Studies On The Low Molecular Weight Thiols Of Mouse Ehrlich Ascites Tumour Cells: Glutathione Content

Author(s): Michael Gronow

The total thiol contents of mouse Ehrlich ascites tumour cells and that of the acid soluble fraction (ASF), released on deproteinization of these cells, were measured using the Ellman reagent, 5,5’ dithio (2-nitrobenzoic acid)(ESSE). It was shown that the ASF constituted 13.2% of the total cellular thiol at an average value of 3.20 + 0.29 femtomoles of RSH per cell and this was confirmed using three different methods of thiol estimation. This extract was shown to contain practically negligible amounts of reducing inorganic sulphur ions, disulphide or vicinal disulphide material. The aromatic mixed disulphides (RSSE) formed by direct addition of the ASF thiols to a three fold excess of Ellman reagent (ESSE) were analysed by ion exchange chromatography and capillary electrophoresis. These analyses revealed only two major components, the glutathione adduct (GSSE) and excess ESSE. Some minor RSSE components were also present and this was confirmed by ³⁵S labelling of these cells. The GSH content, as calculated from the GSSE formed, was 1.280.04 femtomoles/cell which accounts for only 423% of the thiol content of the ASF and 5.3% of the total cellular thiol. Furthermore, the G³⁵S SE isolated from the radioisotope labelled cells contained only13.6% of the ³⁵S incorporated into the ASF. Apart from glutathione, a large proportion, ca 60%, of the thiols present in the ASF of these cells, although reacting with the Ellman reagent do not appear to retain the aromatic thionitrobenzoic acid residue, indicating that they do not form stable aromatic mixed disulphides. These results indicate that caution should be employed in interpreting Ellman values of cellular ASF thiols as attributable solely to the presence of glutathione.

Mouse ascites tumour, non-protein thiols, radio-isotope labelling, Ellman reagent, Ellman thiol adducts, glutathione content, chromatography, capillary electrophoresis

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