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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
Authors:Asamoto Hiromichi  Ichibangase Tomoko  Saimaru Hiroshi  Uchikura Kazuo  Imai Kazuhiro
Institution:Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.
Abstract: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 degrees 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, gamma-glutamylcysteine, reduced glutathione and two other unidentified thiol compounds, confirming the existence of the 'glutathione cycle' in C. elegans similar to the mammalian body.
Keywords:fluorogenic reagent  DAABD‐Cl  HPLC  LC‐MS  low‐molecular‐weight thiols  Caenorhabditis elegans
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