Trypanothione Reductase Activity Assay

This assay, in which generation of reduced trypanothione [T(SH)₂] by TR is coupled with the chemical oxidant 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB; or Ellman’s reagent) (Hamilton et al., 2003; Richardson et al., 2009), was performed in 96-well plates. Reaction mixtures contained 50 mM HEPES pH 8.0, 1 mM EDTA, 30 µM NADP⁺, 1 µM oxidized trypanothione (TS₂), 0.1 mM DTNB, 0.15 mM NADPH and increasing concentrations of EGCG (7.8 – 500 µM). Reactions were started by addition of 7 nM recombinant LiTR.6His (i.e. C-terminally histidine-tagged L. infantum TR), purified from Escherichia coli by nickel chromatography and quantified by the bicinchoninic acid (BCA) protein assay (Pierce), using bovine serum albumin (BSA) as standard. Upon addition of LiTR.6His, the rate of TNB formation was monitored at 410 nm at 25°C for 5 minutes using a UV-2401PC UV–vis recording spectrophotometer (Shimadzu Scientific).