Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

Abstract

: The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate-coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization-Ca²⁺-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme-substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.

Keywords: Ca2+-regulated photoprotein; analytical systems; bioluminescence; coelenterazine; luciferase.

Figure 1

Variety of coelenterazine-dependent bioluminescent systems. (a–d) pathways of coelenterazine oxidation with the release of blue light, found in diverse organisms. Luc—luciferase; apoPhP—apophotoprotein; apoCBP—apo-coelenterazine binding protein; CTZ and CTM—coelenterazine and coelenteramide. Explanations are given in the text.

Figure 2

Chemical structures of coelenterazine and its analogues, mentioned in the review. Imidazopyrazinone core is shown in a circle.

Figure 3

Ca2+ concentration—effect curves for recombinant photoproteins. L—light intensity at the particular Ca2+ concentration; Lint—total light intensity at saturating Ca2+ concentration.

Figure 4

General scheme for bioluminescent solid-phase binding assay. Label is a chemically synthesized conjugate or genetically fused recognition element with a reporter molecule (PhP).

Figure 5

General principle of the BRET-based analytical systems.

Figure 6

General principle of the assay based on the split luciferase complementation effect.

Figure 7

Main principles on the basis of which analytical methods and investigations can be carried out.