CCRF-Mitchell Laboratory, Cambridge Research Laboratories, 181A Huntingdon Rd., Cambridge CB3 ODL, UK.
Cancers (Basel). 2010 Jun 2;2(2):1092-106. doi: 10.3390/cancers2021092.
The low molecular weight thiol (-SH) content of a human prostate carcinoma cell line (LNCap), important to the cellular resistance to drugs and irradiation, was investigated using three forms of thiol assay each utilizing different chemistries. The composition of the mixture was examined by derivatization of the thiols with a three-fold excess of the Ellman reagent to give mixed aromatic disulfides. The components were isolated by chromatography on C18 reverse phase silica gel followed by DE52 anion exchange separation, and then analyzed by capillary electrophoresis against prepared standards. The glutathione adduct (GSSE) and an unknown disulfide (RSSE) were the major components isolated on DE52 together with two minor ones. However, from the absorbance at 325 nm, it was found that the GSSE isolated (1.5 ± 0.2 femtomoles/cell) could only account for 28.5 ± 4.3% of the total ASF thiols. It appeared that the bulk of the thiol material had not formed a stable mixed disulfide with Ellman's reagent, and this was confirmed by 35S labeling of the cells. A large proportion of the 35S labeled components, obtained after reaction of the ASF thiols with the Ellman reagent, did not form mixed aromatic disulfides and could therefore not be identified by this labeling method.
采用三种不同化学性质的巯基检测方法，研究了人前列腺癌细胞系（LNCap）的低分子量巯基（-SH）含量，这对于细胞对药物和辐射的抵抗力很重要。用 Ellman 试剂三倍过量衍生化巯基，生成混合芳香二硫化物，检测混合物的组成。用 C18 反相硅胶进行色谱分离，然后用 DE52 阴离子交换分离，再用对照品进行毛细管电泳分析。在 DE52 上分离出的主要成分是谷胱甘肽加合物（GSSE）和一种未知的二硫化物（RSSE），此外还有两种次要成分。然而，根据 325nm 的吸光度，发现分离出的 GSSE（1.5±0.2 飞摩尔/细胞）仅占总 ASF 巯基的 28.5±4.3%。似乎大部分巯基物质没有与 Ellman 试剂形成稳定的混合二硫化物，这一点通过对细胞进行 35S 标记得到了证实。在 ASF 巯基与 Ellman 试剂反应后，获得的大部分 35S 标记的成分没有形成混合芳香二硫化物，因此无法用这种标记方法进行鉴定。
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