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Oct 1, 2009 (Vol. 29, No. 17)

Studying the S-Glutathionylation of Proteins

Detection Kit Focuses on Improving Qualitative and Quantitative Analysis

  • The S-glutathionylation of proteins is an important post-translational modification that occurs under normal conditions as well as during oxidative stress. This modification is selective, occurring only on cysteine residues positioned in specific contexts on the surface of target proteins. The disulfide linkage between glutathione and protein is reversible, through the action of thiol-disulfide oxidoreductases.

    Glutathionylation substantially alters the functionality of enzymes, receptors, structural proteins, transcription factors, and transport proteins. These changes naturally have far-reaching implications for normal cell biology, response to oxidative stress, and human physiology.

    Glutathione is the most abundant nonprotein thiol in cells. As a major antioxidant, it is maintained in a reduced (GSH) state. In the presence of reactive oxygen species (ROS) and nitrogen species (RNS), GSH donates a reducing equivalent and becomes highly reactive. It can partner with another molecule of reactive glutathione, forming glutathione disulfide (GSSG). Or, it can react with the sulfhydryl group of certain cysteines on proteins.

    Of course, the sulfhydryl group must be accessible for interaction with glutathione. Furthermore, reactivity is strongly enhanced if neighboring residues are basic (positively charged), whereas acidic residues in the vicinity of the sulfhydryl group oppose glutathionylation.

    Oxidized glutathione, GSSG, is reverted to GSH by glutathione reductase, an enzyme that is constitutively active and inducible upon oxidative stress. Similarly, the disulfide linkage between target protein and glutathione (PSSG) is cleaved by thiol-disulfide oxidoreductases, most notably the glutaredoxins. This suggests that glutathionylation normally serves to alter protein function temporarily, in coordination with localized changes in redox tone.

    The effects of glutathionylation on functionality are diverse, as all types of proteins are susceptible. Enzymes involved in energy metabolism are inactivated when glutathionylated, resulting in impaired energy production during oxidative stress. The correlation of oxidative stress and cancer is echoed by the glutathionylation of the tumor suppressor p53, which prevents p53 dimerization necessary for DNA binding.

    Interestingly, glutathionylation protects caspase from cleavage, preventing apoptosis. Glutathionylation activates both p21ras, leading to phosphorylation of ERK and Akt as downstream targets, and ryanodine receptors, causing calcium signals that enhance ERK and CREB phosphorylation.

    S-Glutathionylation occurs extensively in diseases characterized by oxidative stress, including cardiovascular diseases, cancer, lung diseases, neurodegenerative diseases, and cancer. Reversible S-glutathionylation also occurs in cells under normal conditions. A number of important biological questions remain concerning the mechanism and regulation of adding and removing glutathione to proteins, as well as the role(s) of these modifications in various cellular processes.

  • Overview

    Click Image To Enlarge +
    Figure 1. Protocol overview

    Cayman Chemical’s S-Glutathionylated Protein Detection Kit includes reagents to study S-glutathionylation using a variety of approaches. Three simple steps are required. First, free sulfhydryl groups are irreversibly blocked (Figure 1). After washing, glutathione residues are then enzymatically removed from proteins in a reducing reaction.

    This leaves new, exposed free sulfhydryl groups, which are then tagged with biotin-maleimide. Biotinylated proteins can then be evaluated in several ways. The kit includes two avidin-based detection reagents, utilizing either FITC (fluorescent) or HRP (enhanced chemiluminescent, colorimetric). The entire process, from blocking to labeling, requires less than four hours.



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