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Existence of low‐molecular‐weight thiols in Caenorhabditis elegans demonstrated by HPLC‐fluorescene detection utilizing 7‐chloro‐N‐[2‐(dimethylamino)ethyl]‐2,1,3‐benzoxadiazole‐4‐sulfonamide
Author(s) -
Asamoto Hiromichi,
Ichibangase Tomoko,
Saimaru Hiroshi,
Uchikura Kazuo,
Imai Kazuhiro
Publication year - 2007
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.814
Subject(s) - chemistry , chromatography , derivatization , reagent , high performance liquid chromatography , glutathione , electrospray ionization , thiol , tcep , fluorescence , cysteine , caenorhabditis elegans , mass spectrometry , sulfonamide , biochemistry , organic chemistry , enzyme , physics , quantum mechanics , phosphine , catalysis , gene
A highly sensitive and simple method using HPLC‐fluorescence detection with 7‐chloro‐ N ‐[2‐(dimethylamino)ethyl]‐2,1,3‐benzoxadiazole‐4‐sulfonamide (DAABD‐Cl) as a fluorogenic reagent demonstrated the existence of the low‐molecular‐weight thiols in the extract of Caenorhabditis elegans ( C. elegans ). The method includes derivatization of the thiols with DAABD‐Cl at 40°C for 10 min in borate buffer (pH 9.0) containing TCEP, CHAPS and EDTA, separation of the derivatives on an ODS column and fluorometric determination of the derivatives at 510 ± 15 nm with excitation at 400 ± 15 nm. The identification of the thiols was made by HPLC‐electrospray ionization mass spectrometry (LC‐MS) following isolation of the derivatives using HPLC‐fluorescence detection. Low‐molecular‐weight thiols were found to exist in the extract of C. elegans , such as cysteine, cysteinylglycine, γ ‐glutamylcysteine, reduced glutathione and two other unidentified thiol compounds, confirming the existence of the ‘glutathione cycle’ in C. elegans similar to the mammalian body. Copyright © 2007 John Wiley & Sons, Ltd.