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Porasuphatana, Supatra; Weaver, John; Budzichowski, Theodore A.; Tsai, Pei; Rosen, Gerald M.
Analytical biochemistry, 11/2001, Letnik: 298, Številka: 1Journal Article
Nitric oxide synthase (NOS) generates nitric oxide (NO·) by the oxidation of l-arginine. Spin trapping in combination with electron paramagnetic resonance (EPR) spectroscopy using ferro-chelates is considered one of the best methods to detect NO· in real time and at its site of generation. The spin trapping of NO· from isolated NOS I oxidation of l-arginine by ferro-N-dithiocarboxysarcosine (Fe(DTCS)2) and ferro-N-methyl-d-glucamide dithiocarbamate (Fe(MGD)2) in different buffers was investigated. We detected NO–Fe(DTCS)2, a nitrosyl complex, resulting from the reaction of NO· and Fe(DTCS)2, in phosphate buffer. However, Hepes and Tris buffers did not allow formation of NO–Fe(DTCS)2. Instead, both of these buffers reacted with Fe2+, generating sparingly soluble complexes in the absence of molecular oxygen. Fe(DTCS)2 and Fe(MGD)2 were found to inhibit, to a small degree, NOS I activity with a greater effect observed with Fe(MGD)2. In contrast, Fe(MGD)2 was more efficient at spin trapping NO· from the lipopolysaccharide-activated macrophage cell line RAW264.7 than was Fe(DTCS)2. Data suggested that Fe(DTCS)2 and Fe(MGD)2 are efficient at spin trapping NO· but their maximal efficiency may be affected by experimental conditions.
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