Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase.
It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca(2+)-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.
Stepanyuk
Galina A
GA
Photobiology Laboratory, Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia.
Unch
James
J
Malikova
Natalia P
NP
Markova
Svetlana V
SV
Lee
John
J
Vysotski
Eugene S
ES
D000818:Animals; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry*; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049408:Luminescent Agents / Q000737:chemistry*; D009154:Mutation; D011485:Protein Binding; D011719:Pyrazines / Q000737:chemistry*; D048790:Renilla / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D002135:Calcium-Binding Proteins; D007093:Imidazoles; D049408:Luminescent Agents; D011719:Pyrazines; C017144:coelenterazine; D008156:Luciferases; D002118:Calcium