Quantitative Enzymatic Assay for rGSTs

rGSTs of PRMs catalyze the conjugation of GSH to 1-chloro-2,4-dinitrobenzene (CDNB) substrate [44 (link)]. In this study, we measured enzyme activity at different concentrations of each rGST in the presence of a constant concentration of GSH and the CDNB substrate. Briefly, 20 µL of each rGST (1, 2, and 4 µg) in Tris/NaCl buffer (10 mM Tris, 0.5 M NaCl, pH 7.4) were placed in the 96-well microtiter plate in triplicate and the same amount of Tris/NaCl buffer was placed in the control wells. We dissolved 100 µL of 1 mM CDNB in substrate buffer (100 mM potassium dihydrogen phosphate, 1 mM EDTA, pH 6.5, and 2 mM GSH) and placed it in all the wells. Immediately, the absorbance at 340 nm was measured every minute using a microplate reader MTP 900 (Corona Electric Co., Ltd, Ibataki, Japan) for 20 min. To adjust the spontaneous hydrolysis of CDNB, the mean absorbance of the control wells was subtracted from that of the test wells. The specific enzyme activities (μmol/min/mg protein) at different concentrations of each rGST were calculated using the following formula [54 (link)].
$$\frac{\left(At2-At1\right)\times 1000}{A\epsilon \times \left(t2-t1\right)\times b\times m}\times Vtot$$
At1—initial time point
At2—final time point
t1—starting time (min)
t2—end time (min)
Aε—the molar extinction coefficient of CDNB (Aε = 9.6)
b—path length of the spectrophotometer (0.286 cm)
m—the quantity of rGST per well (mg)
Vtot—total volume per well (L).