Measurement of GST enzyme activity

GST activity assays were conducted with 1-chloro-2,4- dinitrobenzene (CDNB) according to previous protocols [20, 26]; with the change in absorbance at 340 nm was measured at 25°C for 6 mins at 30s intervals in a microplate reader. A change in absorbance per min was converted into micromoles of substrate conjugated/min/mg of protein by adjusting the molar extinction coefficient of CDNB ε340 9.6 mM^-1 cm^-1 to 5.3 mM^-1 (path length 0.552 cm). In our hands, the presence or absence of the N-terminal 6xHis tag of rGSTA2 and rGSTA3 did not alter full activity of the enzymes.

Affinity-purified rDdGSTA2 and rDdGSTA3 protein concentrations were determined by A260/280 spectrophotometry, and 5 ug of protein was used in each reaction. Assays were performed at 25°C at pH range from 3–9 with 1 mM GSH and 1mM CDNB as substrates in a 200 μl reaction volume. For pH 3.0–7.0, 100 mM citrate-phosphate buffer; pH 6.0–8.0, 100 mM potassium phosphate buffer and pH 8.0–9.0, 100 mM Tris-HCl buffer was used. The maximal enzyme activity is plotted as 100% relative activity.

GST kinetic assay for calculating K_m and V_max of GSH for rGSTA2 and rGSTA3 enzymes was assayed with CDNB at a saturating concentration and different concentrations of GSH. For calculation of K_m and V_max of CDNB, GSH was used at saturating concentration and CDNB at different concentrations. K_m and V_max were calculated by non-linear regression analysis of hyperbolic plots (V vs. S) using the Michaelis-Menten equation. GraphPad Prism 5 software was used for the analysis.