High-performance liquid chromatography-based assay for glutathione transferase theta 2 activity: Application to...

Human glutathione transferase T2-2 (GSTT2-2) is one of the enzymes considered to play a role in inactivation of toxicants and carcinogens. The expression level of this enzyme is determined by genetic and environmental factors, which may lead to differences in susceptibility. As a specific assay for GSTT2-2 so far a spectroscopical assay based on GSH-conjugation of menaphthyl sulfate (MSu) was used. This spectrophotometric assay, however, appeared too insensitive to accurately quantify the GSTT2-2 activities in a panel of 20 human liver samples. More recently, expression levels of GSTT2-2 in biological samples are quantified by measuring mRNA levels. Since mRNA-levels do not always correlate well with enzyme activity, a specific and sensitive assay is required. In the present study a highly sensitive high-performance liquid chromatography (HPLC)-based method was developed. By applying the new method, firstly, the specificity of GSTT2-2 among 15 recombinant human GST isoforms in catalyzing GSH-conjugation of MSu was confirmed. In addition, a 65-fold inter-individual variation of GSTT2-2 activity was found from the individual liver fractions. By applying the method to individual liver fractions, a 65-fold inter-individual variation of GSTT2-2 activity was found. As a second application, the role of GSTT2-2 in GSH-conjugation of the environmental carcinogen 1-methylpyrene sulfate (MPS) was studied by correlation analysis with GSTT2-2-catalyzed MSu conjugation. The relatively poor correlation suggested that other GSTs also contribute to MPS-conjugation, as confirmed by incubations with recombinant GSTs.