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Assay Tutorials : Oct 1, 2009 (Vol. 29, No. 17)

Studying the S-Glutathionylation of Proteins

Detection Kit Focuses on Improving Qualitative and Quantitative Analysis
  • Thomas Brock
  • ,
  • Micah Doty

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

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.

Experimental Approach and Validation

Preliminary tests indicated significant endogenous protein glutathionylation in murine macrophage RAW 264.7 cells. A sample of these cells were fixed with paraformaldehyde, blocked, reduced, tagged, and FITC labeled. Pronounced fluorescence was observed by fluorescent microscopy in the cytoplasmic compartment, whereas the nucleus was nonfluorescent (Figure 2A).

Omission of the reducing step, which would prevent the release of glutathione residues and the generation of free sulfhydryl groups, resulted in absence of fluorescence (Figure 2B), supporting efficiency of the blocking step. Conversely, when blocking was omitted, intense fluorescence was obtained in both the cytoplasmic and nuclear compartments (Figure 2C).

Untreated RAW 264.7 cells were also evaluated using flow cytometry. One group of cells was fluorescently stained without the reducing step, which gives minimal FITC fluorescence.

By flow cytometry, these cells were found to consist of a major population of low fluorescence cells and a smaller population with higher fluorescence (Figure 3, black). The smaller population most likely reflects cells with high autofluorescence, as is often associated with phagosomes in macrophages. Fluorescent analysis of S-glutathionylation, using standard assay conditions, demonstrated a pronounced right shift of fluorescence intensity for both populations of cells (Figure 3, red).

Biotinylated proteins can be separated by SDS-PAGE, transferred to nitrocellulose, then probed by an avidin overlay technique. Proteins in lysates of RAW 264.7 cells, processed using the S-glutathionylation detection protocol with or without the PSSG reducing step, were separated, transferred, overlaid with avidin-HRP, and developed by enhanced chemiluminescence. Several S-glutathionylated proteins were detected in the sample that was analyzed using the complete protocol.

Flexibility

The Cayman S-Glutathionylated Protein Detection Kit mirrors the versatility of the Cayman S-Nitrosylated Protein Detection Kit. Both can be used on whole cells or on isolated cellular components (e.g., synaptic vesicles). Cells may be fixed before blocking to maximize the retention of soluble proteins or left unfixed for cell fractionation or protein solubilization. Biotin-tagged proteins can be enriched using avidin-coated resin before analysis, e.g., by immunoblot. Similarly, biotin-tagged proteins may be first immunoprecipitated, then analyzed by avidin overlay.

Summary

Cayman’s S-Glutathionylated Protein Detection Assay Kit provides a convenient method for the study of S-glutathionylated proteins in whole cells by flow cytometry and microscopy and in cell lysates by avidin overlay analysis. The assay starts with the blocking of free sulfhydryl groups followed by enzymatic cleavage of protein-SS-glutathione adducts present in the sample.

Biotinylation of the newly formed free sulfhydryl groups provides the basis for visualization using streptavidin-based fluorescence or colorimetric detection. Reagents are provided to test three sets of 10 samples (most convenient) or up to 30 samples total at once if desired.