Detection and characterisation of radicals in biological materials using EPR methodology

• Published in: Biochimica et biophysica acta Vol. 1840; no. 2; pp. 708 - 721
• Main Authors: Hawkins, Clare L., Davies, Michael J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2014
• Subjects: Animals; biophysics; copper; Electron paramagnetic resonance; electron paramagnetic resonance spectroscopy; Electron Spin Resonance Spectroscopy - methods; Free Radicals - analysis; Humans; membrane proteins; metal ions; Nitric oxide; Nitroxide; Radical; reactive oxygen species; Spin trapping; Spin Trapping - methods; Superoxide; temperature; NO; O2; DEPMPO; EPR; EMPO; Nitroxide; Spin trapping; Radical; MNP; CYPMPO; BMPO; Hb; PBN; DTCS; ESR; MS; DMPO; SOD; Superoxide; MGD; POBN; DETC; Nitric oxide; Electron paramagnetic resonance; DBNBS; 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide; N-methyl-d-glucamine; 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide; electron spin resonance; haemoglobin; α-(4-pyridyl-1-oxide)-N-tert-butylnitrone; N-(dithiocarboxy)sarcosine; mass spectrometry; 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide; 3,5-dibromo-4-nitrosobenzene sulfonic acid; N-tert-butyl-α-phenylnitrone; 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide; 5,5-dimethyl-1-pyrroline N-oxide; superoxide radical anion; superoxide dismutase; O(−); diethyldithiocarbamate; 2-methyl-2-nitrosopropane
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2013.03.034

Electron paramagnetic resonance (EPR) spectroscopy (also known as electron spin resonance, ESR, spectroscopy) is widely considered to be the “gold standard” for the detection and characterisation of radicals in biological systems. The article reviews the major positive and negative aspects of EPR spectroscopy and discusses how this technique and associated methodologies can be used to maximise useful information, and minimise artefacts, when used in biological studies. Consideration is given to the direct detection of radicals (at both ambient and low temperature), the use of spin trapping and spin scavenging (e.g. reaction with hydroxylamines), the detection of nitric oxide and the detection and quantification of some transition metal ions (particularly iron and copper) and their environment. When used with care this technique can provide a wealth of valuable information on the presence of radicals and some transition metal ions in biological systems. It can provide definitive information on the identity of the species present and also information on their concentration, structure, mobility and interactions. It is however a technique that has major limitations and the user needs to understand the various pitfalls and shortcoming of the method to avoid making errors. EPR remains the most definitive method of identifying radicals in complex systems and is also a valuable method of examining radical kinetics, concentrations and structure. This article is part of a Special Issue entitled Current methods to study reactive oxygen species — pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. •EPR is the current “gold standard” for detecting radicals in biological systems.•EPR methods for detecting radicals, metal ions and nitric oxide are reviewed.•Advantages and disadvantages of EPR and complementary techniques are discussed.•Kinetic data on EPR spin trapping reactions and adduct decay are presented.•Information is presented on the choice of spin traps and artefact formation.