[10 Detection and Affinity Purification of Oxidant Sensitive Proteins Using Biotinylated Glutathione Ethyl Ester

By Daniel M. Sullivan, Rodney L. Levine, and Toren Finkel Introduction

Effects of oxidative stress in biological systems have long been the subject of intense scientific scrutiny. This work has established at least an ancillary role for oxidants in an astonishingly wide range of human diseases and, more recently, a role in normal signal transduction. Many of these studies have focused on toxicological effects of highly reactive species such as peroxyl, alkoxyl, and hydroxyl radicals, which are capable of modifying a wide range of cellular constituents. In proteins, potent oxidants such as the ones listed above can attack a variety of amino acid side chains and the polypeptide backbone,1'2 inflicting irreversible damage. Such modifications are likely important in pathological processes, such as reperfusion injury and inflammation, in which cells are exposed to high levels of oxidative stress. However, a variety of antioxidant systems are employed by cells to prevent accumulation of highly reactive species in association with normal redox metabolism. Therefore processes associated with lower levels of oxidative stress, such as redox-dependent signal transduction, are more likely mediated by the less reactive species nitric oxide (NO), hydrogen peroxide (H2O2), and superoxide (02 ). The cellular constituents susceptible to attack by these radicals are more limited, and in proteins the sulfur-containing amino acids cysteine and methionine are likely to be the predominant sites of modification.

Among the most oxidant-sensitive proteins are those containing ionized cysteine thiols.3 These thiolate anions, also referred to as reactive cysteines, are a feature of a variety of proteins, including a number of proteins involved in signal transduction. For example, thiolates have been described in transcription factors,4'5 kinases,6'7

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6 R. Gopalakrishna and S. Jaken, Free Radic. Biol. Med. 28, 1349 (2000).

7 n. e. Ward, J. R. Stewart, C. G. Ioannides, and C. A. O'Brian, Biochemistry 39, 10319 (2000).

Fig. 1. Scheme for reversible redox modification of reactive cysteines. A reactive cysteine is a protein thiol (P-SH) that is ionized to the relatively nucleophilic thiolate anion ( P-S ) at physiological pH. The reactive cysteine can be readily oxidized to a thiyl radical (P-S' ) or sulfenic acid (P-SOH) under mild oxidative stress. These oxidized species are frequently unstable under physiological conditions and, if not enzymatically reduced, will react to form more stable intermediates. Further oxidation yields a sulfinic acid (P-SOOH), which is thought to be irreversible in biological systems. Alternatively, the thiyl radical or sulfenic acid can react with a low molecular weight thiol (RSH) to form a stable mixed disulfide (P-S-SR). The mixed disulfide can then be reduced by one of three known enzymes systems [thioredoxin (TRx), glutaredoxin (GRx), or protein disulfide-isomerase (PDI)] to restore the cysteine to its fully reduced state.

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