Differential effect of buffer on the spin trapping of nitric oxide by ironchelates

Nitric oxide synthase (NOS) generates nitric oxide (NO .) by the oxidation Of L-arginine. Spin trapping in combination with electron paramagnetic reso nance (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 spi n trapping of NO . from isolated NOS I oxidation Of L-arginine by ferro-N-d ithiocarboxysarcosine (Fe(DTCS)(2)) and ferro-N-methyl-D-glucamide dithioca rbamate (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 all ow formation of NO-Fe(DTCS)(2). Instead, both of these buffers reacted with Fe2+, generating sparingly soluble complexes in the absence of molecular o xygen. 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 lipopolysacc haride-activated macrophage cell line RAW264.7 than was Fe(DTCS)(2). Data s uggested that Fe(DTCS)(2) and Fe(MGD)(2) are efficient at spin trapping NO . but their maximal efficiency may be affected by experimental conditions. (C) 2001 Academic Press.