Introduction

Nitric oxide (NO) is a free-radical product of cellular metabolism in microorganisms, plants, and animals that is involved in many different physiological processes, such as defense, growth and development, neurotransmission, vasodilation, and inflammation (1-9). Many of the biological functions of NO arise as a direct consequence of chemical reactions between proteins and NO or NO oxides generated as NO/O2 or NO/superoxide reaction products. The reactions of NO with metal ions of heme groups or the forma-

John T. Hancock (ed.), Methods in Molecular Biology, Redox-Mediated Signal Transduction, vol. 476 © 2008 Humana Press, a part of Springer Science + Business Media, Totowa, NJ DOI: 10.1007/978-1-59745-129-1_15

tion of dinitrosyl complexes are demonstrated to play important roles in NO signaling. The best known example is soluble guan-ylate cyclase. This heme protein is activated by NO and catalyses the formation of cyclic GMP, which acts as second messenger in various biological responses (10). However, during the last decade, protein S-nitrosylation—the covalent attachment of NO to sulfhydryl groups of cysteine residues—moved to the center of importance of NO-dependent signaling (11-14). This type of protein modification is a reversible redox-based enzyme-independent mechanism, which is involved in regulation of many physiological processes. S-nitrosylation of cysteine residues can alter the activity of enzymes and transcription factors, can result in translocation of the modified proteins, or can change its physiological function (15-18). However, because of the lability of nitrosothiols, it was difficult to detect and analyze this type of protein modification. Jeffrey and colleagues developed a method, named the biotin switch method, which overcome this limitation (19, 20). The principle behind this method is the substitution of the NO group for a biotin linker (Fig.15.1). In this way, the labile S-NO bond is replaced

Biotin Switch Assay

Fig. 15.1. Schematic presentation of the biotin switch assay. A model protein containing cysteine residues in the disulfide, free thiol, and nitosothiol stage is subjected to the biotin switch assy. First, the free thiol group is blocked with MMTS in the presence of SDS, which ensures the access of buried thiol groups. Then nitrosylated thiols are reduced selectively by ascorbate. In the third step, the re-formed thiol groups are biotinylated with the thiol-modifying agent biotin-HPDP.

Fig. 15.1. Schematic presentation of the biotin switch assay. A model protein containing cysteine residues in the disulfide, free thiol, and nitosothiol stage is subjected to the biotin switch assy. First, the free thiol group is blocked with MMTS in the presence of SDS, which ensures the access of buried thiol groups. Then nitrosylated thiols are reduced selectively by ascorbate. In the third step, the re-formed thiol groups are biotinylated with the thiol-modifying agent biotin-HPDP.

by a more stabile disulfide bridge. This biotin-labeling allows the detection and purification of the previously S-nitrosylated proteins using anti-biotin antibodies or immobilised streptavidin, respectively. The biotin switch method was successfully used to identify candidates for S-nitrosylation in different organisms and tissues, such as rat brain lysate, mouse mesangial cells, rat liver mitochondria, endothelia cells, Mycobacterium tuberculosis and Arabidopsis thaliana leaves and cell cultures (19, 21-25) (Figs. 15.2, 15.3 and 15.4). However, there are also some reports doubting the specificity of the biotin switch method (26-28).

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