Electron paramagnetic resonance (EPR) is one of the few methods that allows for the unambiguous detection of nitric oxide (NO). However, the dithiocarbamate-iron spin traps employed with this method inhibit the activity of nitric oxide synthase and catalyze NO production from nitrite. These disadvantages limit EPR's application to biological NO detection. We present a liposome-encapsulated spin-trap (LEST) method for the capture and in situ detection of NO by EPR. The method shows a linear response for [NO]≥4µM and can detect [NO]≥40nM in a 500µL sample (≥20 pmol). The kinetics of NO production can be followed in real time over minutes to hours. LEST does not inhibit the activity of inducible nitric oxide synthase or nitrate reductase and shows minimal abiotic NO production in the presence of nitrite and NADH. Nitrate reductase-like activity is detected in cell lysates of the coccolithophore Emiliania huxleyi and is elevated in virus-infected culture. This method shows particular promise for NO detection in cell lysates and crude preparations of NO-producing tissues.
Keywords: Algae; Coccolithophore; Dithiocarbamate-iron; EPR; ESR; Electron paramagnetic resonance; Liposomes; Nitrate reductase; Nitric oxide; Nitric oxide synthase; Spin trap.
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