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Fig. 5. Affinity purification of proteins labeled with BioGEE. (A) Diagram of the protocol devised for labeling and purification of S-glutathiolated proteins. Abbreviations: S, Sulfur; SG, glutathione; SA, streptavidin; DTT, dithiothreitol. (B) A soluble protein extract was obtained, as outlined in (A) and text, from HeLa cells treated with H2O2. Biotin-containing proteins were extracted from 0.5 mg of soluble protein with streptavidin-agarose. The beads were washed and eluted sequentially, first with PBS containing 0.1% (w/v) SDS (-DTTeluate) and then with PBS containing 0.1 % (w/v) SDS and 10 mM DTT (+DTT eluate). The eluates were resolved by SDS-PAGE and proteins were detected by silver staining the gel.

11. Pellet the agarose and transfer the supernatant to a fresh tube.

12. Wash the beads with 1 volume of PBS-0.1% (w/v) SDS and combine the wash with the DTT eluate.

13. Add DTT to the —DTT eluate to a final concentration of 10 mM and transfer both the —DTT and DTT eluates to Centricon-10 ultrafiltration units. Concentrate proteins according to the manufacturer protocol.

14. Add SDS-PAGE sample buffer to the concentrated proteins and separate by SDS-PAGE.

15. Detect proteins by Coomassie or silver staining the gel, using standard protocols.

Interpretation of Data Obtained with Biotinylated Glutathione Ethyl Ester

The dynamic incorporation of BioGEE into protein in association with oxidative stress, its structural similarity to glutathione and apparent selectivity for reactive cysteines make BioGEE an excellent marker for reversible redox modification of proteins. However, as mentioned above, many details regarding the metabolism of BioGEE remain to be established. In particular, it is not known whether the enzyme systems involved in removing GSH from protein are effective at removing BioGEE. Even if BioGEE is completely deesterified to biotinylated GSH, it is conceivable that the remaining biotinyl moiety could affect the ability of enzymes, such as glutaredoxin, to use biotinylated GSH as a substrate. If that were the case, BioGEE might be expected to have a slower off rate and therefore accumulate in protein to a greater extent than endogenous glutathione. Likewise, the extent of BioGEE incorporation into protein is dependent on the unknown ratio of BioGEE to endogenous glutathione in the cell. It should therefore be stressed that the extent to which BioGEE is incorporated into a given protein cannot at this point be directly correlated with the extent to which that protein is glutathiolated. A finding that BioGEE is incorporated into a protein should therefore be taken as evidence of the existence of a redox-active cysteine that is sufficiently exposed to accept a molecule of glutathione. Such information is a crucial first step in understanding the effects of oxidants on the modified protein and ultimately on the cell.

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