| Autor: |
Kudryavtsev AN; Institute of Biophysics, Federal Research Center 'Krasnoyarsk Science Center SB RAS', 660036 Krasnoyarsk, Russia., Krasitskaya VV; Institute of Biophysics, Federal Research Center 'Krasnoyarsk Science Center SB RAS', 660036 Krasnoyarsk, Russia., Efremov MK; School of Fundamental Biology and Biotechnology, School of Non-Ferrous Metals and Material Science, Siberian Federal University, pr. Svobodny 79, 660041 Krasnoyarsk, Russia., Zangeeva SV; School of Fundamental Biology and Biotechnology, School of Non-Ferrous Metals and Material Science, Siberian Federal University, pr. Svobodny 79, 660041 Krasnoyarsk, Russia., Rogova AV; School of Fundamental Biology and Biotechnology, School of Non-Ferrous Metals and Material Science, Siberian Federal University, pr. Svobodny 79, 660041 Krasnoyarsk, Russia.; Kirensky Institute of Physics, Federal Research Center 'Krasnoyarsk Science Center SB', 660036 Krasnoyarsk, Russia., Tomilin FN; School of Fundamental Biology and Biotechnology, School of Non-Ferrous Metals and Material Science, Siberian Federal University, pr. Svobodny 79, 660041 Krasnoyarsk, Russia.; Kirensky Institute of Physics, Federal Research Center 'Krasnoyarsk Science Center SB', 660036 Krasnoyarsk, Russia., Frank LA; Institute of Biophysics, Federal Research Center 'Krasnoyarsk Science Center SB RAS', 660036 Krasnoyarsk, Russia.; School of Fundamental Biology and Biotechnology, School of Non-Ferrous Metals and Material Science, Siberian Federal University, pr. Svobodny 79, 660041 Krasnoyarsk, Russia. |
| Abstrakt: |
Ca 2+ -triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca 2+ . NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca 2+ -triggering "character". |
