5-(Pentafluorobenzoylamino)fluorescein: A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity.
5-(Pentafluorobenzoylamino)fluorescein (PFB-F), a new thiol-reactive molecule 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 simultaneous analysis of multiple samples is possible because the glutathione adduct, GS-TFB-F, is separated from PFB-F by thin-layer chromatography (TLC) 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/microl and 1 ng/microl using equine liver GST, respectively. Determination of GSH concentration or GST activity in bovine pulmonary artery endothelial (BPAE) cell lysates gave a linear response for samples corresponding to 500-2500 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 GST isozymes and other sources of mammalian GST. The selectivity of PFB-F with GSH was proven by comparing trace amount of the adducts that formed with cysteine and beta-galactosidase to that formed with GSH. The HPLC profile of a reaction mixture where cell lysate was used in place of purified GST, also 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 cells to dl-buthionine-[S,R]-sulfoximine (BSO). Our results of GS-TFB-F determination indicate that 12-, 24-, or 36-h incubations with BSO caused 2-, 6-, or 7.6-fold reductions in GSH levels, respectively.
Arttamangkul
S
S
Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, Oregon 97402-9165, USA.
Bhalgat
M K
MK
Haugland
R P
RP
Diwu
Z
Z
Liu
J
J
Klaubert
D H
DH
Haugland
R P
RP
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D004730:Endothelium, Vascular / Q000737:chemistry / Q000201:enzymology; D005452:Fluoresceins / Q000138:chemical synthesis; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D005978:Glutathione / Q000032:analysis*; D005982:Glutathione Transferase / Q000032:analysis*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013379:Substrate Specificity; D005466:Fluorocarbons
D005452:Fluoresceins; D005456:Fluorescent Dyes; D005978:Glutathione; D005982:Glutathione Transferase; D011993:Recombinant Fusion Proteins; C119286:5-(pentafluorobenzoylamino)fluorescein; D005466:Fluorocarbons