Fig. 1
Biochemical and preliminary cell-based characterization of naphthyl-luciferin bioluminescence substrates and different luciferases. a Chemical structures of NH2-NpLH2, OH-NpLH2, and D-LH2. The naphthyl moieties (i.e., naphtho[2,1]thiazole) provide additional π conjugation. b Homology model of CBR highlighting residues R334 and G351. c Bioluminescence emission spectra of D-LH2, NH2-NpLH2, and OH-NpLH2 produced by purified CBR and CBR2 (data presented as means (n = 3) ± S.D.). Peak values for CBR are reported in Table 1. For CBR2 the peak values are as follows: D-LH2, 614 nm; NH2-NpLH2, 730 nm; OH-NpLH2, 743 nm. The spectrum for Luc2/D-LH2 (peak emission 559 nm) is shown for reference. d Live cell (HEK-293) bioluminescence intensity (RLUmax) for different combinations of substrate and luciferase (n = 3). CBR2opt is a gene encoding the same CBR2 enzyme as the CBR2 gene, but it uses codons optimized for expression in mammalian cells. There was no detectable signal for Luc2/OH-NpLH2. e Physical stability of CBR, CBR2 (encoded by CBR2opt) and Luc2 in HEK-293 lysates at 37 °C (n= 3). f Bioluminescence emission spectra of D-LH2 and NH2-NpLH2 produced by CBR2opt and Luc2 cells (HEK-293). Emission peaks: Luc2/D-LH2, 608 nm; CBR2opt/D-LH2, 617 nm; CBR2opt/NH2-NpLH2, 728 nm. The signal for Luc2/NH2-NpLH2 was too low to generate meaningful spectral data. Attempts to collect spectra for OH-NpLH2 were also unsuccessful due to insufficient signal. Error bars represent standard deviation (S.D.)
