TY - JOUR
T1 - 5-(pentafluorobenzoylamino)fluorescein
T2 - A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity
AU - Arttamangkul, Seksiri
AU - Bhalgat, Mahesh K.
AU - Haugland, Rosaria P.
AU - Diwu, Zhenjun
AU - Liu, Jixiang
AU - Klaubert, Dieter H.
AU - Haugland, Richard P.
PY - 1999/5/1
Y1 - 1999/5/1
N2 - 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/μl and 1 ng/μl 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 β-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.
AB - 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/μl and 1 ng/μl 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 β-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.
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U2 - 10.1006/abio.1999.4044
DO - 10.1006/abio.1999.4044
M3 - Article
C2 - 10222018
AN - SCOPUS:0345643415
SN - 0003-2697
VL - 269
SP - 410
EP - 417
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -