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.
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.
Asamoto
Hiromichi
H
Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.
Ichibangase
Tomoko
T
Saimaru
Hiroshi
H
Uchikura
Kazuo
K
Imai
Kazuhiro
K
D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry*; D029742:Caenorhabditis elegans Proteins; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D010069:Oxadiazoles / Q000737:chemistry / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry*; D013449:Sulfonamides / Q000737:chemistry / Q000302:isolation & purification*
C491139:4-(dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole; D029742:Caenorhabditis elegans Proteins; D004151:Dipeptides; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D013438:Sulfhydryl Compounds; D013449:Sulfonamides; C028505:cysteinylglycine; D005978:Glutathione; D003545:Cysteine; C017341:gamma-glutamylcysteine