DETERMINATION OF TOTAL GLUTATHIONE IN CELL LYSATES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH O-PHTHALALDEHYDE PRECOLUMN DERIVATIZATION IN THE PRESENCE OF TRIS(2-CARBOXYETHYL)-PHOSPHINE

Abstract

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 with o-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.

ACKNOWLEDGMENTS

This work was supported by the Swiss Science Foundation Grant No. 3100-040807.94/1 and by the Kanton of Zurich/Switzerland.