A simple biochemical assay for glutathione S-transferase activity and its possible field application for screening glutathione S-transferase-based insecticide resistance

Glutathione S-transferase (GST) activity assays in insects are usually perf ormed by spectrophotometric kinetic measurements of conjugated product form ation with substrates such as reduced glutathione (GSH) and 1-chloro-2,4-di nitrobenzene (CDNB). This requires a spectrophotometer that can measure abs orbance in the UV range and microcentrifugation to remove the particulates from crude homogenates which absorb light at 340 nm. Such an assay is not i deal for detecting elevated levels of GST activity in insects under field c onditions, which is a requirement in, for example, insecticide resistance m anagement programs. We have developed a simple quantitative assay for Visua lly determining GST activity in individual insects. The substrates GSH and CDNB are used in this assay. After the linear enzyme reaction has run for a fixed time, free GSH is determined stoichiometrically by iodometric titrat ion. The results can be determined visually from the discrete color change. We demonstrate the equivalence of this iodometric end point assay and the standard kinetic assay for a five-fold range of purified recombinant Anophe les gambiae agGST1-6 enzyme concentrations and for crude homogenates of ind ividual insects. Results of the application of this test in the diagnosis o f GST-based insecticide resistance are presented, demonstrating its practic ality for field use. (C) 2000 Academic Press.