DETERMINATION OF TOTAL GLUTATHIONE IN CELL LYSATES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITHO-PHTHALALDEHYDE PRECOLUMN DERIVATIZATION IN THE PRESENCE OF TRIS(2-CARBOXYETHYL)-PHOSPHINE
作者:
Ragna Sack,
Annemarie Willi,
PeterE. Hunziker,
期刊:
Journal of Liquid Chromatography & Related Technologies
(Taylor Available online 2000)
卷期:
Volume 23,
issue 19
页码: 2947-2962
ISSN:1082-6076
年代: 2000
DOI:10.1081/JLC-100101835
出版商: Taylor & Francis Group
数据来源: Taylor
摘要:
A very sensitive HPLC method for the determination of total glutathione (GSH) and γ-glutamyl-cysteine (γ-Glu-Cys) content in cell lysates is presented. It is based on a precolumn derivatization witho-phthalaldehyde (OPA) using tris(2-carboxyethyl)-phosphine (TCEP) as a reducing agent. Separation of the peptide derivatives is carried out by reversed-phase chromatography on a C18 column, followed by spectrofluorimetric detection. The fluorescence response of GSH and γ-Glu-Cys derivatives is linear over a range of 0.05 to 100 μM and 1 to 25 μM, respectively, with good precision. The detection limits approach 26 fmol and 60 fmol on column, respectively. In contrast the detection limit for GSH in the presence of DTT, which is often used as the reducing agent, is 250 fmol GSH on column. The results for the GSH concentration of cell lysates are in agreement with a radiotracer method based on metabolic labeling of GSH with [35S]-Cys.
点击下载:
PDF (213KB)
返 回