TY - JOUR
T1 - para-Sulfobenzoyloxybromobimane
T2 - A new membrane-impermeable reagent useful for the analysis of thiols and their export from cells
AU - Newton, Gerald L.
AU - Aguilera, Joseph A.
AU - Fahey, Robert C.
AU - Ward, John F.
AU - Radkowsky, Annette E.
AU - Kosower, Edward M.
N1 - Funding Information:
This research was supported by Grant CA-35982 from the National Cancer Institute and Grant 87-00091 from the US-Israel Binational Science Foundation. We thank Professor Nechama S. Kosower for advice on the use of dichloromethane extraction for removal of excess mBBr and nonionic bimane derivatives. We also thank Tonghyun Kim for assistance with the production of the figures.
PY - 1992/2/14
Y1 - 1992/2/14
N2 - para-Sulfonylbenzoyloxybromobimane (sBBr) was shown to be similar to the fluorescent labeling agent monobromobimane (mBBr) in reacting rapidly and selectively with thiols to produce stable derivatives which are readily separated by HPLC. Chromatography of the sBBr derivative provides a useful means of confirming the identification of an unknown thiol based upon the chromatography of its mBBr derivative and can be useful for quantitative determination of polycationic thiols for which chromatography of the mBBr derivative is unsatisfactory. Unlike mBBr, which readily penetrates cells, sBBr was found not to be taken up by cells. These characteristics allow sBBr to be used, in conjunction with mBBr, to quantify the export of thiols from cells, as illustrated for GSH and the radioprotective drug WR1065, from V79 cells. Simultaneous determination of GSH and glutathione disulfides in cell culture medium could be achieved by labeling of thiols with sBBr followed by reduction of disulfides with dithiothreitol, labeling of the resulting thiols with mBBr, and HPLC analysis for both glutathione derivatives.
AB - para-Sulfonylbenzoyloxybromobimane (sBBr) was shown to be similar to the fluorescent labeling agent monobromobimane (mBBr) in reacting rapidly and selectively with thiols to produce stable derivatives which are readily separated by HPLC. Chromatography of the sBBr derivative provides a useful means of confirming the identification of an unknown thiol based upon the chromatography of its mBBr derivative and can be useful for quantitative determination of polycationic thiols for which chromatography of the mBBr derivative is unsatisfactory. Unlike mBBr, which readily penetrates cells, sBBr was found not to be taken up by cells. These characteristics allow sBBr to be used, in conjunction with mBBr, to quantify the export of thiols from cells, as illustrated for GSH and the radioprotective drug WR1065, from V79 cells. Simultaneous determination of GSH and glutathione disulfides in cell culture medium could be achieved by labeling of thiols with sBBr followed by reduction of disulfides with dithiothreitol, labeling of the resulting thiols with mBBr, and HPLC analysis for both glutathione derivatives.
UR - http://www.scopus.com/inward/record.url?scp=0026527841&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(92)90170-C
DO - 10.1016/0003-2697(92)90170-C
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AN - SCOPUS:0026527841
SN - 0003-2697
VL - 201
SP - 30
EP - 42
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -