Enzyme kinetics of optimized RLUC proteins. (A) Derivation of KM values. The KM for wild-type RLUC was similar to previously published data such as 210 nM (coelenterazine h)5 and 300 nM23. Also shown are wild-type RLUC, RLUC+ and SuperRLUC, which were purified by nickel affinity chromatography and run on a polyacrylamide gel. (B) Luminescence spectra for RLUC, RLUC+ and Super-RLUC. (C) Time course of luminescence activity. Note the increased half life of activity of the RLUC+ and SuperRLUC mutants. (D) Inhibition of RLUC activity by high substrate concentration.