmingham, UK
Cysteine (Cys-SH) readily undergoes oxidation by reactive oxygen
species (ROS) to form sulfenic (Cys-OH), sulfinic (Cys-SO2H)
and sulfonic (Cys-SO3H) acids. Thiol modifications of cysteine
have been implicated as modulators of cellular processes and
represent significant biological modifications that occur during
oxidative stress and cell signalling. However, the different oxidation
states are difficult to monitor in a physiological setting due to
the limited availability of experimental tools. Therefore it is of
interest to synthesise a range of chemical probes that selectively recognise specific oxidation states of cysteine. The current
study is aimed at investigating a synthetic approach for novel
fluorescent probe synthesis, for the specific detection of cysteine
sulfenic acids by fluorescence spectroscopy. N-[2-(anthracen2-ylamino)-2-oxoethyl]-3,5-dioxocyclohehanecarboxamide,
fluoresceneamine-5-N-(3,5-dioxoxyxlohexane)carboxamide and
3,5-dioxo-N-(2-phenylethyl)cyclohexanecarboxamide have successfully been synthesised and confirmed by nuclear magnetic
resonance spectroscopy. Optimal conditions for testing the novel
probes, using the oxidation of cysteine moieties to produce sulfenic
acid by hydrogen peroxide mediated oxidation of human serum
albumin, are described. Sulfenic acid was detected using the
commercial, non-fluorescent probe DCP-Bio1. Jurkat T cells have
been used as a model to investigate sulfenic acid modifications
in a cell lysate and membrane fraction pre- and post- stimulus.
Membrane proteins of resting Jurkat T cells have sulfenic acid modifications and 2 prominent bands are detected by SDS-PAGE and
western blotting with molecular weights of approximately 25kDa
and 50 kDa. Their identity is under investigation by LC-MS/MS.