Quantitative evaluation of luminescence intensity from enzymatic luminescence reaction of coelenterazine and analogues

Recent advances in bioluminescence technology rely on the discovery of luciferin-luciferase pairs having excellent luminescence activity. Here, we focus on coelenterazine (CTZ) and its analogues and quantitatively investigate reaction factors, including bioluminescence reaction quantum yield (φ_BL) and enzyme reaction kinetics (turnover rate, k_cat), determining the luminescence intensity to design brighter reaction systems. Initially, it was confirmed that the CTZ was kept stable during the storage and the φ_BL value measuring experiment in terms of comparative study using benzyl-protected CTZ. Using a luminometer whose absolute responsivity is calibrated for each luminescence reaction spectrum, we absolutely measured φ_BL and other reaction factors of CTZ and 3 types of blue-shifted CTZ analogues, CTZ 400a, 6-pi-H-CTZ and 6-pi-Ph-CTZ. The results suggested that there was no significant difference in reaction kinetics, but a clear difference in φ_BL (up to 22-fold), which was presumably due to an improvement in fluorescence quantum yield (φ_FL) of the corresponding coelenteramide (CTMD), oxidized form of CTZ. Our evaluation suggests that the design of CTZ analogues considering φ_FL is important for developing a bright luminescent reaction system.