Quantitative acetyltransferase assay (DTNB based)

DTNB assay or Ellman’s assay is a colorimetric assay that detects generation of free sulfhydryl group. In an acetyltransferase reaction, the products formed are acetylated substrate and CoASH. This free CoASH reacts with DTNB to form mixed disulfide and TNB which gives a yellow color at 412 nm. Thus, enzyme assays were set up according to published protocol^42,43 along with control reactions a) Substrate control (Peptide + acetyl coA) b) Enzyme control (acetyl coA + RimI^Mtb). Briefly, reaction components were added in 50 mM Tris Buffer pH 8.0, 4 mM peptide substrate, 0.5 mM acetyl coA in a total reaction volume of 0.1 ml. Reactions were initiated by addition of 8 μM of RimI^Mtb and samples incubated at 25 °C for one hour. Enzyme activity was quenched by addition of 60 μl of 3.2 M Gn-Hcl and 10 μl of 2 mM DTNB was added. The reactions were vortexed and allowed to stand for 5 min before absorbance was measured at 412 nm in a plate reader. Absorbance of enzyme control was subtracted from enzyme assay readouts. For each peptide, the amount of product formed was calculated from CoASH calibration curve (0.1 mM–1 mM) and specific activity of the enzyme calculated. The mean average value and standard deviation for triplicate experiments were plotted.