5-(pentafluorobenzoylamino)fluorescein: A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity

5-(Pentafluorobenzoylamino)flluorescein (PFB-F), a new thiol-reactive molec ule was synthesized to improve the detection limits and specificity of the assays for glutathione S-transferase (GST) activity and glutathione (GSH). A rapid assay method to measure GSH concentration or GST activity and the s imultaneous analysis of multiple samples is possible because the glutathion e adduct, GS-TFB-F, is separated from PFB-F by thin-layer chromatography (T LC) and can be quantitated by a fluorescence scanner. The detection limits for GSH and for GST activity using TLC were found to be as low as 10 pmol/m u l and 1 ng/mu l using equine liver GST, respectively. Determination of GS H concentration or GST activity in bovine pulmonary artery endothelial (BPA E) cell lysates gave a linear response for samples corresponding to 500-250 0 cells. PFB-F could also measure GST activities of GST fusion proteins and prove to be a suitable substrate for determining the activities of human G ST isozymes and other sources of mammalian GST. The selectivity of PFB-F wi th GSH was proven by comparing trace amount of the adducts that formed with cyst-eine and P-galactosidase to that formed with GSH, The HPLC profile of a reaction mixture where cell lysate was used in place of purified GST, al so shows only two main peaks, corresponding to GS-TFB-F and unreacted PFB-F . The selectivity of PFB-F for GSH was further confirmed by exposing BPAE c ells to DL-buthionine-[S,R]-sulfoximine (BSO). Our results of GS-TFB-F dete rmination indicate that 12-, 24-, or 36-h incubations with ESO caused 2-, 6 -, or 7.6-fold reductions in GSH levels,respectively. (C) 1999 Academic Pre ss.