Decay properties of RLUC enzyme activity. (A) A titration assay indicates that 1 molecule of RLUC can turn over ~100 molecules of coelenterazine. A 1 μM solution of native coelenterazine substrate was incubated with increasing amounts of RLUC enzyme (1 nM to 100 nM) and the reaction was allowed to go to completion. Once light emission had ceased, the reaction was split in half and supplemented with fresh enzyme (squares) to examine whether all substrate had been depleted; or with fresh substrate (circles) to check for residual enzyme activity. Note, if an excess of substrate was added, the loss of RLUC activity was not reversible by adding fresh substrate. (B) Spectrophotometric confirmation of substrate turnover by RLUC. Absorbance spectra were recorded for 10 μM coelenterazine before (black trace) and after (blue trace) turnover by 100 nM RLUC. Two traces of partially completed reactions are shown for comparison (red and green traces). (C) Half life of RLUC activity in the in vitro assay as measured by luminescence decay kinetics.