Differential Effect of Buffer on the Spin Trapping of Nitric Oxide by Iron Chelates

✍ Scribed by Supatra Porasuphatana; John Weaver; Theodore A. Budzichowski; Pei Tsai; Gerald M. Rosen

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 113 KB
• Volume: 298
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5389

✦ Synopsis

Nitric oxide synthase (NOS) generates nitric oxide (NO⅐) 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 NO⅐ in real time and at its site of generation. The spin trapping of NO⅐ from isolated NOS I oxidation of L-arginine by ferro-Ndithiocarboxysarcosine (Fe(DTCS) 2 ) and ferro-Nmethyl-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 NO⅐ 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 Fe 2؉ , 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 NO⅐ 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 NO⅐ but their maximal efficiency may be affected by experimental conditions.