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Analysis of the bioactivity of magnetically immunoisolated peroxisomes

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Abstract

Peroxisomes produce reactive oxygen species which may participate in biotransformations of innate biomolecules and xenobiotics. Isolating functional peroxisomes with low levels of contaminants would be a useful tool to investigate biotransformations occurring in these organelles that are usually confounded with biotransformations occurring in other co-isolated organelles. Here, we immunoisolate peroxisomes and demonstrate that the impurity level after isolation is low and that peroxisomes retain their biological activity. In this method, an antibody targeting a 70-kDa peroxisomal membrane protein was immobilized to silanized magnetic iron oxide beads (1–4 μm in diameter) coated with Protein A. Peroxisomes from L6 rat myoblast homogenates were magnetically captured, washed, and then analyzed for subcellular composition using enzymatic assays. Based on the ratio of peroxisomal to lysosomal activity, the retained fraction is 70-fold enriched relative to the unretained fraction. Similarly, the ratio of peroxisomal activity to mitochondrial content suggests that the retained fraction is >30-fold enriched relative to the unretained fraction. H2O2 production from the β-oxidation of palmitoyl-CoA demonstrated that the isolated peroxisomal fraction was biologically active. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) analysis confirmed that the immunopurified fractions were capable of transforming the anticancer drug doxorubicin and the fatty acid analog, BODIPY 500/510 C1C12. Besides its use to investigate peroxisome biotransformations in health and disease, the combination of magnetic immunoisolation with CE-LIF could be widely applicable to investigate subcellular-specific biotransformations of xenobiotics occurring at immunoisolated subcellular compartments.

Peroxisomes are immunoisolated using Protein A-coated magnetic beads. The isolated peroxisomes are highly enriched relative to other contaminating organelles. Capillary electrophoretic analysis shows that isolated peroxisomes can biotransform doxorubicin, a common anti-cancer drug.

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Acknowledgments

YW acknowledges support through a 2008–2009 Merck Research Laboratories Fellowship in Analytical/Physical Chemistry. THT acknowledges support from NIH grant T32GM008700. EAA acknowledges support from NIH grant AG020866.

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  1. Department of Chemistry, University of Minnesota, Minneapolis, MN, 55455, USA

    Yaohua Wang, Thane H. Taylor & Edgar A. Arriaga

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  1. Yaohua Wang
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  2. Thane H. Taylor
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  3. Edgar A. Arriaga
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Correspondence to Edgar A. Arriaga.

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Published in the 10th Anniversary Issue.

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Wang, Y., Taylor, T.H. & Arriaga, E.A. Analysis of the bioactivity of magnetically immunoisolated peroxisomes. Anal Bioanal Chem 402, 41–49 (2012). https://doi.org/10.1007/s00216-011-5476-3

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  • DOI: https://doi.org/10.1007/s00216-011-5476-3

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