Study of cytosolic glutathione s-tranferase activity with styrene oxide by electromigration methods
| Authors | |
|---|---|
| Year of publication | 2004 |
| Type | Article in Proceedings |
| Conference | Book of abstract 14 th International Symposium on Capillary Electroseparation Techniques ITP 2004 |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | GST; capillary electrochromatography; MEKC |
| Description | Styrene-7,8-oxide is an industrial chemical used in the manufacture of epoxy resins; as an intermediate in the preparation of various agricultural chemicals, cosmetics and plastics; and in the processing of textile and fibres. SO is also the major cytochrome P450 metabolite of styrene, used in plastic and resin manufacture. As epoxide, SO is highly reactive and able to undergo reactions with various nucleophilic groups in tissue components, causing radiomimetic effects such as growth inhibition, cytotoxicity and mutagenicity. In addition SO has been classified as carcinogenic in animals and is probably carcinogenic in humans. Its detoxification may occur due to glutathione S-transferase (GST) catalysed conjugation with cytosolic glutathione. As people working in a number of industries are exposed to styrene and SO, it is important to examine the role of glutathione conjugation versus other competing detoxication pathways. Similar studies have often been hampered by the lack of reliable methods for quantitating the glutathione adducts. In this study two electromigration methods - capillary electrochromatography (CEC) in silica-based polymeric monolithic columns and micellar electrokinetic capillary chromatography (MEKC) with SDS as pseudostationary phase were tested for this purpose. Because of relatively hydrophilic nature of given conjugates no separation was achieved by CEC whereas good separation of conjugates was obtained by MEKC. The method based on MEKC was further optimised and used for measuring GST activity with SO. Unlike previous methods, proposed assay did not require the use of radiolabelled substrates, and was sufficiently sensitive to detect conjugate formation in tissues with low GST activity. In addition it could be applied for determination of kinetic parameters of GST - pH and temperature optima of enzymatic reaction, and Michaelis constants (Km) for SO. |
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