Fig. 2

Secondary enzyme screening assays at nicotine concentrations found in smokers: Activity of purified enzyme variants. (a) His-tagged wt NicA2 enzyme (circles) or mutant NicA2A107R (squares) were captured from E. coli lysates by anti-His-tag mAb in assay wells. Buffer containing 250 nM S-nicotine was added, samples retrieved, and heat inactivated at the time points indicated. Samples were run on the biosensor instrument, and Response Units were plotted as a function of time. (b) Purified enzyme or mutant NicA2A107R were added to rat serum containing 40 ng/mL S-(−)-nicotine, and samples were withdrawn and activity quenched by MeOH addition at the time points indicated. Residual nicotine concentration was measured by GC. (c) Enhanced activity of 10 variants over wt NicA2 at 40 ng/mL nicotine in serum. Each purified variant (1.5 μg/mL) was added to rat serum containing 40 ng/mL nicotine at 37 °C, and activity quenched with methanol at various timepoints. Open symbols indicate increased activity compared to wt of > 3-fold in this assay. Nicotine concentrations were measured by GC