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Differential Effect of Buffer on the Spin Trapping of Nitric Oxide by Iron Chelates
Authors: Porasuphatana S.1; Weaver J.2; Budzichowski T.A.2; Tsai P.1; Rosen G.M.1, 3, 4, 5
Source: Analytical Biochemistry, Volume 298, Number 1, November 2001 , pp. 50-56(7)
Publisher: Academic Press
Abstract:
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. Copyright 2001 Academic Press.
Keywords: nitric oxide; nitric oxide synthase; spin trapping
Language: English
Document Type: Research article
Affiliations: 1: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland, 21201 2: Department of Chemistry, University of Maryland Baltimore County, Baltimore, Maryland, 21228 3: Medical Biotechnology Center, University of Maryland Biotechnology Institutes, Baltimore, Maryland, 21201 4: Center for Low Frequency EPR for In Vivo Physiology, University of Chicago, Chicago, Illinois, 60637 5: University of Maryland, Baltimore, Maryland, 21201
Publication date: 2001-11-01
- In this: publication
- By this: publisher
- In this Subject: Anatomy & Physiology , Biology , Organic Chemistry , Biochemistry
- By this author: Porasuphatana S. ; Weaver J. ; Budzichowski T.A. ; Tsai P. ; Rosen G.M.
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