[26] Trypanothione and N1-glutathionylspermidine:Isolation and determination

Publisher Summary Trypanothione [T(SH) 2 , N 1 , N 8 -bis(glutathionyl)spermidine] and monoglutathionylspermidine (Gsp) are conjugates of the tripeptide glutathione and the polyamine spermidine. When determining the relative amounts of glutathione (GSH), Gsp, and T(SH) 2 in trypanosomatids, the integrity of the cells is crucial. In cell lysates, the glutathionylspermidine conjugates are rapidly broken down to free glutathione (GSH), which explains the varying recovery of Gsp and T(SH) 2 . N 1 -Monoglutathionylspermidine also occurs in bacteria. When entering the stationary phase, E. coli cells convert spermidine nearly quantitatively to this glutathione conjugate. The chapter describes the procedures for the chemical synthesis of glutathionylspermidine conjugates. Gsp ox can be isolated from E. coli cells harvested in stationary phase. The thiol forms of the glutathionylspermidines can be quantitatively determined with Ellman's reagent and the disulfides TS 2 and Gsp ox can be quantitated enzymatically by end-point measurements. Glutathione, monoglutathionylspermidine, trypanothione, and the corresponding disulfides can be separated by reversed-phase high-performance liquid chromatography (HPLC).