Elsevier

Analytical Biochemistry

Volume 283, Issue 2, 1 August 2000, Pages 214-221
Analytical Biochemistry

Regular Article
Identification of Proteins Containing Cysteine Residues That Are Sensitive to Oxidation by Hydrogen Peroxide at Neutral pH

https://doi.org/10.1006/abio.2000.4623Get rights and content

Abstract

A procedure for detecting proteins that contain H2O2-sensitive cysteine (or selenocysteine) residues was developed as a means with which to study protein oxidation by H2O2 in cells. The procedure is based on the facts that H2O2 and biotin-conjugated iodoacetamide (BIAM) selectively and competitively react with cysteine residues that exhibit a low pKa, and that the decrease in the labeling of cell lysate proteins with BIAM caused by prior exposure of cells to H2O2 or to an agent that induces H2O2 production can be monitored by streptavidin blot analysis. This procedure was applied to rat pheochromocytoma PC12 cells directly treated with H2O2, mouse hippocampal HT22 cells in which H2O2 production was induced by glutamate, and human erythroleukemia K562 cells in which H2O2 production was induced by phorbol myristate acetate. It revealed that several cell proteins contain cysteine or selenocysteine residues that are selectively oxidized by H2O2. Three of these H2O2-sensitive proteins were identified as a member of the protein disulfide isomerase family, thioredoxin reductase, and creatine kinase, all of which were previously known to contain at least one reactive cysteine or selenocysteine at their catalytic sites. This procedure should thus prove useful for the identification of proteins that are oxidized by H2O2 generated in response to a variety of extracellular agents.

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    1

    Both authors contributed equally to this work.

    2

    Present address: Department of Biochemistry, College of Medicine, Youngnam University, Taegu 705-003, Korea.

    3

    Present address: Department of Biochemistry, College of Natural Sciences, Kyounsang National University, Chinju 660-701, Korea.

    4

    To whom correspondence should be addressed at Building 3, Room 122, National Institutes of Health, Bethesda, MD 20892. Fax: 301-480-0357. E-mail: [email protected].

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