The earthworm Lumbricus rubellus (Hoffmeister, 1843) is a terrestrial pollution sentinel. Enzyme activity and transcription of phase II detoxification superfamily glutathione transferases (GST) is known to respond in earthworms after soil toxin exposure, suggesting GST as a candidate molecular-based pollution biomarker. This study combined sub-proteomics, bioinformatics and biochemical assay to characterise the L. rubellus GST complement as pre-requisite to initialise assessment of the applicability of GST as a biomarker. L. rubellus possesses a range of GSTs related to known classes, with evidence of tissue-specific synthesis. Two affinity-purified GSTs dominating GST protein synthesis (Sigma and Pi class) were cloned, expressed and characterised for enzyme activity with various substrates. Electrospray ionisation mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) following SDS-PAGE were superior in retaining subunit stability relative to two-dimensional gel electrophoresis (2-DE). This study provides greater understanding of Phase II detoxification GST superfamily status of an important environmental pollution sentinel organism.\ud \ud This study currently provides the most comprehensive view of the Phase II detoxification enzyme superfamily of glutathione transferases within the important environmental pollution sentinel earthworm Lumbricus rubellus.\ud \u
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