Gsh

Fig. 2. Structure of BioGEE. The reagent BioGEE is composed of the glutathione tripeptide with a molecule of biotin incorporated at the y-glutamyl amine and ethyl esters at one (glycyl) or both of the carboxyl groups.

glutathiolated.21 In this chapter we present detailed protocols for the synthesis, purification, and use of BioGEE.

Preparation of Biotinylated Glutathione Ethyl Ester

BioGEE (Fig. 2) consists of the glutathione tripeptide modified to include ethyl esters at one or both of the carboxylates, and a molecule of biotin attached at the y-glutamyl amine. The use of ethyl ester derivatives of glutathione is based on data indicating that the GSH mono(glycyl) or diethyl esters cross the plasma membrane much more efficiently than does GSH.22-24 Incorporation of biotin into the molecule allows for simple and highly specific streptavidin-based detection and purification of proteins into which BioGEE is incorporated.

In our initial publication, establishing the utility of BioGEE, synthesis was accomplished by simply biotinylating a commercially available preparation of glutathione ethyl ester.21 The crude biotinylation reaction was then added directly to cells in culture. Although this approach is simple and ultimately proved effective,

21 D. M. Sullivan, N. B. Wehr, M. M. Fergusson, R. L. Levine, and T. Finkel, Biochemistry 39, 11121 (2000).

22 E. J. Levy, M. E. Anderson, and A. Meister, Methods Enzymol. 234, 499 (1994).

23 A. Meister, in "Glutathione: Chemical Biochemical and Medical Aspects" (D. Dolphin, R. Poulson, and O. Auramovic, eds.), p. 22. John Wiley & Sons, New York, 1989.

24 M. E. Anderson, E. J. Levy, and A. Meister, Methods Enzymol. 234, 492 (1994).

0 0

Post a comment