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 performed by spectrophotometric kinetic measurements of conjugated product formation with substrates such as reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). This requires a spectrophotometer that can measure absorbance in the UV range and microcentrifugation to remove the particulates from crude homogenates which absorb light at 340 nm. Such an assay is not ideal for detecting elevated levels of GST activity in insects under field conditions, which is a requirement in, for example, insecticide resistance management programs. We have developed a simple quantitative assay for visually 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 titration. 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 Anopheles gambiae agGST1-6 enzyme concentrations and for crude homogenates of individual insects. Results of the application of this test in the diagnosis of GST-based insecticide resistance are presented, demonstrating its practicality for field use.