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Detection of Nitric Oxide by Electron Paramagnetic Resonance Spectroscopy: Spin-Trapping with Iron-Dithiocarbamates

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1424)

Abstract

Electron paramagnetic resonance (EPR) spectroscopy is the ideal methodology to identify radicals (detection and characterization of molecular structure) and to study their kinetics, in both simple and complex biological systems. The very low concentration and short life-time of NO and of many other radicals do not favor its direct detection and spin-traps are needed to produce a new and persistent radical that can be subsequently detected by EPR spectroscopy.

In this chapter, we present the basic concepts of EPR spectroscopy and of some spin-trapping methodologies to study NO. The “strengths and weaknesses” of iron-dithiocarbamates utilization, the NO traps of choice for the authors, are thoroughly discussed and a detailed description of the method to quantify the NO formation by molybdoenzymes is provided.

Key words

Nitric oxide radical Electron paramagnetic resonance (EPR) Spin-trap Iron-dithiocarbamate Nitrite Xanthine oxidoreductase Aldehyde oxidoreductase 

Abbreviations

AOR

Aldehyde oxidoreductase

CPTIO

2-(4-Carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide

DETC

Diethyldithiocarbamate

DMPO

5,5-Dimethyl-1-pyrroline N-oxide

EPR

Electron paramagnetic resonance

Fe-(TC)2

Iron-bis-dithiocarbamates

Fe2+-(TC)2

Ferrous-bis-dithiocarbamate

Fe3+-(TC)2

Ferric-bis-dithiocarbamate

Hb

Hemoglobin

MGD

N-methyl-d-glucamine-dithiocarbamate

MNIC

Mononitrosyl-iron complex

MNP

2-Methyl-2-nitrosopropane

NO

Nitric oxide radical (NO)

ST

Spin-trap molecule

TEMPO

2,2,6,6-Tetramethylpiperidinyl-N-oxyl

XO

Xanthine oxidase

Notes

Acknowledgments

This work was supported by the Unidade de Ciências Biomoleculares Aplicadas-UCIBIO which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728.

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© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.UCIBIO, REQUIMTE, Departamento Química, Faculdade de Ciências e TecnologiaUniversidade Nova de LisboaCaparicaPortugal

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