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J. Biol. Chem., Vol. 282, Issue 19, 13977-13983, May 11, 2007

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Assessment and Application of the Biotin Switch Technique for Examining Protein S-Nitrosylation under Conditions of Pharmacologically Induced Oxidative Stress^*

Michael T. Forrester, Matthew W. Foster^¶, and Jonathan S. Stamler^¶1

From the Department of Biochemistry, Medical Scientist Training Program, and the ^¶Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

Protein S-nitrosylation has emerged as a principal mechanism by which nitric oxide exerts biological effects. Among methods for studying protein S-nitrosylation, the biotin switch technique (BST) has rapidly gained popularity because of the ease with which it can detect individual S-nitrosylated (SNO) proteins in biological samples. The identification of SNO sites by the BST relies on the ability of ascorbate to generate a thiol from an S-nitrosothiol, but not from alternatively S-oxidized thiols (e.g. disulfides, sulfenic acids). However, the specificity of this reaction has recently been challenged, prompting several claims that the BST may produce false-positive results and raising concerns about the application of the BST under oxidizing conditions. Here we perform a comparative analysis of the BST using differentially S-oxidized and S-nitrosylated forms of protein tyrosine phosphatase 1B, as well as intact and lysed human embryonic kidney 293 cells treated with S-oxidizing and S-nitrosylating agents, and verify that the assay is highly specific for SNO. Strikingly, exposure of samples to indirect sunlight from a laboratory window resulted in artifactual ascorbate-dependent signals that are likely promoted by the semidehydroascorbate radical; protection from sunlight eliminated the artifact. In contrast, exposure of SNO proteins to a strong ultraviolet light source (SNO photolysis) prior to the BST provided independent verification of assay specificity. By combining BST with photolysis, we have shown that anti-cancer drug-induced oxidative stress facilitates the S-nitrosylation of the major apoptotic effector glyceraldehyde-3-phosphate dehydrogenase. Collectively, these experiments demonstrate that SNO-dependent signaling pathways can be modulated by oxidative conditions and suggest a potential role for S-nitrosylation in antineoplastic drug action.

Received for publication, October 13, 2006 , and in revised form, February 26, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Box 2612, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-6933; Fax: 919-684-6998; E-mail: staml001{at}mc.duke.edu.

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