Differential effect of buffer on the spin trapping of nitric oxide by iron chelates.
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.
Porasuphatana
S
S
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, USA.
Weaver
J
J
Budzichowski
T A
TA
Tsai
P
P
Rosen
G M
GM
D000818:Animals; D001120:Arginine / Q000378:metabolism; D002021:Buffers; D002460:Cell Line / Q000166:cytology; D004578:Electron Spin Resonance Spectroscopy / Q000295:instrumentation; D004731:Endotoxins / Q000494:pharmacology; D005296:Ferrous Compounds / Q000737:chemistry*; D006531:HEPES / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007502:Iron Chelating Agents / Q000737:chemistry*; D007668:Kidney / Q000166:cytology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D019097:Spin Trapping / Q000379:methods; D014325:Tromethamine / Q000737:chemistry
D002021:Buffers; D004731:Endotoxins; D005296:Ferrous Compounds; D007502:Iron Chelating Agents; C446401:ferro-N-dithiocarboxysarcosine; C446399:ferro-N-methyl-d-glucamide dithiocarbamate; D014325:Tromethamine; D009569:Nitric Oxide; D001120:Arginine; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; C496321:Nos1 protein, mouse; D006531:HEPES