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44091-B4
Biotinylated Glutathione Derivatives and Aromatic S-Nitroso Compounds as Tools to Study Protein Thiol Modifications
Our recent results show that brain microtubule proteins including tubulin, tau and MAP2 are very sensitive to changes in 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.
One undergraduate student worked in the lab during the 2007 10 week summer session on this project. We chose to begin with the fluorescein-derivatives of GSH and GSSG because the intense yellow color of fluorescein would provide a quick assessment of product formation during purification steps.
We began by following a published procedure for the synthesis of biotinylated GSSG. However, it soon became clear that fluorescein-labeled GSSG (*F-GSSG-F*) behaved differently during purification. Thus, the summer was dominated by our attempts to develop our own protocol for the synthesis and purification of *F-GSSG-F*.
We successfully prepared some *F-GSSG-F* by reacting GSSG with fluorescein isothiocyanate (FITC). FITC was separated from products, a mixture of singly and doubly fluorescein-labeled GSSG, on a solid phase C18 column. The successful separation was confirmed by HPLC. However we still see two product peaks by HPLC. Subsequent steps to separated *F-GSSG from *F-GSSG-F* have been difficult. We are using several different chromatographic approaches including ion-exchange and reverse-phase C18 and C8 columns.
Preliminary experiments using our mixture of products, *F-GSSG and *F-GSSG-F*, and several proteins have been successful. Glyceraldehyde-3-phosphate dehydrogenase, tubulin and MAPs react with *F-GSSG and *F-GSSG-F* to yield fluorescein-labeled proteins. We have performed both simple dot blots as well as SDS-PAGE separation of modified proteins. Addition of a disulfide reducing agent removes the fluorescein-label from all protein samples tested.
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 2007-08 academic year, two undergraduate students are working on this project for academic credit.
