Liquid chromatography–electrospray mass spectrometry study of cysteine-10 S-glutathiolation in recombinant glutathione S-transferase of Ochrobactrum anthropi

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 787; no. 2; pp. 405 - 413
• Main Authors: Celli, Nicola, Motos-Gallardo, Agata, Tamburro, Antonio, Favaloro, Bartolo, Rotilio, Domenico
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 25.04.2003
• Subjects: Amino Acid Sequence; Base Sequence; Chromatography, Affinity - methods; Cysteine; Cysteine - metabolism; DNA, Bacterial; Glutathione S-transferase; Glutathione Transferase - metabolism; Molecular Sequence Data; Ochrobactrum anthropi - enzymology; Recombinant Proteins - metabolism; Spectrometry, Mass, Electrospray Ionization - methods; Ochrobactrum anthropi; Glutathione S-transferase; Cysteine
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/S1570-0232(02)00706-7

Glutathione S-transferase of Ochrobactrum anthropi (OaGST), a bacterium isolated from soils contaminated by xenobiotic pollutants, was recently purified, cloned and characterised in our laboratories. The recombinant OaGST (rOaGST), highly expressed in Escherichia coli, when purified by glutathione-affinity chromatography and then analysed by electrospray ionisation mass spectrometry (ESI-MS), has evidenced a disulphide bond with glutathione ( S-glutathiolation), which was removable by reduction with 2-mercaptoethanol. Enzymatic digestion of rOaGST with endoproteinase Glu-C, followed by liquid chromatography (LC)–ESI-MS analyses of the peptide mixtures under both reducing and not reducing conditions, have shown that glutathione was covalently bound to the Cys10 residue of rOaGST. Furthermore, LC–ESI-MS analyses of overexpressed rOaGST in Escherichia coli crude extracts, with and without incubation with glutathione, have not shown any S-glutathiolation of the recombinant enzyme.