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

Seksiri Arttamangkul, Mahesh K. Bhalgat, Rosaria P. Haugland, Zhenjun Diwu, Jixiang Liu, Dieter H. Klaubert, Richard P. Haugland

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)410-417
Number of pages8
JournalAnalytical Biochemistry
Volume269
Issue number2
DOIs
StatePublished - May 1 1999
Externally publishedYes

Fingerprint

Glutathione Transferase
Glutathione
Substrates
Thin layer chromatography
Endothelial cells
Thin Layer Chromatography
Pulmonary Artery
Limit of Detection
Assays
Endothelial Cells
5-(pentafluorobenzoylamino)fluorescein
Galactosidases
Sulfhydryl Compounds
Human Activities
Liver
Isoenzymes
Horses
Cysteine
Fusion reactions
Fluorescence

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

5-(pentafluorobenzoylamino)fluorescein : A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity. / Arttamangkul, Seksiri; Bhalgat, Mahesh K.; Haugland, Rosaria P.; Diwu, Zhenjun; Liu, Jixiang; Klaubert, Dieter H.; Haugland, Richard P.

In: Analytical Biochemistry, Vol. 269, No. 2, 01.05.1999, p. 410-417.

Research output: Contribution to journalArticle

Arttamangkul, Seksiri ; Bhalgat, Mahesh K. ; Haugland, Rosaria P. ; Diwu, Zhenjun ; Liu, Jixiang ; Klaubert, Dieter H. ; Haugland, Richard P. / 5-(pentafluorobenzoylamino)fluorescein : A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity. In: Analytical Biochemistry. 1999 ; Vol. 269, No. 2. pp. 410-417.
@article{c312e4e2b25c4f549e1979253cb49ea1,
title = "5-(pentafluorobenzoylamino)fluorescein: A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity",
abstract = "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.",
author = "Seksiri Arttamangkul and Bhalgat, {Mahesh K.} and Haugland, {Rosaria P.} and Zhenjun Diwu and Jixiang Liu and Klaubert, {Dieter H.} and Haugland, {Richard P.}",
year = "1999",
month = "5",
day = "1",
doi = "10.1006/abio.1999.4044",
language = "English (US)",
volume = "269",
pages = "410--417",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

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.

UR - http://www.scopus.com/inward/record.url?scp=0345643415&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0345643415&partnerID=8YFLogxK

U2 - 10.1006/abio.1999.4044

DO - 10.1006/abio.1999.4044

M3 - Article

C2 - 10222018

AN - SCOPUS:0345643415

VL - 269

SP - 410

EP - 417

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -