FIGURE 5.
Analysis of deacylation reaction by TLC and LC/MS. A, reaction products of [35S]Cys-tRNAPro deacylation by H. influenzae YbaK detected by cellulose TLC. Eluent and carrier solvents were n-butanol/acetic acid/water (4:1:1, v/v/v) and methanol, respectively. A unique product band (Rf = 0.21, lanes 3–5, Product) distinct from free cysteine (Rf = 0.15, lane 6) is observed. Lane 1, deacylation reaction in the absence of YbaK; lanes 2–5, deacylation reaction in the presence of YbaK at 0, 10, 20, and 30 min; lane 6, authentic [35S]cysteine; lane 7, [35S]cystine. B, LC traces of crude reaction mixtures following H. influenzae YbaK-catalyzed Cys-tRNAPro deacylation (black) or a control reaction in the absence of YbaK (gray). Free cysteine eluted at 1 min, and cysteine methyl ester eluted at 3.5 min.