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44091-B4
Biotinylated Glutathione Derivatives and Aromatic S-Nitroso Compounds as Tools to Study Protein Thiol Modifications

Lisa M. Landino, College of William and Mary

Results from our laboratory demonstrate that brain microtubule proteins including tubulin, tau and MAP2 are very sensitive to changes in thiol redox state. In addition to oxidation of cysteine residues of these cytoskeletal proteins, in preliminary experiments, we detected S-glutathionylation using an anti-glutathione antibody. In the current work, we seek better tools to probe these reactions that are easy to use, safe and reliable. To study S-glutathionylation, we are in the process of preparing fluorescein-labeled derivatives of reduced (GSH) and oxidized (GSSG) glutathione.

Two undergraduate students worked on this project lab during the 2008 10 week summer session. Considerable progress has been made to synthesize and purify singly (F-GSSG) and doubly fluorescein-labeled GSSG (F-GSSG-F). First, we altered the reaction conditions from a neutral phosphate buffer to 0.1 M ammonium bicarbonate (pH 8-8.5) so that fluorescein isothiocyanate (FITC) reacted completely thereby eliminating the need to remove unreacted FITC in our purification. 

F-GSSG, F-GSSG-F and unreacted GSSG were successfully separated using a solid phase C8. Surprisingly attempts to reduce F-GSSG-F to F-GSH were unsuccessful perhaps owing to the steric hindrance of the two fluoresceins in proximity to the disulfide bond. However F-GSSG was reduced to form F-GSH and GSH. Again using a C8 column, F-GSH was purified from GSH and excess reducing agent and quantitated using a fluorescein standard curve.

With both F-GSSG-F and F-GSH at our disposal, we began to study the reactions of these glutathione analogs with proteins including tubulin, a mixture of heat stable MAPs (predominantly MAP2 and tau), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH).

Reduced proteins (PSH) can under thiol-disulfide exchange with F-GSSG-F to yield oxidized protein (PSSP) and some F-GSH. During this process, some mixed disulfide between protein and F-GSH was detected by dot blot and by SDS-PAGE under nonreducing conditions. Addition of a disulfide reducing agent removes the fluorescein-label from all protein samples tested.

2PSH (red) + F-GSSG-F (ox) → PSSP (ox) + 2F-GSH (red) + PSSG-F

This exchange experiment yielded only very low levels of glutathionylated proteins. This is not unexpected because the desired mixed disulfide is merely an intermediate in the thiol-disulfide exchange process.

Much more significant labeling of protein thiols with F-GSH was accomplished by combining reduced protein, F-GSH and an oxidizing agent such as H₂O₂. Labeling was especially pronounced for GAPDH and tubulin.

PSH + F-GSH + H₂O₂ → PSSG-F + 2H₂O

            Thus, our concept is valid. Fluorescein-labeled glutathione derivatives will react with proteins and therefore we are encouraged to continue our efforts in this area. During the 2008-09 academic year, the two undergraduate students who worked on the project during the summer are continuing their work. Among the areas of current study: assaying the effect of F-GSH labeling on enzymatic function (for GAPDH, LDH and ADH) and performing protein digestions to determine if all, or only select cysteines are labeled with F-GSH. Two other undergraduate students are working on the synthesis and purification of biotinylated GSH.

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