Immuno-spin trapping from biochemistry to medicine: advances, challenges, and pitfalls. Focus on protein-centered radicals

Abstract

Background: Immuno-spin trapping (IST) is based on the reaction of a spin trap with a free radical to form a stable nitrone adduct, followed by the use of antibodies, rather than traditional electron paramagnetic resonance spectroscopy, to detect the nitrone adduct. IST has been successfully applied to mechanistic in vitro studies, and recently, macromolecule-centered radicals have been detected in models of drug-induced agranulocytosis, hepatotoxicity, cardiotoxicity, and ischemia/reperfusion, as well as in models of neurological, metabolic and immunological diseases.

Scope of the review: To critically evaluate advances, challenges, and pitfalls as well as the scientific opportunities of IST as applied to the study of protein-centered free radicals generated in stressed organelles, cells, tissues and animal models of disease and exposure.

Major conclusions: Because the spin trap has to be present at high enough concentrations in the microenvironment where the radical is formed, the possible effects of the spin trap on gene expression, metabolism and cell physiology have to be considered in the use of IST and in the interpretation of results. These factors have not yet been thoroughly dealt with in the literature.

General significance: The identification of radicalized proteins during cell/tissue response to stressors will help define their role in the complex cellular response to stressors and pathogenesis; however, the fidelity of spin trapping/immuno-detection and the effects of the spin trap on the biological system should be considered. 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.

Keywords: 5,5-dimethyl-1-pyrroline N-oxide; AG; Anti-DMPO; BSA; DMPO; Disease/exposure model; ESR; HPLC; I/R; IST; Immuno-spin trapping; LC; LPS; MRI; MS; OA; Protein radical; Reactive chemical species; SOD; Spin trap; TPO; aminoglutethimide; bovine serum albumin; electron spin resonance; high performance liquid chromatography; hoMb; horse myglobin; huHb/Mb; human hemoglobin/myoglobin; immuno-spin trapping; ischemia/reperfusion; lipopolysaccharide; liquid chromatography; magnetic resonance imaging; mass spectrometry; octanoic acid; superoxide dismutase; thyroid peroxidase.

Figure 1. Spin trapping and fate of protein-DMPO adducts

A protein radical (a radical site in a protein) reacts with DMPO to form a radical adduct. Depending on microenvironment conditions and structural characteristics of the target protein, the radical adduct can be reduced to hydroxylamine or oxidized to a stable nitrone adduct. It can also disproportionate to generate both the hydroxylamine and nitrone adducts. In cells and in vivo there are a number of competing reactions that can affect the yield of DMPO-protein adducts. Reduced glutathoine (GSH) and L-ascorbate (ASCH) [28] can react with protein radicals faster than the rate of reaction with DMPO, resulting in reduced yield of protein-DMPO nitrone adducts, a repaired protein and a less reactive radical (i.e., GS^• and ASC^•). Oxygen, the best spin trap in nature, can also react with protein radicals to form protein-peroxyl radicals, which very slowly react with DMPO. Protein radicals can react with other radical sites in the same or different proteins to form cross links (e.g., Tyr-Tyr, His-His or Trp-Trp). Protein radicals can also react with lipid radicals, ^•NO₂/^•NO or drug/toxicant radicals, thus resulting in protein-lipid or protein-drug/toxicant adducts (P-R).

Figure 2. Principle of immuno-spin trapping of protein radicals

Immuno-spin trapping can be applied to investigate protein radicals in purified systems, cells, tissues and in the whole animal. The production of protein radicals is caused by one-electron-mediated oxidation of specific residues in a protein. These residues can be primary or secondary targets. Protein radicals are trapped in situ by DMPO to form protein-DMPO radical adducts. With time a radical adduct decays to form a stable DMPO-protein nitrone adduct. The DMPO motif of a protein-DMPO nitrone adduct can be detected with an anti-DMPO antibody using immunoassays. Protein-DMPO nitrone adducts can be pulled down by immunoprecipitation from complex mixtures such as homogenates of organelles, whole cells or tissues and then characterized using MS. To preserve tissue architecture, protein-nitrone adducts can be observed using the anti-DMPO antibody and fluorescent or immunogold techniques. Protein-DMPO nitrone adducts can also be detected using non-invasive techniques such as molecular magnetic resonance imaging (mMRI).

Figure 3. In vivo molecular MR imaging (mMRI) of DMPO-nitrone adducts

A) Schematic representation of the anti-DMPO mMRI probe used to detect free radicals forming membrane-associated-DMPO nitrone adducts; B) scheme of the experimental design to form membrane-associated radicals and detection of these radicals using the probe shown in A with mMRI. See reference [78] for further details.

Figure 4. Localization and identification of protein-DMPO nitrone adducts in RAW 264.7 macrophages treated with LPS and DMPO

A) Single-plane confocal images of nitrone adducts formed in cells treated with 1 ng/ml LPS and 12.5 or 50 mM DMPO for 24 h. Green indicates nitrone adducts and blue indicates nuclei. Insert is a high-power magnification of the image of a single and representative cell. The white arrowhead indicates the perinuclear localization of most nitrone adducts. B) Western blot analysis of protein-DMPO nitrone adducts in homogenates of cells treated with LPS and/or DMPO for 24 h. Right panel, coomassie blue staining of the homogenate of cells treated with 1 ng/ml LPS and 50 mM DMPO for 24 h, separated in a reducing gel and showing 7 representative bands that correspond to anti-DMPO-positive bands in the Western blot. C) Schematic representation of an anti-DMPO molecular “catcher”-protein-DMPO nitrone adduct complex used to pull-down proteins labeled with DMPO. M indicates Magic Mark Western XP molecular weight marker (Invitrogen). Modified from [29].