Asamoto Hiromichi, Ichibangase Tomoko, Saimaru Hiroshi, Uchikura Kazuo, Imai Kazuhiro
Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.
Biomed Chromatogr. 2007 Oct;21(10):999-1004. doi: 10.1002/bmc.814.
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.
一种高灵敏度且简便的方法，使用以7-氯-N-[2-(二甲氨基)乙基]-2,1,3-苯并恶二唑-4-磺酰胺（DAABD-Cl）作为荧光试剂的高效液相色谱-荧光检测法，证明了秀丽隐杆线虫提取物中存在低分子量硫醇。该方法包括在含有三(2-羧乙基)膦（TCEP）、3-[(3-胆酰胺丙基)二甲基铵]-1-丙磺酸（CHAPS）和乙二胺四乙酸（EDTA）的硼酸盐缓冲液（pH 9.0）中，于40℃用DAABD-Cl将硫醇衍生化10分钟，在十八烷基硅烷键合硅胶（ODS）柱上分离衍生物，并在激发波长为400±15 nm时于510±15 nm处对衍生物进行荧光测定。硫醇的鉴定是在使用高效液相色谱-荧光检测分离衍生物后，通过高效液相色谱-电喷雾电离质谱（LC-MS）进行的。发现秀丽隐杆线虫提取物中存在低分子量硫醇，如半胱氨酸、半胱氨酰甘氨酸、γ-谷氨酰半胱氨酸、还原型谷胱甘肽以及另外两种未鉴定的硫醇化合物，证实了秀丽隐杆线虫中存在类似于哺乳动物体内的“谷胱甘肽循环”。
