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J. R. Burgoyne*, J. P. Brennan, P. Eaton.King’s College London, Department of Cardiology, The Rayne Institute, St Thomas’ Hospital, London, SE1 7EH, UK

Nitric oxide (NO) has many well-defined effects on the cardiovascular system, including regulation of vascular tone, excitation contraction coupling, and injury during ischaemia and reperfusion. NO mediates many of its biological responses by the well-defined and important role of cGMP production and signalling through protein kinase G. However, in recent years there has been increasing awareness that many of the functional effects of NO may be a result of protein S-nitrosylation, a reversible coupling of NO to a reactive cysteine thiol. A novel method for detecting and purifying S-nitrosylated proteins was recently described, which utilises the ascorbate selective reduction of S-nitrosylated proteins and their subsequent labelling with biotin. The S-nitroso group on the thiol group of cysteine is thus ‘switched’ for biotin, which allows detection on Western blots probed with streptavidin-HRP and purification on streptavidin-agarose columns.

We utilised this method of measuring protein S-nitrosylation in isolated rat hearts. Preliminary studies with homogenates treated in vitro with the nitric oxide donor (100µM SNAP for 30 min) were used to generate the positive controls. Indeed, when samples treated in vitro with an NO donor were analysed by the biotin-switch method, a number of S-nitrosylated proteins were detected on non-reducing Westerns. Other control experiments, highlight there is a propensity for the spontaneous decomposition of S-nitrosylated proteins, which yield small ascorbate independent signals. Attempts to limit ascorbate independent labelling, which may be due to redox active copper or iron, using the metal chelators Neocuproine (0.2 mM) or DTPA (1 mM) was only partially successful. Currently, we are assessing the ability of the method to detect changes in protein S-nitrosylation in intact isolated hearts administered different classes of NO-donor, as well …

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