Simple methods for the detection and quantification of thiols from Crithidia fasciculata and for the isolation of trypanothione

Abstract

Methods for the qualitative and quantitative analysis of thiols by means of the fluorogenic reagent 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin are described, with particular reference to the trypanosomatid metabolites glutathionylspermidine (GSH-spermidine) and trypanothione. Second-order rate constants for the derivatization of seven different thiols under defined experimental conditions and at 21 degrees C varied between 619 +/- 34 and 10,560 +/- 236 M-1.s-1.T.l.c. of the thiols from Crithidia fasciculata was used to monitor the purification of trypanothione from this organism in three steps involving adsorption, ion-exchange and reversed-phase chromatography. The yield was approx. 50 mg of pure trypanothione from 100 g (wet wt.) of trypanosomatids. The method for the quantitative analysis of biological thiols is based on fluorometric detection after separation by reversed-phase or ion-paired chromatography on a phenyl-silica column. Analysis of the thiol composition of cell lysates prepared under nondenaturating conditions point to the rapid degradation of the GSH-spermidine conjugates. In addition to GSH, GSH-spermidine and trypanothione, at least one other prominent thiol was detected, and the contribution of this thiol to the total thiol content in the various growth phases of C. fasciculata was investigated.